GB2437281A

GB2437281A - Linum transformation method using acetohydroxyacid synthase gene selection marker

Info

Publication number: GB2437281A
Application number: GB0607937A
Authority: GB
Inventors: Beata Dedicova
Original assignee: BASF Plant Science GmbH
Current assignee: BASF Plant Science GmbH
Priority date: 2006-04-21
Filing date: 2006-04-21
Publication date: 2007-10-24
Also published as: GB0607937D0

Abstract

A method for transforming a plant of the genus Linum such as Linseed (L. usitatissium) wherein the selectable marker is an acetohydroxyacid synthase gene. Preferably the transformation method uses hypocotyls expants and is mediated by Rhzobiaceae bacteria such as Agrobacterium. The acetohydroxyacid synthase gene may be isolated from Arabidopsis and be under the control of its own promoter. Constructs comprising the acetohydroxyacid synthase gene and its promoter are claimed, as are methods of multiple rounds of plant transformation.

Description

Acetohydroxyacid synthase inhibiting herbicide as a selectable marker

for the introduction of a nucleic acid sequence into the genome of a plant of the genus Linum The present invention relates to improved methods for the introduction of a nucleic acid sequence into the genome of a plant of the genus Linum, preferably linum usitatissimum based on selection with an acetohydroxyacid synthase inhibiting herbicide. Preferably, the introduction, e.g. transformation, is mediated by Agrobacterium.

The selection of transgenic cells and later on transgenic tissues and plants by employing adequate selection system is an essential step in a successful transformation experiment. The selection system furthermore has to be suitable for the particular crop. Moreover the selection system has to have public acceptance, as it is employed in food production.

Although several different selection systems are known, for linseed only the selectable marker systems based on antibiotic resistance is used, for a survey see e.g. WO 01/05221.

Transgenic linseed plants were selected under antibiotic selection pressure even for the introduction of herbicide resistence genes, see e.g. Mc Hughen, development and preliminary field testing a glucosinate-ammonium tolerant transgenic flax, Can. J. Plant Science 75 (1995), 117-120.

Multiple subsequent transformations of a plant of the genus Linum with more than one construct (necessary for some of the more complicated high-value traits and for gene stacking) is complicated due to the limited availability of suitable selection markers.

This situation is becoming compounded as antibiotic resistance markers (such as hygromycin or kanamyciri resistance) become less viable options as a result of tightened regulatory requirements and environmental concerns.

Accordingly, the object of the present invention is to provide an improved, reliable, efficient method for regeneration and selection of stably transformed plants of the genus Linum, preferably linum usitatissimum. This objective is achieved by the present invention.

The present inventions provides a method for generating a transgenic plant of the genus Linum comprising the following steps a. introducing into a cell of a plant of the genus linum a construct comprising i) a first nucleic acid sequence encoding an acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide operably linked to a promoter active in said cell and ii) optionally a second nucleic acid sequence, preferably conferring to said plant an agronomically valuable trait operably linked to a promoter active in said plant, and b. incubating the cell of step a) on a selection medium comprising an acetohydroxyacid synthase inhibiting herbicide in a concentration of 0.1 pM or more for a time period of 5 days or more, and c. transferring the cell of step b) to a regeneration medium and regenerating and optionally selecting the plant comprising said construct.

20050987 Wil 19.04.2006 7 Fig + Seq The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides or polymers or hybrids thereof in either single-or double-stranded, sense or antisense form.

A nucleic acid sequence refers to a consecutive list of abbreviations, letters, characters or words, which represent nucleotides. The coding region or open reading frame of said nucleic acid is the portion of the nucleic acid, which is transcribed and translated in a sequence-specific manner to produce into a particular polypeptide or protein when placed under the control of appropriate regulatory sequences, such as a promoter.

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e. g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term "nucleic acid" is used interchangeably herein with "gene", "cDNA and "mRNA".

A construct refers to a nucleic acid sequence at least partly created by recombinant methods. The term construct is referring to a RNA or DNA. The construct may be single-or -preferably -double stranded. The construct may be circular or linear. The skilled worker is familiar with a variety of ways to obtain the construct of the present invention.

The person skilled in the art is aware of numerous nucleic acid sequences encoding an acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide. The term "acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide" means preferably an enzyme, whose catalytic activity is relatively more resistant to the presence of an acetohydroxyacid synthase inhibiting herbicide compared to a wild type acetohydroxyacid synthase.

The acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide of the present invention need not have the total catalytic activity necessary to maintain the viability of the cell of a plant of the genus linum, but must have some catalytic activity in an amount, alone or in combination with the catalytic activity of additional copies of the same acetohydroxyacid synthase to maintain the viability of a cell of a plant of the genus linum if exposed to an acetohydroxyacid synthase inhibiting herbicide. For example, catalytic activity may be increased to minimum acceptable levels for resisitance to acetohydroxyacid synthase inhibiting herbicides by introducing multiple copies of a nucleic acid sequence encoding a acetohydroxyacid synthase resistant to acetohydroxyacid synthase inhibiting herbicides into the cell or by introducing said nucleic acid sequence operably linked to a strong promoter to enhance the production of the acetohydroxyacid synthase resistant to acetohydroxyacid a synthase inhibiting herbicide.

More resistant means that the catalytic activity of the variant is diminished by the acetohydroxyacid synthase inhibiting herbicide, if at all, to a lesser degree than the wild-type, e.g. by 5 %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% less than the wild-type when measured as described in Us 5,928,937 (structure-based designed herbicide resistant products), column 13, lines 22 to 30, which is incorporated herewith by reference.

Preferably the catalytic activity of an acetohydroxyacpd synthase resistant to an acetohydroxyacid synthase inhibiting herbicide of the present invention retains sufficient to maintain the viability of a cell of a plant of the genus linum wherein at the same concentration of the same herbicide, wild-type acetohydroxyacid synthase would not retain sufficient catalytic activity to maintain the viability of the cell. Preferably the enzymatic properties are measured as described in US 5,928,937 (structure-based designed herbicide resistant products), column 13, lines 19 to 52, which is incorporated herewith by reference.

Preferably, the acetohydroxyacid synthase (E.C. 4.1.3.18) resistant to acetohydroxyacid synthase inhibiting herbicides is as disclosed by G.W Haughn, J. Smith, B.J. Mazur, C. Somerville, Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistance to sulfonylurea herbicides. Mol. Gen. Genet 204 (1986) 430-434 or B. 3. Mazur, C.F. Chui, J.K. Smith, isolation and characterization of plant gene for acetolactate synthase, the target enzyme for two classes of herbicides. Plant Physiology 85 (1987) 1110-111, which are incorporated herewith by reference. Especially preferred is the nucleic acid sequence as described by SEQ ID NO: 9.

A suitable acetohydroxyacid synthase resistant to acetohydroxyacid synthase inhibiting herbicides also includes fragments, mutants, derivatives, variants and alleles of the polypeptides exemplified above. Suitable fragments, mutants, derivatives, variants and alleles are those, which retain the functional characteristics of acetohydroxyacid synthase resistant to acetohydroxyacid synthase inhibiting herbicides as defined above.

Changes to a sequence, to produce a mutant, variant or derivative, may due to addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide. Of course, changes to the nucleic acid that make no difference to the encoded amino acid sequence are included.

Mutants and derivatives of the specified sequences can also comprise enzymes, which are improved in one or more characteristics (Ki, substrate specificity etc.) but still are resistant to acetohydroxyacid synthase inhibiting herbicides.

More preferably for the method of the invention, the acetohydroxyacid synthase is selected from the group consisting of i) an acetohydroxyacid synthase with an amino acid sequence as described by SEQ ID NO: 10, ii) an acetohydroxyacid synthase having an amino acid sequence identity of 8O%, preferably 85%, more preferably 90%, even more preferably 91%, 92%, 93%, 94% or 95%, most preferably 96%, 97%, 98% or 99% or more to the amino acid sequence as described by SEQ ID NO: 10, and iii) an acetohydroxyacid synthase encoded by a nucleic acid sequence capable to hybridize to the nucleic acid sequence as described by SEQ ID NO: 9 or its corn plement.

Even more preferably for the method of the invention, the acetohydroxyacd synthase is selected from the group consisting of i) an acetohydroxyacici synthase with an amino acid sequence as described by SEQ ID NO: 10, ii) an acetohydroxyacid synthase having an amino acid sequence identity of 80%, preferably 85%, more preferably 90%, even more preferably 91%, 92%, 93%, 94% or 95%, most preferably 96%, 97%, 98% or 99% or more to the amino acid sequence as described by SEQ ID NO: 10, and iii) an acetohydroxyacid synthase encoded by a nucleic acid sequence capable to hybridize to the nucleic acid sequence as described by SEQ ID NO: 9 or its corn plement and selection is done on a medium comprising an acetohydroxyacid synthase inhibiting herbicide in a concentration from 0.25 to 2 pM, more preferably from 0.5 to 1,75 pmM, even more preferably from 0.75 to 1,5 pM, most preferably from 1 to 1,5 pM and further most proferably from about 1 to 1,25 pM.

For the purposes of the invention hybridzation means preferably hybridization under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 C with washing in 2 X SSC, 0. 1% SDS at 50 C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 C with washing in 1 X SSC, 0.1% SDS at 50 C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 C with washing in 0.5 X SSC, 0. 1% SDS at 50 C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 C with washing in 0.1 X SSC, 0.1% SDS at 50 C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 C with washing in 0.1 X SSC, 0.1% SDS at 65 C to a nucleic acid comprising 50 to 200 or more consecutive nucleotides.

For the purposes of the invention, the percent sequence identity between two nucleic acid or polypeptide sequences is determined using the Vector NTI 7.0 (PC) software package (InforMax, 7600 Wisconsin Ave., Bethesda, MD 20814). A gap-opening penalty of 15 and a gap extension penalty of 6.66 are used for determining the percent identity of two nucleic acids. A gap-opening penalty of 10 and a gap extension penalty of 0.1 are used for determining the percent identity of two polypeptides. All other parameters are set at the default settings. For purposes of a multiple alignment (Clustal W algorithm), the gap-opening penalty is 10, and the gap extension penalty is 0.05 with blosum62 matrix. It is to be understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymidine nucleotide sequence is equivalent to an uracil nucleotide.

The acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide of the invention may be expressed in the cytosol, peroxisome, or other intracellular compartment of the plant cell.

The term "promoter as used herein is intended to mean a DNA sequence that directs the transcription of a DNA sequence (e.g., a structural gene). Typically, a promoter is located in the 5' region of a gene, proximal to the transcriptional start site of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter. Also, the promoter may be regulated in a tissue-specific or tissue preferred manner such that it is only active in transcribing the associated coding region in a specific tissue type(s) such as leaves, roots or meristem.

The term "promoter active in a cell of a plant of the genus Linum" means any promoter, whether plant derived or not, which is capable to induce transcription of an operably linked nucleotide sequence in at least one cell, tissue, organ or plant of the genus Linum at at least one time point in development or under dedifferentiated conditions. Such promoter may be a non-plant promoter (e.g., derived from a plant virus or agrobacter-lum) or a plant promoter, perferably a dicotyledonous plant promoter.

The person skilled in the art is aware of several promoter which might be suitable for use in a plant of the genus Linum. In this context, expression can be constitutive, inducible or development-dependent.

The promoter of step i) of claim 1 and the promoter of step ii) of claim 1 can be the same or different. The promoter of step i) of claim 1 and the promoter of step ii) of claim 1 are preferably different. This applies also for the other embodiments of the present invention.

The promoter operably linked to the agronomically valuable trait of the present invention is preferably active in the tissue of the plant of the genus linum where the agronomically valuable trait is expressed.

Preferred are constitutive promoters. Most preferred is the Arabidopsis thaliaria AHAS acetohydroxyacid synthase promoter.

The Arabidopsis thaliana AHAS acetohydroxyacid synthase promoter is preferably selected from the group consisting of a) a nucleic acid sequence comprising the nucleic acid sequence as described by SEQ ID NO: 8, and b) a nucleic acid sequence having promoter activity in a cell of a plant of the genus linum comprising at least one fragment of at least 50, 75, 100, 125, 150, 175, 200, 225 or 250 consecutive base pairs of the nucleic acid sequence as described by SEQ ID NO: 8, and, c) a nucleic acid sequence having promoter activity in a cell of a plant of the genus linum comprising a nucleic acid sequence having at least 60%, especially 80%, preferably 85%, more preferably 90%, even more preferably 91%, 92%, 93%, 94% or 95%, most preferably 96%, 97%, 98% or 99% or more identity to the nucleic acid sequence as described by SEQ ID NO: 8, and, d) a nucleic acid sequence having promoter activity in a cell of a plant of the genus linum comprising a nucleic acid sequence hybridizing to the nucleic acid sequence as described by SEQ ID NO: 8 or its complement.

The construct of the invention (or the vectors in which these are comprised) may comprise further functional elements and genetic control sequences in addition to the promoter active in plants of the genus Linum. The terms "functional elements" or "genetic control sequences" are to be understood in the broad sense and refer to all those sequences, which have an effect on the materialization or the function of the nucleic acid sequence according to the invention. For example, genetic control sequences modify transcription and translation of a gene. Genetic control sequences are described for example by Gruber (1993) "Vectors for Plant Transformation," in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY; CRC Press, Boca Raton, Florida, eds.: Glick and Thompson, Chapter 7, pp.89-119 and the references cited therein.

Functional elements which may be comprised in a vector of the invention include I) Origins of replication which ensure replication of the construct according to the invention in, for example, E. coli. Examples which may be mentioned are ORI (origin of DNA replication), the pBR322 on or the P1SA on (Sam brook et al.: Molecular Cloning. A Laboratory Manual, 2' ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), ii) Multiple cloning sites (MCS) to enable and facilitate the insertion of one or more nucleic acid sequences, iii) Sequences which make possible homologous recombination, marker deletion, or insertion into the genome of a host organism. Available marker deletion methods are shown for example on p. 3, I. 1 to 27 of the international patent application WO 2005/090581 (Improved constructs for marker excision based on dual-fnction selection marker), which is incorporated herewith in its entirety, iv) Elements, for example border sequences, which make possible the Agrobacterium-mediated transfer in plant cells for the transfer and integration into the plant genome, such as, for example, the right or left border of the T-DNA or the vir region.

The term "transgene" as used herein refers to any nucleic acid sequence, which is introduced into the genome of a cell or which has been manipulated by experimental manipulations by man. Preferably, said sequence is resulting in a genome which is different from a naturally occurring organism (e.g., said sequence, if endogenous to said organism, is introduced into a location different from its natural location, or its copy number is increased or decreased).

The term "transgenic" or "recombinant" when used in reference to a cell or an organism (e.g., with regard to a barley plant or plant cell) refers to a cell or organism which contains a transgene, or whose genome has been altered by the introduction of a transgene. A transgenic organism or tissue may comprise one or more transgenic cells. Preferably, the organism or tissue is substantially consisting of transgenic cells (i.e., more than 80%, preferably 90%, more preferably 95%, most preferably 99% of the cells in said organism or tissue are transgenic).

A further object of the present invention are the above and below described constructs. The constructs of the present invention comprise a) a first nucleic acid sequence encoding an enzyme as defined in claim 5 linked to the promoter as defined in claim 10 or 11 and b) optionally a second nucleic acid sequence conferring to a plant of the genus linum an agronomically valuable trait operably linked to a promoter active in a plant of the genus linum.

A further embodiment of the present invention is a plant or a part of a plant comprising a construct comprising a) a first nucleic acid sequence encoding an enzyme as defined in claim 5 linked to the promoter as defined in claim 10 or 11 and b) optionally a second nucleic acid sequence conferring to said plant an agronomically valuable trait operably linked to a promoter active in a cell of a plant of the genus linum.

Preferably, the construct inserted into the genome of the target plant comprises at least one second construct, which confers to a plant of the genus Linum an agronomically valuable trait. This can be achieved by expression of selection markers, trait genes, antisense RNA or double-stranded RNA. The person skilled in the art is aware of numerous sequences which may be utilized in this context, e.g. to increase quality of food and feed, to produce chemicals, fine chemicals or pharmaceutica's e.g., vitamins, oils, carbohydrates, conferring resistance to herbicides, or conferring male sterility. Furthermore, growth, yield, and resistance against abiotic and biotic stress factors (like e.g., fungi, viruses or insects) may be enhanced.

A construct according to the invention may advantageously be introduced into cells using vectors into which said construct is inserted. Examples of vectors may be plasmids, cosmids, phages, viruses, retroviruses or Agrobacteria. In an advantageous embodiment, the construct is introduced by means of plasmid vectors. Preferred vectors are those, which enable the stable integration of the construct into the host genome, i.e. the binary vector pBPSMM192a (Fig. 1.) as described by SEQ ID NO: 1.

The construct can be introduced into the target plant cells or organisms by any of the several means known to those of skill in the art. Various transformation procedures suitable for a plant of the genus Linum have been described, eg. WO 01/05221 Al, page 6 to 7 (Novel methods for the generation and selection of transgenic linseed/flax plants) which is incorporated herewith by reference.

Especially preferred is the introduction of the construct with Agrobacterium. It is known in the art that not only Agrobacteriurn but also other soil-borne bacteria are capable to mediate T-DNA transfer provided that the relevant functional elements for the T-DNA transfer of an Ti-or Ri-plasmid (van Veen RJM et al. (1988) Mol Plant Microb Interact 1(6):23i.-234) are available. More preferably, transformation is mediated by a Rhizobiaceae bacterium selected from the group of disarmed Agrobacterium tumefaciens or Agrobacterium rhizogenes bacterium strains. Especially preferred is Agrobacterium tumefaciens. Most preferred is the Agrobacterium strain LBA4404.

Another preferred embodiment of the present method for generating a transgenic plant of the genus Linum, preferably linum usitatissimum, is comprising the steps of a. a. isolating a hypocotyl segment of a plant of the genus linum and b. introducing the construct of step a) of claim 1 with the aid of a bacterium of the genus Rhizobiaceae into said hypocotyl segment by co-cultivating said hypocotyl segment with said bacterium and, c. optionally transferring the co-cultivated hypocotyl segment to a regeneration medium, said regeneration medium lacking a phytotoxic effective amount of an acetohydroxyacicj synthase inhibiting herbicide prior to selection, d. inducing formation of a callus and selecting transgenic callus on a medium comprising, i. an effective amount of at least one auxin corn pound, and ii. an acetohydroxyacici synthase inhibiting herbicide in a concentration of 0.1 pM or more, and e. regenerating and optionally selecting plants containing the construct from the said transgenic callus..

Another embodiment is the above described method, wherein the construct of step a) of claim 1 is part of a T-DNA.

For preperation of the Agrobacterium inoculum preferably two days prior to inoculation Agrobacterium is taken from a frozen glycerol stock onto about 5 ml of LB liquid medium containing about 100mg/I rifampicillin and about 50mg/I kanamycin and placed on a shaker (250 rpm) overnight at 28 C. One Day before inoculation the Agro bacterium culture is preferably subcultured by placing 1 ml in a tube containing 4 ml of fresh LB medium with antibiotics which is placed again on a shaker (250 rpm) at 28 C overnight. On the day of transformation Agrobacterium is preferably spin down (10 min./ 5000 rpm) and pellet is re-suspended in 10 ml MS liquid medium (Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiologa Plantarum, 15 (1962) 473-479) to an O.D. 660 of 0.5.

The cell of a plant of the genus linum can be a differentiated cell or an undifferentiated cell. The cell of a plant of the genus linum can be isolated, in the form of tissue or in plantae. Various tissue can be employed for the transformation procedure disclosed herein. Such plant material may include but is not limited to for example leaf, root, immature and mature embryos, pollen, meristematic tissues, hypocotyl or inflorescences but also callus, protoplasts or suspensions of plant cells. Preferably, the plant material is hypocotyl. The material can be pre-treated (e.g., by inducing dedifferentiation prior to transformation) or not pre-treated.

The plant material for transformation (e.g., the hypocotyl explants) can be obtained or isolated from virtually any variety or plant of the genus Linum. Especially preferred are all linum species, e.g. linum usitatissiumum, linum sulcatum, linum perenne, linum narbonense, linum grundiflorum, linum flavum, linum elegans, linem catharticum, linum bienne, linum austriacum, linum arboreum especially of the linaceae family more especially preferred is linum usitatissimum. The method of the invention can be preferably used to produce transgenic plants from linum usitatissimum varieties such as the commercial varieties Flanders, SW0144403 (Svalöf Weibull), Gemini, McGregor, Ed 45 or Solin and especially preferred with Flanders Sw 00-4402.

However, it should be pointed out, that the method of the invention is not limited to certain varieties but is highly genotype-independent. Plants of the genus Linum for isolation of hypocotyl are grown as known in the art, preferably as described below in

the examples.

Hypocotyl is defined as a part of a germinating seedling of a seed plant. As the plant embryo grows at germination, it sends out a shoot called a radicle that becomes the primary root and penetrates down into the soil. After emergence of the radicle, the hypocotyl emerges and lifts the growing tip above the ground, bearing the embryonic leaves (cotyledons) and the plumule that gives rise to the first true leaves. The hypocotyl is the primary organ of extension of the young plant and develops into the stem. Especially preferred are segments of the hypocotyl of the size of about 2 to 4 mm.

Preferably hypocotyl segments for transformation are produced by the following procedure. Seeds of the genus linum are preferably rinsed with 70% ethanol for about 1 mm followed by about 20 mm sterilisation using 25% Klorin with one drop of Tween 80, rinsing four times in sterile deionised water. The germination medium preferably contains MS macro and microelements including vitamins, 3% sucrose and 0.8% agar. Seeds are preferably sown in Petri dishes and germinated in a vertical position in a growth chamber at about 24 C and about 16 h day/night photoperiod.

Seed material coming from field is preferably sterilised with about 0.02% HgCl2 instead of Klorin . Preferably, hypocotyl segments with a size of about 2-4 mm of about four days old linseed seedlings are used.

The term "about" is used for the purposes of this invention to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent, preferably 10 percent, more preferably 5 percent up or down (higher or lower).

The inoculation of the hypocotyl explants with Agrobacterium ranges preferably from about 45 to 140, more preferably for about 120 minutes preferably on MS-liquid medium. The inoculated explants are generally placed on a plant compatible co-cultivation medium preferably containing MS medium with vitamins, about 1.5% sucrose, supplemented with about 40 mM acetosyringone and about 1 mg/I 2,4-Dichlorophenoxyacetjc acid.

A range of co-cultivation periods from a few hours to 10 days may be employed. The co-cultivation of Agrobacterium with hypocotyl is in general carried out for about 12 hours to about 7 days, preferably about 3 days to about 4 days at about 20 C to about 26 C, preferably at about 22 C to about 24 C. The Co-culture plates preferably contains MS medium with vitamins, about about 40 mM acetosyringone, about 3% sucrose, about pH5.4, solidified with about 0.8% agar and plates are preferably incubated at about 24 C in dark.

Transformed cells can be selected from untransformed cells preferably using the selection method of the invention.

Prior to a transfer to a regeneration or a selection medium, especially in case of Agrobacterjum-rnedja transformation, certain other intermediate steps may be employed. Selection medium is defined as medium suitable for regeneration and selection of a transformed plant or part of a plant of the genus linum. For example, any Agrobacteria remaining from the co-cultivation step may be removed e.g., by a washing step. To prevent re-growth of said bacteria, the subsequently employed regeneration and! or selection medium preferably comprises a bacteriocide (antibiotic) suitable to prevent Agrobacteriurn growth. Preferred bactericidal antibiotics to be employed are e.g., carbenicillin, cefotaxime or TimentinTh (GlaxoSmithKline; a mixture of ticarcillin disodium and clavulanate potassium; 0.8 g TimentinTh contains 50 mg clavulanic acid with 750 mg ticarcillin. Chemically, ticarcillin disodium is N-(2-Carboxy-3,3-dimethyl-7-oxo-1-thia-1 -aza bicyclo[3.2. O]hept-6-yl)-3-thio-phenernalonarn ic acid disodjum salt. Chemically, clavulanate potassium is potassium (Z)-(2R, 5R)-3-(2-hyd roxyethylidene)-7-oxo_40 1 -azabicyclo{3.2.0] heptane-2-carboxylate).

Preferably, after the co-culture period, the hypocotyl segments are preferably washed two times about 15 minutes with sterile water.

In a further step the inoculated and washed hypocotyl explants can be cultived without selection pressure before including selection pressure. Preferably the inoculated and washed hypocotyl explants are cultivated on callus inducing and shoot regeneration medium including selection pressure of phytotoxic amount of an acetohydroxyacici synthase inhibiting herbicide, preferably about 1.5 pM imidazolinone, preferably Imazamox. These plates preferably contain MS medium with vitamins, about 2 mg/I

IC

BAP and about 0.1mg/I NM, about 200 mg/I cefotaxime, about 3% sucrose, pH 5.4- 5.6, solidified with 0.8% agar and plates are incubated preferably at about 24 C at a aboutl6/8h photoperiod with light intensity of about 40 pEm2.s1.

The term "phytotoxic" as used herein is intended to mean any measurable, negative effect on the physiology of a plant or plant cell resulting in symptoms including reduced growth, reduced photosynthesis, reduced cell division, reduced regeneration (e.g., of a mature plant from a cell culture, callus, or shoot etc.), reduced fertility.

Phytotoxicfty may further include effects like e.g., necrosis or apoptosis. A preferred embodiment results in an reduction of growth or regenerability of at least 50%, preferably at least 80%, more preferably at least 90% in comparison with a plant which was not treated with said phytotoxic compound.

The specificacetohydroxyacid synthase inhibiting herbicide employed for selection is chosen depending on which marker protein is expressed. The an acetohydroxyacid synthase inhibiting herbicide is generally present in a phytotoxic concentration.

In a preferred embodiment acetohydroxyacd synthase inhibiting herbicide is selected from the group consisting of imidazolinones, sulfonylureas, triazolopyrimicline sulfonamides, pyrim idyloxy-benzoic acids, sulfamoyl ureas, sulfonylca rboximides and corn binations thereof.

The optimal concentration of the acetohydroxyacid synthase inhibiting herbicide, may vary depending on the target tissue employed for transformation but in general the total concentration (i.e. the sum in case of a mixture) of the acetohydroxyacid synthase inhibiting herbicide ranges from 0,1 pM or more, preferably 0,25 pM or more and most preferably 1 pM or more.

Preferably the acetohydroxyacid synthase inhibiting herbicide is present in a concentration from 0.25 to 2 pM, more preferably from 0.5 to 1,75 pmM, even more preferably from 0.75 to 1,5 pM, most preferably from 1 to 1,5 pM and further most proferably from 1 to 1,25 pM.

Also the selection time may vary depending on the target tissue used and the regeneration protocol employed. In general a selection time is at least 5, preferably at least 10 days. More specifically the total selection time is from 1 to 30 weeks, preferably, 3 to 25 weeks, more preferably 6 to 20 weeks.

In between the selection period the callus may be transferred to fresh selection medium one or more times. Preferably selection is done in several steps, both during callus growth and during shoots regeneration and even under rooting conditions.

In an even more preferred embodiment, the selection of step b) of claim 1 of the present method is done in two steps, using a first selection step for a time period of 1 to 20, preferably for 1 to 16 and most preferably from 6 to 16 weeks and in a further embodiment from 15 to 21 days then transferring the surviving cells or tissue to a second selection medium with essentially the same composition than the first selection medium for additional from 2 to 7, more preferable from 3 to 4 weeks.

Preferably said selection medium is -for part of the selection period -also a dedifferentiation medium comprising at least one suitable plant growth regulator for

I

induction of embryogenic callus formation. The term "plant growth regulator" (PGR) as used herein means naturally occurring or synthetic (not naturally occurring) compounds that can regulate plant growth and development. PGRs may act singly or in consort with one another or with other compounds (e.g., sugars, amino acids). More specifically the medium employed for embryogenic callus induction and selection corn prises i. an effective amount of at least one auxin compound, and ii. an effective amount of a selection agent allowing for selection of cells comprising the transgenic.

Furthermore the callus induction medium may optionally comprise an effective amount of at least one antibiotic that inhibits or suppresses the growth of the soil-borne bacteria (as defined above).

The term "auxin" or "auxin compounds" comprises compounds which stimulate cellular elongation and division, differentiation of vascular tissue, fruit development, formation of adventitious roots, production of ethylene, and -in high concentrations -induce dedifferentiation (callus formation). The most common naturally occurring auxin is indoleacetic acid (IAA), which is transported polarly in roots and stems. Synthetic auxins are used extensively in modern agriculture. Synthetic auxin compounds comprise indole-3-butyric acid (IBA), naphthylacetic acid (NM), and 2,4-dichlorphenoxyacetic acid (2,4-D), Dicamba, especially preferred is NM. Furthermore, combination of different auxins can be employed, for example a combination of 2,4-D and Picloram or Dicamba.

Preferably, in one embodiment when used as the sole auxin compound, NM in a concentration of about 0.05 mg/I to about 6 mg/I, more preferably about 0.1 to about 0.5 mg/I, most preferably about 0.1 mg/I is employed.

The medium may be optionally further supplemented with one or more additional plant growth regulator, like e.g., cytokinin compounds (e.g., 6-benzylaminopurine) or other auxin compounds. Such compounds include, but are not limited to, IAA, NM, IBA, cytokinins, auxins, kinetins, and thidiazuron. Cytokinin compounds comprise, for example zeati n, 6-isopentenyladeni ne (IPA) and 6-benzyladenine/6-benzylarni nopurine (BAP). Especially preferred is BAP. BAP is generally applied in concentrations in the range from 0,1 to 10 mg/I, preferably from 0,5 to 6 mg/I and even more preferred from ito 4 mg/I and most preferred of about 2 mg/I.

In an most preferred embodiment the selection medium during callus growth is MS medium with vitamins, preferably Gamborg B5 vitamins, SAP preferably in a concentration from about 0.5 to about 10 mg/L, more preferably from about 1.0 to about 5 mg/L, even more preferred about 2 mg/I and about 0.1mg/I NM, about 150 mg/I cefotaxime, about 3% sucrose, pH 5.8, solidified with about 0.8% agar and supplemented with a selective agent, e.g. imidazolinone in a concentration of about 0,5 to 1,5 pM depending on the germplasm used within transformation for a time period of about 150 days.

The callus tissue is preferably isolated after about 14 to 21 days and further subcultured onto fresh MS medium supplemented with preferably supplemented with about 2 mg/I BAP and about 0.1mg/I NM, about 150 mg/I cefotaxime, about 3% sucrose, pH 5.8, solidified with about 0.8% agar and supplemented with imidazolinone l2 in a concentration of about 0,5 to 1,5 pM depending on the germplasm used within transformation for a time period of about 150 days.

Green shoots of the above mentioned callus tissue are preferably placed onto elongation medium carrying out the second selection step (MS micro & macro elements with vitamins, about 250 mg/I timentin, about 1% sucrose, pH 5.8, solidified with about 0.8% agar and supplemented with the selective agent, preferably imidzolinone, preferably in a concentration of 1 to 5 pm at about 23 C under a photoperiod of about 16 hours light intensity about 40 pEm2.s' for about 3 to 4 weeks.

In a further step shoots, which were elongated as described above and remained green, are generally further cultured on root inducing medium preferably at about 24 C under about 16/8 h photoperiod with light intensity of about 40 pEm2.s1. Preferably the rood inducing medium is MS hormone free und comprises about 150 mg/I Cefotaxime, about 3% sucrose, pH about 5.4-5.6, 0.8% agar, and 0,5 to 1,5, preferably 0,75 -1,25.0 pM of the acetohydroxyacid synthase inhibiting herbicide, preferably Imazamox. At this step a plant sample can be taken to determine the presence of the T-DNA, e.g. by applying Tagman analyses. Methods for said identification are well known in the art, e.g. PCR analysis, Northern blot, Southern blot, or phenotypic screening.

Generally depending on the germplasm after about 3 to 4 weeks the first small roots can be observed. Rooted shoots are generally transferred into soil and kept moistened, covered and shaded until acclimatized. Transgenic plants after acclimatisation are generally growing for about 12 weeks in the greenhouse (19-21 C) till ripe seeds can be collected. Two or more generations should be grown in order to ensure that the genomic integration is stable and hereditary For example transgenic events in Ti or T2 generations could be involved in pre breeding hybridization program for combining different transgenes (gene stucki ng).

The presence of acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide does not rule out that additional markers are employed.

Other important aspects of the invention include the progeny of the transgenic plants prepared by the disclosed methods, as well as the cells derived from such progeny, and the seeds obtained from such progeny.

Descendants may comprise one or more copies of the agronomically valuable trait gene. Preferably, descendants are isolated which only comprise one copy of said trait gene.

Other embodiments of the invention relate to parts, organs, cells, fruits, and other reproduction material of a plant of the genus Linum, preferably linum usitatissimum of the invention. Preferred parts are selected from the group consisting of tissue, cells, pollen, ovule, roots, leaves, seeds, microspores, and vegetative parts.

Another object of the present invention is the use of the plants according to the present invention for the production of food, pharmaceuticals or fine chemicals.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification are incorporated herein in their entirety by reference. Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figure described below.

The methods and compositions of the invention allow for subsequent transformation.

The selection with an acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide of the present invention is compatible and does not interfere with other selection marker and selection systems. It is therefore possible to transform existing transgenic plants comprising another selection marker with the constructs of the invention or to subsequently transform the plants obtained by the method of the invention (and comprising the expression constructs for acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide) with another marker. Thus, another embodiment of the invention relates to a method for subsequently introducing at least two constructs into a plant of the genus Linum, preferably linum usitatissinium comprising the steps of a) introducing a first construct comprising at least one nucleic acid encoding an acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide operably linked to a promoter active in said cell, and b) introducing a second construct comprising a second nucleic acid sequence encoding a marker protein which is not conferring resistance against imidazolinone operably linked to a promoter active in said cell.

Preferably said second marker gene is a negative selection marker conferring a resistance to a biocidal compound such as a metabolic inhibitor (e.g., 2-deoxyglucose- 6-phosphate, WO 98/45456), antibiotics (e.g., kanamycin, G 418, bleomycin or hygromycin) or herbicides (e.g., phosphinothricin or glyphosate). Examples are: -Phosphinothricin acetyftransferases (PAT; also named Bialophos resistance; bar; US 4,975,374) -S-enolpyruvylshikimate3phosphate synthase (EPSPS) conferring resistance to Glyphosate (N-(phosphonomethyl)glycine) (Shah et al. (1986) Science 233: 478) -Glyphosate degrading enzymes (Glyphosate oxidoreductase; gox), -Dalapon inactivating dehalogenases (deh) -Bromoxynil degrading nitrilases (bxn) -Kanamycin-or. geneticin (G418) resistance genes (NPTII; NPTI) coding e.g., for neomycin phosphotransferases (Fraley 1983; Nehra 1994) -hygromycin phosphotransferase (HPT), which mediates resistance to hygromycin (Vanden Elzen et al. (1985) Plant Mol Biol. 5:299).

-dihydrofolate reductase (Eichholtz et al. (1987) Somatic Cell and Molecular Genetics 13: 67-76) Preferably, said second marker gene is defined as above and is most preferably conferring resistance against at least one compound select from the group consisting of phosphi notricin, glyphosate, phosphinotrici n, glyphosate-type herbicides.

A further object of the present inventions relates to a plant or a part of a plant of the genus linum comprising a) a nucleic acid sequence encoding an acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide operably linked to a promoter active in said plant and optionally b) a second nucleic acid sequence conferring to said plant of the genus linum an agronomically valuable trait operably linked a promoter active in said plant.

The present invention relates furthermore to a plant or a part of a plant of the genus Li num, preferably linum usitatissimum comprising a) a first nucleic acid sequence encoding an acetohydroxyacid synthase resistant to an acetohydroxyacici synthase inhibiting herbicide operably linked to a promoter active in said plant, and b) a second nucleic acid sequence encoding a selection marker protein, which is not conferring resistance against imidazolinone.

The invention also relates to a plant or the part of a plant of claim 16, wherein the acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide is defined by claims S or 6.

The invention also relates to a plant or the part of a plant comprising claim 13.

The present invention provides a further improved, reliable, efficient, publicly acceptable method for the generation and selection of stably transforming a plant of the genus Linum. It offers a minimized escape rate without interfering with hypocotyl callus formation and high number of transgenic shoots regeneration of plants of the genus Linum..

Sequences SEQ ID NO: 1 binary vector containing AtAHAS expression cassette and GUS cassette SEQ ID NO: 8 Arabidopsis thaliana acetohydroxyacid synthase promoter SEQ ID NO: 9 Arabidopsis thaliana acetohydroxyacici synthase large subunit gene SEQ ID NO: 10 Arabidopsis thaliana acetohydroxyacid synthase large subunit Figures Fig.1. Map of the binary vector pBPSMM192a.

Fig. 2. Nucleic acid sequence encoding an acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide Fig. 3. Linseed hypostyles in v/tro culture.

Fig. 4. Shoots formation from callus tissues.

Fig. 5. Gus assay on leaves material from plants in the greenhouse.

Fig. 6. Gus assay on leaves material from in vitro plants.

Fig. 7. Transgenic linseed plants in greenhouse.

Examples

Unless indicated otherwise, chemicals and reagents in the Examples were obtained from Duchefa or Sigma-Aldrich AB, . The cloning steps carried out for the purposes of the present invention, such as, for example, transformation of E. coli cells, growing bacteria, multiplying phages and sequence analysis of recombinant DNA, are carried out as described by Sambrook (1989). The following examples are offered by way of illustration and not by way of limitation.

Abbreviations MS Murashige and Skoog micro and macro elements md. vitamins O.D. Optical Density BAP 6-benzyladenine/6-benzylami nopurine NM naphthylacetic acid 1. Plant material Linseed seeds variety Flanders (batch n. 00-44002 Swalöf Weibull, AB) were rinsed with 70% ethanol for 1 mm following by 20 mm sterilisation using 25% Klorin with one drop of Tween 80, rinsing four times in sterile deionised water. The germination medium contained MS macro and microelements including vitamins, 3% sucrose and 0.8% agar. Seeds were sown in Petri dishes and germinated in a vertical position in a growth chamber at 24 C and 16 h day/night photoperiod. Seed material coming from field was sterilised with 0.02% HgCI2 instead of Klorin , following the same procedure.

2. Preparation of Agrobacterium Inoculum Two days prior to inoculation one loop of Agrobacterium was taken from a frozen glycerol stock onto 5 ml of LB liquid medium containing 100mg/I rifampicillin and 50mg/I kanamycin and placed on a shaker (250 rpm) overnight at 28 C. Day before inoculation the Agro bacterium culture is subculture by placing 1 ml in a tube containing 4 ml of fresh LB medium with antibiotics which was placed again on a shaker (250 rpm) at 28 C overnight. On the day of transformation Agrobacterium was spin down (10 mm.! 5000 rpm) and pellet was re-suspended in 10 ml MS liquid medium (47) to an O.D. 660 of 0.5.

Transformation was performed using the bacterial strain LBA4404 carried constructs pBPSMM192a (Fig. 1.) harbouring mutate Arab/dopsis t/ia/iana gene (G.W Haughn, 3.

Smith, B.J. Mazur, C. Somerville, Transformation with a mutant Arab/dopsis acetolactate synthase gene renders tobacco resistance to sulfonylurea herbicides. Mol. Gen. Genet 204 (1986) 430-434, B. 3. Mazur, C.F. Chui, 1K. Smith, isolation and characterization of plant gene for acetolactate synthase, the target enzyme for two classes of herbicides. Plant Physiology 85 (1987) 1110-111), 3. Inoculation Hypocotyls segments (2-4 mm) of four days old hypocotyls linseed seedlings were cut using sterile scalpel under the surface of S ml MS medium which was later replaced by 24 hour grown culture of Agrobacterium for 2 h. After twohour infection period the Agrobactermum solution was aspirated off the hypocotyls segments. The segments were then blotted dry between two pieces of sterile filter paper and transferred to co-culture plates. Co-culture plates containing MS medium with vitamins, 1 mg/I 2,4-D, 40 mM acetosyringone, 3% sucrose, pH 5.4, solidified with 0.8% agar and plates were incubated at 24 C in dark.

4. Tissue Culture and Regeneration After three days hypocotyl segments were rinsed three times with sterile distilled water and transferred to plates with regeneration medium. These plates contained MS medium with vitamins, 2 mg/I BAP and 0.1mg/I NM, 200 mg/I cefotaxime 3% sucrose, pH 5.4-5.6, solidified with 0.8% agar and plates are incubated at 24 C 16/8h photoperiod with light intensity 40 pEm2.s'.

5. Callus growth and shoots regeneration under selection After sixteen days the hypocotyls segments were moved onto MS medium with 2 mg/I BAP and 0.1mg/I NM, 150 mg/I cefotaxime 3% sucrose, 0.8% agar or 0.1 mg/I Kinetin and 1.5 pM Imazamox 14 to 21 days. The response of transgenic plant tissues on imidazolinones was visible, callus was growing and expanding rapidly and green shoots were developed. Green shoots were then placed in rooting MS hormone free medium with 150 mg/I Cefotaxime, 3% sucrose, pH 5.4-5.6, 0.8% agar, and carried out the Second Selection Step 1.0 pM Imazamox at 24 C under 16/8 h photoperiod with light intensity 40 pEm2.s1 for 21 days.

Leaves from in vitro regenerated plants are screened for the Gus expression (R.A.

Jefferson, K.J. Wilson, The Gus gene fusion system. Plant Mol. Biol. (1991) B14: 1-33) and dark blue reaction in the leaf tissues showed transgenic origin of the regenerated shoots (Fig. 4). Later on the same test was performed with transgenic plants adapted and growing in vivo in greenhouse.

Rooted shoots were placed in moistened Jiffy pots and kept enclose plastic boxes until acclimatized at 20 C, 80% humidity and with 16/8 photoperiod with 60 pEm2.s'light intensity. Shoots were hardened off 5-10 days and they were growing 2 weeks under the following conditions 24 C, with 16/8 photoperiod with 60 pEm2.s1light intensity.

The presence of T-DNA introduced through the transformation process is determined by polymerase chain reaction (PCR). PCR was carried out with AHAS primers and confirmed transgenic origin of tested plants. Determination of copy. number of transgene was done by Taqman and these results indicated the integration of one or two copies of the nucleic acid sequence as described in figure 2 into the original plant genome. Transgenic plants after acclimatisation have been grown for 12 weeks in greenhouse (24 C, with 16/8 photoperiod) till maturity when seeds were collected.

Figures 3 to 7 show the results of different stages of the regeneration and selection process according to the present invention as well as the transgenic linseed plants obtainable by the process of the present invention.

pbpsmml92a.st25.txt

SEQUENCE LISTING

<110> BAsF Plant Science GmbH <120> Acetohydroxyac-jd synthase inhibiting herbicide as a selectable marker for the introduction of a nucleic acid sequence into the genome of a plant of the genus Linum <130> PF 57661 <140> AE 20041075 <141> 2006-04-19 <160> 10 <170> Patentln version 3.3 <210> 1 <211> 16064 <212> DNA <213> Artificial <220> <223> binary vector containing AtAHAS expression cassette and GUS cassette <220> <221> promoter <222> (7786).. (8766) <223> promoter region of Arabidopsis acetohydroxyac-jd synthase large subunit <220> <221> CDS <222> (8839).. (10851) <223> Arabidopsis acetohydroxyac-jd synthase large subunit gene <220> <221> terminator <222> (8839).. (12067) <223> AtAHASL terminator including 3' UTR <220> <221> promoter <222> (12224).. (13335) <223> Super promoter <22 0> <221> CDS <222> (13361).. (15358) <223> CDS of GUS including potato ST-LS]. intron [PIV-2J <22 0> <221> terminator <222> (15429).. (15681) <223> Nopal-ine synthase terminator region from Agrobacterium tumefacj ens <400> 1 actttgatcc aacccctccg ctgctatagt gcagtcggct tctgacgttc agtgcagccg 60 tcttctgaaa acgacatgtc gcacaagtcc taagttacgc gacaggctgc cgccctgccc 120 ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa agtagaatac ttgcgactag 180 Page 1 cit pbpsmml92a.st25.txt aaccggagac attacgccat gaacaagagc gccgccgctg gcctgctggg ctatgcccgc 240 gtcagcaccg acgaccagga cttgaccaac caacgggccg aactgcacgc ggccggctgc 300 accaagctgt tttccgagaa gatcaccggc accaggcgcg accgcccgga gctggccagg 360 atgcttgacc acctacgccc tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc 420 cgcagcaccc gcgacctact ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg 480 cgtagcctqg cagagccgtg ggccgacacc accacgccgg ccggccgcat ggtgttgacc 540 gtgttcgccg gcattgccga gttcgagcgt tccctaatca tcgaccgcac ccggagcggg 600 cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc gccctaccct caccccggca 660 cagatcgcgc acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa agaggcggct 720 gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcagcgaggaagtg 780 acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac 840 gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg caccaggacg 900 gccaggacga accgtttttc attaccgaag agatcgaggc ggagatgatc gcggccgggt 960 acgtgttcga gccgcccgcg cacgtctcaa ccgtgcggct gcatgaaatc ctggccggtt 1020 tgtctgatgc caagctggcg gcctggccgg ccagcttggc cgctgaagaa accgagcgcc 1080 gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt 1140 atatgatgcg atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt 1200 acttaaccag aaaggcgggt caggcaagac gaccatcgca acccatctag cccgcgccct 1260 gcaactcgcc ggggccgatg ttctgttagt cgattccgat ccccagggca gtgcccgcga 1320 ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat 1380 tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg gagcgcccca 1440 ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca 1500 gccaagccct tacgacatat gggccaccgc cgacctggtg gagctggtta agcagcgcat 1560 tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac 1620 gcgcatcggc ggtgaggttg ccgaggcgct ggccgggtac gagctgccca ttcttgagtc 1680 ccgtatcacg cagcgcgtga gctacccagg cactgccgcc gccggcacaa ccgttcttga 1740 atcagaaccc gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc 1800 aaaactcatt tgagttaatg aggtaaagag aaaatgagca aaagcacaaa cacgctaagt 1860 gccggccgtc cgagcgcacg cagcagcaag gctgcaacgt tggccagcct ggcagacacg 1920 ccagccatga agcgggtcaa ctttcagttg ccggcggagg atcacaccaa gctgaagatg 1980 tacgcggtac gccaaggcaa gaccattacc gagctgctat ctgaatacat cgcgcagcta 2040 ccagagtaaa tgagcaaatg aataaatgag tagatgaatt ttagcggcta aaggaggcgg 2100 catggaaaat caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac 2160 gggcggttgg ccaggcgtaa gcggctgggt tgtctgccgg ccctgcaatg gcactggaac 2220 Page 2 pbpsmmlg2a. st25.txt ccccaagccc gaggaatcgg cgtgacggtc gcaaaccatc cggcccggta caaatcggcg 2280 cggcgctggg tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac 2340 gcatcgaggc agaagcacgc cccggtgaat cgtggcaagc ggccgctgat cgaatccgca 2400 aagaatcccg gcaaccgccg gcagccggtg cgccgtcgat taggaagccg cccaagggcg 2460 acgagcaacc agattttttc gttccgatgc tctatgacgt gggcacccgc gatagtcgca 2520 gcatcatgga cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga 2580 tccgctacga gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca 2640 gtgtgtggga ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc 2700 gataccggga agggaaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg 2760 tactcaagtt ctgccggcga gccgatggcg gaaagcagaa agacgacctg gtagaaacct 2820 gcattcggtt aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc 2880 gcctggtgacggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg 2940 aaaccgggcg gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca 3000 cagaaggcaa gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg 3060 gcatcggccg ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat 3120 ggttgttcaa gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt 3180 tcaccgtgcg caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggagg 3240 cggggcaggc tggcccgatc ctagtcatgc gctaccgcaacctgatcgag ggcgaagcat 3300 ccgccggttc ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag 3360 gtcgaaaagg tctctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg 3420 ggaaccggaa cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt 3480 aagtgactga tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactta 3540 ttaaaactct taaaacccgc ctggcctgtg cataactgtc tggccagcgc acagccgaag 3600 agctgcaaaa agcgcctacc cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc 3660 ggcctatcgc ggccgctggc cgctcaaaaa tggctggcct acggccaggc aatctaccag 3720 ggcgcggaca agccgcgccg tcgccactcg accgccggcg cccacatcaa ggcaccctgc 3780 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc 3840 acagcrtgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt 3900 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact 3960 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa 4020 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgcr tcctcgctca 4080 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4140 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4200 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4260 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4320 Page 3 pbpsmmlg2a. st25.txt tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4380 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 4440 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4500 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4560 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 4620 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 4680 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 4740 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 4800 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 4860 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgcat tctaggtact 4920 aaaacaattc atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca 4980 gtaagtcaaa aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga 5040 cgcagaaggc aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc 5100 cacttacttt gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga 5160 caagttcctc ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa 5220 atggagtgtc ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt 5280 aatccaattc ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga 5340 tggagtgaaa gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt 5400 catcttcata ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc 5460 agccatcatg ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata 5520 gcatcatgtc cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca 5580 tttttaaata taggttttca ttttctccca ccagcttata taccttagca ggagacattc 5640 cttccgtatc ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc 5700 tcattttagc catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga 5760 agctaattat aacaagacga actccaattc actgttcctt gcattctaaa accttaaata 5820 ccagaaaaca gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga 5880 gccgattttg aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca 5940 tgctaccctc cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg 6000 aatagcatcg gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg 6060 tcccggactg atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg 6120 agctgttggc tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa 6180 cttaataaca cattgcggac gtttttaatg tactgaatta acgccgaatt aagctcaatt 6240 gactagtggc qcgcccacgt gttaattaac ggtccgaggc ctcctcagca agctgttaac 6300 gcgatcgcgc tgaggcggac cgcacgtgga attcgagctc ggtacccggg gatcctctag 6360 Page 4 pbpsnimlg2a. st25.txt attatgtatt tccaactttc attaacaata taatcgcata taaatgaaaa atcgtttcca 6420 ggataatatt ttgatgaaat ctcatattat tgttcgtact cggattgatg ttgaaggctt 6480 gaagcgcttc aaattataga ccagattatt taagtttttc ttttgtttac tccatatcaa 6540 tttgatccat tatactacct aagaaaattt aggtaacata gaattattta ttgttatagt 6600 aaaaaaaagg aaaaccacaa aaataatcta cttttacgta tatactattt tcatgacata 6660 agtaattaag ttgtacaact tttttttaat gaaaagagag agtaaatrta tcatgttcat 6720 gtgtagttac ctcgtgaata accgacggtt atatagacgc ctaacatgaa ttgttcagtt 6780 gaagacagtt caaaacatgt gtttcactct aaaatcctca acaaaaaaaa agtgttaaaa 6840 tttgtaaacc tctttcaagc aaaaaaagaa aaagtgttag aatcccaaga ttctttcata 6900 atccggaatc ttggctgaaa acgtataaaa gagattgacg tagtaacaag gagtcttggt 6960 atgcttccat gctttttatc cttttttgtc atggaaccat gatttggtta ccatttatta 7020 tgtaaccgaa attttcattg taataatgaa tatttaaatt tttagcaaaa aaaaacaaaa 7080 aaaaacaagg agtcttgtct tcgttctcaa atttcagagc tcttgcactt ttcaagagtt 7140 ttactttgat gagtgagaca tttgtctttt tagtgtttat tttctaaact taaaatagta 7200 gcatcaacat cactcaatta taattcttaa gatgttgtag aaaaatattt tatagatgga 7260 aagtaatcga tattaagaca aataagaaac caaaccggac tttgtgttca gaccgaatca 7320 aatctgaatt ggagaaatta tggtggaggc gaaagtcaac ggaactaaag tataaaacca 7380 aatgtcaaaa ataaaaccca attttcatcc ttaaacgaac ctgctgaaac cctaatttcg 7440 attaccaatt ccgatctaaa aagaagtcat ggaagccatt gattccgcaa tcgatcctct 7500 cagagatttc gctaagagca gtgttcgtct cgtccagcgc tgtcacaaac ccgatcgcaa 7560 gggtaacgcc ttttctcaaa aaaatctcat ttccgatttt tgatctgtag attagggttt 7620 tctgaaattt tgatatcatt tgtaattgaa ttggttatca gaattcacga aagtagctgt 7680 gcgtacggcg attggatttg tggtgatggg attcgttgga ttcttcgtga agctcgtttt 7740 catcccaatc aacaacatca tcgttggatc ttcttagtgt agtactttct ttacgaggta 7800 attgatctcg cattatatat ctacattttg gttatgttac ttgacatata gtcattgatt 7860 caatagttct gttaattcct ttaaagatca ttttgactag accacattct tggttcattc 7920 ctcaataatt tgtaatcata ttggtggata tagaagtaga ttggttatag atcagatagt 7980 ggaagacttt aggatgaatt tcagctagtt tttttttttg gcttattgtc tcaaaagatr 8040 agtgctttgc tgtctccatt gcttctgcta tcgacacgct tctgtctcct tgtatcttta 8100 ttatatctat tcgtcccatg agttttgttt gttctgtatt cgttcgctct ggtgtcatgg 8160 atggagtctc tgttccatgt ttctgtaatg catgttgggt tgtttcatgc aagaaatgct 8220 gagataaaca ctcatttgtg aaagtttcta aactctgaat cgcgctacag gcaatgctcc 8280 gaggaqtagg aggagaagaa cgaaccaaac gacattatca gccctttgag qaagctctta 8340 gttttgttat tgtttttgta gccaaattct ccattcttat tccattttca cttatctctt 8400 gttccttata gaccttataa gttttttatt catgtataca aattatattg tcatcaagaa 8460 Page 5 pbpsmmlg2a. st25.txt gtatctttaa aatctaaatc tcaaatcacc aggactatgt ttttgtccaa ttcgtggaac 8520 caacttgcag cttgtatcca ttctcttaac caataaaaaa agaaagaaag atcaatttga 8580 taaatttctc agccacaaat tctacattta ggttttagca tatcgaaggc tcaatcacaa 8640 atacaataga tagactagag attccagcgt cacgtgagtt ttatctataa ataaaggacc 8700 aaaaatcaaa tcccgagggc attttcgtaa tccaacataa aacccttaaa cttcaagtct 8760 catttttaaa caaatcatgt tcacaagtct cttcttcttc tctgtttctc tatctcttgc 8820 tcatctttct cctgaacc atg gcg gcg gca aca aca aca aca aca aca tct 8871 Met Ala Ala Ala Thr Thr Thr Thr Thr Thr Ser 1 5 10 tct tcg atc tcc ttc tcc acc aaa cca tct cct tcc tcc tcc aaa tca 8919 Ser Ser lie Ser Phe Ser Thr Lys Pro Ser Pro Ser Ser Ser Lys Ser 20 25 cca tta cca atc tcc aga ttc tcc ctc cca ttc tcc cta aac ccc aac 8967 Pro Leu Pro lie Ser Arg Phe Ser Leu Pro Phe Ser Leu Asn Pro Asn 35 40 aaa tca tcc tcc tcc tcc cgc cgc cgc ggt atc aaa tcc agc tct ccc 9015 Lys Ser Ser Ser Ser Ser Arg Arg Arg Gly lie Lys Ser Ser Ser Pro 50 55 tcc tcc atc tcc gcc gtg ctc aac aca acc acc aat gtc aca acc act 9063 Ser Ser lie Ser Ala Val Leu Asn Thr Thr Thr Asn Val Thr Thr Thr 65 70 75 ccc tct cca acc aaa cct acc aaa ccc gaa aca ttc atc tcc cga ttc 9111 Pro Ser Pro Thr Lys Pro Thr Lys Pro Glu Thr Phe lie Ser Arg Phe 85 90 gct cca gat caa ccc cgc aaa ggc gct gat atc ctc gtc gaa gct tta 9159 Ala Pro Asp Gin Pro Arg Lys Gly Ala Asp lie Leu Val Giu Ala Leu 100 105 gaa cgt caa ggc gta gaa acc gta ttc gct tac cct gga ggt gca tca 9207 Glu Arg Gin Gly val Giu Thr Val Phe Ala Tyr Pro Gly Gly Ala Ser 115 120 atg gag att cac caa gcc tta acc cgc tct tcc tca atc cgt aac gtc 9255 Met Glu lie His Gin Ala Leu Thr Arg Ser Ser ser lie Arg Asn Vai 130 135 ctt cct cgt cac gaa caa gga ggt gta ttc gca gca gaa gga tac gct 9303 Leu Pro Arg His Glu Gin Gly Gly Val Phe Ala Ala Glu Giy Tyr Ala 145 150 155 cga tcc tca ggt aaa cca ggt atc tgt ata gcc act tca ggt ccc gga 9351 Arg Ser Ser Gly Lys Pro Gly lie cys lie Ala Thr Ser Gly Pro Gly 165 170 gct aca aat ctc gtt agc gga tta gcc gat gcg ttg tta gat agt gtt 9399 Ala Thr Asn Leu Vai Ser Giy Leu Ala Asp Ala Leu Leu Asp Ser Vai 180 185 cct ctt gta gca atc aca gga caa gtc cct cgt cgt atg att ggt aca 9447 Pro Leu Val Ala lie Thr Gly Gin Val Pro Arg Arg Met lie Gly Thr 195 200 gat gcg ttt caa gag act ccg att gtt gag gta acg cgt tcg att acg 9495 Asp Ala Phe Gin Glu Thr Pro lie Val Glu Val Thr Arg Ser lie Thr 205 210 215 Page 6 pbpsmmlg2a. st25.txt aag cat aac tat ctt gtg atg gat gtt gaa gat atc cct agg att att 9543 Lys His Asn Tyr Leu Val Met Asp Val Giu Asp lie Pro Arg lie lie 220 225 230 235 gag gaa gct ttc ttt tta gct act tct ggt aga cct gga cct gtt ttg 9591 Giu Glu Ala Phe Phe Leu Ala Thr Ser Gly Arg Pro Gly Pro vai Leu 240 245 250 gtt gat gtt cct aaa gat att caa caa cag ctt gcg att cct aat tgg 9639 val Asp val Pro Lys Asp lie Gin Gin Gin Leu Ala lie Pro Asn Trp 255 260 265 gaa cag gct atg aga tta cct ggt tat atg tct agg atg cct aaa cct 9687 Giu Gin Ala Met Arg Leu Pro Gly Tyr Met Ser Arg Met Pro Lys Pro 270 275 280 ccg gaa gat tct cat ttg gag cag att gtt agg ttg att tct gag tct 9735 Pro Giu Asp Ser His Leu Giu Gin lie Vai Arg Leu lie Ser Glu Ser 285 290 295 aag aag cct gtg ttg tat gtt ggt ggt ggt tgt ttg aat tct agc gat 9783 Lys Lys Pro Va] Leu Tyr Val Giy Giy Gly Cys Leu Asn Ser Ser Asp 300 305 310 315 gaa ttg ggt agg ttt gtt gag ctt acg ggg atc cct gtt gcg agt acg 9831 Glu Leu Gly Arg Phe vai Glu Leu Thr Gly lie Pro Vai Ala Ser Thr 320 325 330 ttg atg ggg ctg gga tct tat cct tgt gat gat gag ttg tcg tta cat 9879 Leu Met Gly Leu Gly Ser Tyr Pro Cys Asp Asp Giu Leu Ser Leu His 335 340 345 atg ctt gga atg cat ggg act gtg tat gca aat tac gct gtg gag cat 9927 Met Leu Gly Met His Giy Thr Vai Tyr Ala Asn Tyr Ala Val Giu His 350 355 360 agt gat ttg ttg ttg gcg ttt ggg gta agg ttt gat gat cgt gtc acg 9975 Ser Asp Leu Leu Leu Ala Phe Giy vai Arg Phe Asp Asp Arg Val Thr 365 370 375 ggt aag ctt gag gct ttt gct agt agg gct aag att gtt cat att gat 10023 Gly Lys Leu Giu Aia Phe Ala Ser Arg Ala Lys lie vai His lie Asp 380 385 390 395 att gac tcg gct gag att ggg aag aat aag act cct cat gtg tct gtg 10071 lie Asp Ser Ala Giu lie Giy Lys Asn Lys Thr Pro His Va] Ser Val 400 405 410 tgt ggt gat gtt aag ctg gct ttg caa ggg atg aat aag gtt ctt gag 10119 Cys Gly Asp vai Lys Leu Ala Leu Gin Gly Met Asn Lys Va] Leu Glu 415 420 425 aac cga gcg gag gag ctt aag ctt gat ttt gga gtt tgg agg aat gag 10167 Asn Arg Ala Glu Giu Leu Lys Leu Asp Phe Gly Val Trp Arg Asn Giu 430 435 440 ttg aac gta cag aaa cag aag ttt ccg ttg agc ttt aag acg ttt ggg 10215 Leu Asn Vai Gin Lys Gin Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly 445 450 455 gaa gct att cct cca cag tat gcg att aag gtc ctt gat gag ttg act 10263 Glu Ala lie Pro Pro Gin Tyr Ala lie Lys Va] Leu Asp Giu Leu Thr 460 465 470 475 gat gga aaa gcc ata ata agt act ggt gtc qgg caa cat caa atg tgg 10311 Asp Gly Lys Ala lie lie Ser Thr Gly Vai Gly Gin His Gin Met Trp 480 485 490 gcg gcg cag ttc tac aat tac aag aaa cca agg cag tgg cta tca tca 10359 Page 7 pbpsmmlg2a. st25.txt Ala Ala Gin Phe Tyr Asn Tyr Lys Lys Pro Arg Gln Trp Leu Ser Ser 495 500 505 gga ggc ctt gga gct atg gga ttt gga ctt cct gct gcg att gga gcg 10407 Gly Giy Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala lie Gly Ala 510 515 520 tct gtt gct aac cct gat gcg ata gtt gtg gat att gac gga gat gga 10455 ser val Ala Asn Pro Asp Ala lie Val Val Asp lie Asp Giy Asp Gly 525 530 535 agc ttt ata atg aat gtg caa gag cta gcc act att cgt gta gag aat 10503 Ser Phe lie Met Asn Val Gin Glu Leu Ala Thr lie Arg Vai Glu Asn 540 545 550 555 ctt cca gtg aag gta ctt tta tta aac aac cag cat ctt ggc atg gtt 10551 Leu Pro Val Lys Vai Leu Leu Leu Asn Asn Gin His Leu Gly Met Val 560 565 570 atg caa tgg gaa gat cgg ttc tac aaa gct aac cga gct cac aca ttt 10599 Met Gin Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Phe 575 580 585 ctc ggg gat ccg gct cag gag gac gag ata ttc ccg aac atg ttg ctg 10647 Leu Gly Asp Pro Ala Gin Giu Asp Giu lie Phe Pro Asn Met Leu Leu 590 595 600 ttt gca gca gct tgc ggg att cca gcg gcg agg gtg aca aag aaa gca 10695 Phe Ala Ala Ala Cys Gly lie Pro Ala Ala Arg Val Thr Lys Lys Ala 605 610 615 gat ctc cga gaa gct att cag aca atg ctg gat aca cca gga cct tac 10743 Asp Leu Arg Glu Ala lie Gin Thr Met Leu Asp Thr Pro Gly Pro Tyr 620 625 630 635 ctg ttg gat gtg att tgt ccg cac caa gaa cat gtg ttg ccg atg arc 10791 Leu Leu Asp Val lie Cys Pro His Gin Giu His Vai Leu Pro Met lie 640 645 650 ccg aat ggt ggc act ttc aac gat gtc ata acg gaa gga gat ggc cgg 10839 Pro Asn Giy Giy Thr Phe Asn Asp Val lie Thr Glu Gly Asp Gly Arg 655 660 665 att aaa tac tga gagatgaaac cggtgattat cagaaccttt tatggtcttt 10891 lie Lys Tyr gtatgcatat ggtaaaaaaa cttagtttgc aatttcctgt ttgttttggt aatttgagtt 10951 tcttttagtt gttgatctgc ctgctttttg gtttacgtca gactactact gctgttgttg 11011 tttggtttcc tttctttcat tttataaata aataatccgg ttcggtttac tccttgtgac 11071 tggctcagtt tggttattgc gaaatgcgaa tggtaaattg agtaattgaa attcgttatt 11131 agggttctaa gctgttttaa cagtcactgg gttaatatct ctcgaatctt gcatggaaaa 11191 tgctcttacc attggttttt aattgaaatg tgctcatatg ggccgtggtt tccaaattaa 11251 ataaaactac gatgtcatcg agaagtaaaa tcaactgtgt ccacattatc agttttgtgt 11311 atacgatgaa atagggtaat tcaaaatcta gcttgatatg ccttttggtt cattttaacc 11371 ttctgtaaac attttttcag attttgaaca agtaaatcca aaaaaaaaaa aaaaaaatct 11431 caactcaaca ctaaattatt ttaatgtata aaagatgctt aaaacatttg gcttaaaaga 11491 aagaagctaa aaacatagag aactcttgta aattgaagta tgaaaatata ctgaattggg 11551 Page 8 pbpsmmlg2a. st25.txt tattatatga atttttctga tttaggattc acatgatcca aaaaggaaat ccagaagcac 11611 taatcagaca ttggaagtag gaatatttca aaaagttttt tttttttaag taagtgacaa 11671 aagcttttaa aaaatagaaa agaaactagt attaaagttg taaatttaat aaacaaaaga 11731 aattttttat attttttcat ttctttttcc, agcatgaggt tatgatggca ggatgtggat 11791 ttcatttttt tccttttgat agccttttaa ttgatctatt ataattgacg aaaaaatatt 11851 agttaattat agatatattt taggtagtat tagcaattta cacttccaaa agactatgta 11911 agttgtaaat atgatgcgtt gatctcttca tcattcaatg gttagtcaaa aaaataaaag 11971 cttaactagt aaactaaagt agtcaaaaat tgtactttag tttaaaatat tacatgaata 12031 atccaaaacg acatttatgt gaaacaaaaa caatatctag agtcgacctg caggcatgca 12091 agcttggcgc gccttaatta aaggcctgtt aacagcgctg ggcccagcgc tgttaacagg 12151 cctttaatta aggcgcgcca agcttgcatg cctgcaggtc gactctagat tcgacggtat 12211 cgataagctc gcggatccct gaaagcgacg ttggatgtta acatctacaa attgcctttt 12271 cttatcgacc atgtacgtaa gcgcttacgt ttttggtgga cccttgagga aactggtagc 12331 tgttgtgggc ctgtggtctc aagatggatc attaatttcc accttcacct acgatggggg 12391 gcatcgcacc ggtgagtaat attgtacggc taagagcgaa tttggcctgt aggatccctg 12451 aaagcgacgt tggatgttaa catctacaaa ttgccttttc ttatcgacca tgtacgtaag 12511 cgcttacgtt tttggtggac ccttgaggaa actggtagct gttgtgggcc tgtggtctca 12571 agatggatca ttaatttcca ccttcaccta cgatgggggg catcgcaccg gtgagtaata 12631 ttgtacggct aagagcgaat ttggcctgta ggatccctga aagcgacgtt ggatgttaac 12691 atctacaaat tgccttttct tatcgaccat gtacgtaagc gcttacgttt ttggtggacc 12751 cttgaggaaa ctggtagctg ttgtgggcct gtggtctcaa gatggatcat taatttccac 12811 cttcacctac gatggggggc atcgcaccgg tgagtaatat tgtacggcta agagcgaatt 12871 tggcctgtag gatccgcgag ctggtcaatc ccattgcttt tgaagcagct caacattgat 12931 ctctttctcg atcgagggag atttttcaaa tcagtgcgca agacgtgacg taagtatccg 12991 agtcagtttt tatttttcta ctaatttggt cgtttatttc ggcgtgtagg acatggcaac 13051 cgggcctgaa tttcgcgggt attctgtttc tattccaact ttttcttgat ccgcagccat 13111 taacgacttt tgaatagata cgctgacacg ccaagcctcg ctagtcaaaa gtgtaccaaa 13171 caacgcttta cagcaagaac ggaatgcgcg tgacgctcgc ggtgacgcca tttcgccttt 13231 tcagaaatgg ataaatagcc ttgcttccta ttatatcttc ccaaattacc aatacattac 13291 actagcatct gaatttcata accaatctcg atacaccaaa tcgaagatct cccgggtggt 13351 cagtccctt atg tta cgt cct gta gaa acc cca acc cgt gaa atc aaa aaa 13402 Met Leu Arg Pro Val Glu Thr Pro Thr Arg Glu lie Lys Lys 675 680 ctc gac gyc ctg tgg gca ttc agt ctg gat cgc gaa aac tgt gga att 13450 Leu Asp Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly lie 685 690 695 700 gat cag cgt tgg tgg gaa agc gcg tta caa gaa agc cgg gca att gct 13498 Page 9 pbpsmml92a. st25.txt Asp Gin Arg Trp Trp Glu Ser Ala Leu Gin Glu Ser Arg Ala lie Ala 705 710 715 gtg cca ggc agt ttt aac gat cag ttc gcc gat gca gat att cgt aat 13546 Val Pro Gly Ser Phe Asn Asp Gin Phe Ala Asp Ala Asp lie Arg Asn 720 725 730 tat gcg ggc aac gtc tgg tat cag cgc gaa gtc ttt ata ccg aaa ggt 13594 Tyr Ala Gly Asn Val Trp Tyr Gin Arg Glu Vai Phe lie Pro Lys Gly 735 740 745 tgg gca ggc cag cgt atc gtg ctg cgt ttc gat gcg gtc act cat tac 13642 Trp Ala Gly Gin Arg Tie val Leu Arg Phe Asp Ala Val Thr His Tyr 750 755 760 ggc aaa gtg tgg gtc aat aat cag gaa gtg atg gag cat cag ggc ggc 13690 Gly Lys Va] Trp Val Asn Asn Gin Glu Val Met Glu His Gin Gly Gly 765 770 775 780 tat acg cca ttt gaa gcc gat gtc acg ccg tat gtt att gcc ggg aaa 13738 Tyr Thr Pro Phe Glu Ala Asp Val Thr Pro Tyr Vai lie Ala Gly Lys 785 790 795 agt gta cgt aag ttt ctg ctt cta cct ttg ata tat ata taa taa tta 13786 Ser Vai Arg Lys Phe Leu Leu Leu Pro Leu Tie Tyr lie Leu 800 805 810 tca tta att agt agt aat ata ata ttt caa ata ttt ttt tca aaa taa 13834 Ser Leu lie Ser Ser Asn lie lie Phe Gin lie Phe Phe Ser Lys 815 820 825 aag aat gta gta tat agc aat tgc ttt tct gta gtt tat aag tgt gta 13882 Lys Asn Vai Val Tyr Ser Asn Cys Phe Ser Vai Val Tyr Lys Cys Val 830 835 840 tat ttt aat tta taa ctt ttc taa tat atg acc aaa att tgt tga tgt 13930 Tyr Phe Asn Leu Leu Phe Tyr Met Thr Lys lie Cys Cys 845 850 gca ggt atc acc gtt tgt gtg aac aac gaa ctg aac tgg cag act atc 13978 Ala Gly lie Thr Val Cys Va] Asn Asn Glu Leu Asn Trp Gin Thr lie 855 860 865 870 ccg ccg gga atg gtg att acc gac gaa aac ggc aag aaa aag cag tct 14026 Pro Pro Gly Met Va] lie Thr Asp Glu Asn Gly Lys Lys Lys Gin Ser 875 880 885 tac ttc cat gat ttc ttt aac tat gcc gga atc cat cgc agc gta atg 14074 Tyr Phe His Asp Phe Phe Asn Tyr Ala Gly lie His Arg Ser Val Met 890 895 900 ctc tac acc acg ccg aac acc tgg gtg gac gat atc acc gtg gtg acg 14122 Leu Tyr Thr Thr Pro Asn Thr Trp Va] Asp Asp lie Thr Val Val Thr 905 910 915 cat gtc gcg caa gac tgt aac cac gcg tct gtt gac tgg cag gtg gtg 14170 His vai Ala Gin Asp Cys Asn His Ala Ser vai Asp Trp Gin Va] Va] 920 925 930 gcc aat ggt gat gtc agc gtt gaa ctg cgt gat gcg gat caa cag gtg 14218 Ala Asn Gly Asp Va] Ser Va] Glu Leu Arg Asp Ala Asp Gin Gin Va] 935 940 945 950 gtt gca act gga caa ggc act agc ggg act ttg caa gtg gtg aat ccg 14266 Val Ala Thr Gly Gin Gly Thr Ser Gly Thr Leu Gin Va] Val Asn Pro 955 960 965 cac ctc tgg caa ccg ggt gaa ggt tat ctc tat gaa ctg tgc gtc aca 14314 His Leu Trp Gin Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Cys vai Thr Page 10 lc pbpsmmlg2a. st25.txt 970 975 980 gcc aaa agc cag aca gag tgt gat atc tac ccg ctt cgc gtc ggc atc 14362 Ala Lys Ser Gin Thr Glu Cys Asp lie Tyr Pro Leu Arg Val Gly lie 985 990 995 cgg tca gtg gca gtg aag ggc caa cag ttc ctg att aac cac aaa 14407 Arg Ser Val Ala Val Lys Gly Gin Gin Phe Leu lie Asn His Lys 1000 1005 1010 ccg ttc tac ttt act ggc ttt ggt cgt cat gaa gat gcg gac tta 14452 Pro Phe Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala Asp Leu 1015 1020 1025 cgt ggc aaa gga ttc gat aac gtg ctg atg gtg cac gac cac gca 14497 Arg Gly Lys Gly Phe Asp Asn Val Leu Met Val His Asp His Ala 1030 1035 1040 tta atg gac tgg att ggg gcc aac tcc tac cgt acc tcg cat tac 14542 Leu Met AspTrp lie Giy Ala Asn Ser Tyr Arg Thr Ser His Tyr 1045 1050 1055 cct tac gct gaa gag atg ctc gac tgg gca gat gaa cat ggc atc 14587 Pro Tyr Ala Glu Giu Met Leu Asp Trp Ala Asp Giu His Gly lie 1060 1065 1070 gtg gtg att gat gaa act gct gct gtc ggc ttt aac ctc tct tta 14632 Val Va] lie Asp Giu Thr Ala Ala Val Giy Phe Asn Leu Ser Leu 1075 1080 1085 ggc att ggt ttc gaa gcg ggc aac aag ccg aaa gaa ctg tac agc 14677 Gly lie Gly Phe Giu Ala Gly Asn Lys Pro Lys Glu Leu Tyr Ser 1090 1095 1100 gaa gag gca gtc aac ggg gaa act cag caa gcg cac tta cag gcg 14722 Giu Giu Ala Val Asn Gly Giu Thr Gin Gin Ala His Leu Gin Ala 1105 1110 1115 att aaa gag ctg ata gcg cgt gac aaa aac cac cca agc gtg gtg 14767 lie Lys Giu Leu lie Ala Arg Asp Lys Asn His Pro Ser Val Va] 1120 1125 1130 atg tgg agt att gcc aac gaa ccg gat acc cgt ccg caa gtg cac 14812 Met Trp Ser lie Ala Asn Glu Pro Asp Thr Arg Pro Gin Val His 1135 1140 1145 ggg aat att tcg cca ctg gcg gaa gca acg cgt aaa ctc gac ccg 14857 Gly Asn lie Ser Pro Leu Ala Giu Aia Thr Arg Lys Leu Asp Pro 1150 1155 1160 acg cgt ccg atc acc tgc gtc aat gta atg ttc tgc gac gct cac 14902 Thr Arg Pro lie Thr Cys Val Asn Val Met Phe Cys Asp Ala His 1165 1170 1175 acc gat acc atc agc gat ctc ttt gat gtg ctg tgc ctg aac cgt 14947 Thr Asp Thr lie Ser Asp Leu Phe Asp Va] Leu Cys Leu Asn Arg 1180 1185 1190 tat tac gga tgg tat gtc caa agc ggc gat ttg gaa acg gca gag 14992 Tyr Tyr Gly Trp Tyr Vai Gin Ser Gly Asp Leu Glu Thr Ala Glu 1195 1200 1205 aag gta ctg gaa aaa gaa ctt ctg gcc tgg cag gag aaa ctg cat 15037 Lys Val Leu Glu Lys Glu Leu Leu Ala Trp Gin Giu Lys Leu His 1210 1215 1220 cag ccg att atc atc acc gaa tac ggc gtg gat acg tta gcc ggg 15082 Gin Pro lie lie lie Thr Glu Tyr Gly Val Asp Thr Leu Aia Giy 1225 1230 1235 Page 11 pbpsnimlg2a. st25.txt ctg cac tca atg tac acc gac atg tgg agt gaa gag tat cag tgt 15127 Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu Glu Tyr Gin Cys 1240 1245 1250 gca tgg ctg gat atg tat cac cgc gtc ttt gat cgc gtc agc gcc 15172 Ala Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg Val Ser Ala 1255 1260 1265 gtc gtc ggt gaa cag gta tgg aat ttc gcc gat ttt gcg acc tcg 15217 Val Val Gly Glu Gin Val Trp Asn Phe Ala Asp Phe Ala Thr Ser 1270 1275 1280 caa ggc ata ttg cgc gtt ggc ggt aac aag aaa ggg atc ttc act 15262 Gin Gly Tie Leu Arg Val Gly Gly Asn Lys Lys Gly Tie Phe Thr 1285 1290 1295 cgc gac cgc aaa ccg aag tcg gcg gct ttt ctg ctg caa aaa cgc 15307 Arg Asp Arg Lys Pro Lys Ser Ala Ala Phe Leu Leu Gin Lys Arg 1300 1305 1310 tgg act ggc atg aac ttc ggt gaa aaa ccg cag cag gga ggc aaa 15352 Trp Thr Gly Met Asn Phe Gly Glu Lys Pro Gin Gin Gly Gly Lys 1315 1320 1325 caa tga atcaacaact ctcctggcgc accatcgtcg gctacagcct cgggaattgc 15408 Gin taccgagctc gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga 15468 atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg 15528 taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc 15588 cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat 15648 tatcgcgcgc ggtgtcatct atgttactag atcgggaatt ggcatgcaag cttggcactg 15708 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 15768 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 15828 tcccaacagt tgcgcagcct gaatggcgaa tgctagagca gcttgagctt ggatcagatt 15888 gtcgtttccc gccttcagtt taaactatca gtgtttgaca ggatatattg gcgggtaaac 15948 ctaagagaaa agagcgttta ttagaataac ggatatttaa aagggcgtga aaaggtttat 16008 ccgttcgtcc atttgtatgt gcatgccaac cacagggttc ccctcgggat caaagt 16064 <210> 2 <211> 670 <212> PRT <213> Artificial <220> <223> Synthetic Construct <400> 2 Met Ala Ala Ala Thr Thr Thr Thr Thr Thr Ser Ser Ser Tie Ser Phe 1 5 10 15 Ser Thr Lys Pro Ser Pro Ser Ser Ser Lys Ser Pro Leu Pro Tie Ser 25 30 Page 12 pbpsmml92a. st25.txt Arg Phe Ser Leu Pro Phe Ser Leu Asn Pro Asn Lys Ser Ser Ser Ser 40 45 Ser Arg Arg Arg Gly lie Lys Ser Ser Ser Pro Ser Ser lie Ser Ala 55 60 Val Leu Asn Thr Thr Thr Asn Val Thr Thr Thr Pro Ser Pro Thr Lys 70 75 80 Pro Thr Lys Pro Glu Thr Phe lie Ser Arg Phe Ala Pro Asp Gin Pro 90 95 Arg Lys Gly Ala Asp lie Leu Vai Glu Ala Leu Glu Arg Gin Gly Val 105 110 Giu Thr Val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile His Gin 120 125 Ala Leu Thr Arg Ser Ser Ser lie Arg Asn Val Leu Pro Arg His Giu 135 140 Gin Gly Gly Val Phe Ala Ala Glu Giy Tyr Ala Arg Ser Ser Gly Lys 150 155 160 Pro Gly lie Cys lie Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu Val 170 175 Ser Gly Leu Ala Asp Ala Leu Leu Asp Ser val Pro Leu Val Ala lie 185 190 Thr Gly Gin val Pro Arg Arg Met lie Gly Thr Asp Ala Phe Gin Glu 200 205 Thr Pro lie Val Giu Vai Thr Arg Ser Ile Thr Lys His Asn Tyr Leu 210 215 220 Val Met Asp Val Giu Asp lie Pro Arg lie lie Giu Glu Ala Phe Phe 225 230 235 240 Leu Ala Thr Ser Gly Arg Pro Gly Pro vai Leu Val Asp val Pro Lys 245 250 255 Asp lie Gin Gin Gin Leu Ala lie Pro Asn Trp Glu Gin Ala Met Arg 260 265 270 Leu Pro Gly Tyr Met Ser Arg Met Pro Lys Pro Pro Glu Asp Ser His 275 280 285 Leu Giu Gin lie val Arg Leu lie Ser Glu Ser Lys Lys Pro val Leu 290 295 300 Page 13 pbpsmml92a. st25.txt Tyr Val Gly Gly Gly Cys Leu Asn Ser Ser Asp Glu Leu Gly Arg Phe 305 310 315 320 Val Glu Leu Thr Gly lie Pro Val Ala Ser Thr Leu Met Gly Leu Gly 325 330 335 Ser Tyr Pro Cys Asp Asp Glu Leu Ser Leu His Met Leu Gly Met His 340 345 350 Gly Thr Val Tyr Ala Asn Tyr Ala Val Glu His Ser Asp Leu Leu Leu 355 360 365 Ala Phe Gly val Arg Phe Asp Asp Arg Val Thr Gly Lys Leu Glu Ala 370 375 380 Phe Ala Ser Arg Ala Lys lie Val His lie Asp lie Asp Ser Ala Glu 385 390 395 400 lie Gly Lys Asn Lys Thr Pro His vai Ser Val Cys Gly Asp Val Lys 405 410 415 Leu Ala Leu Gln Gly Met Asn Lys vai Leu Glu Asn Arg Ala Glu Glu 420 425 430 Leu Lys Leu Asp Phe Giy Val Trp Arg Asn Glu Leu Asn val Gin Lys 435 440 445 Gln Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala lie Pro Pro 450 455 460 Gln Tyr Ala lie Lys Val Leu Asp Glu Leu Thr Asp Gly Lys Ala lie 465 470 475 480 Ile Ser Thr Gly Val Gly Gln His Gin Met Trp Ala Ala Gin Phe Tyr 485 490 495 Asn Tyr Lys Lys Pro Arg Gin Trp Leu Ser Ser Gly Gly Leu Gly Ala 500 505 510 Met Gly Phe Gly Leu Pro Ala Ala Ile Giy Ala Ser Val Aia Asn Pro 515 520 525 Asp Ala lie Val Val Asp Tie Asp Gly Asp Giy Ser Phe Ile Met Asn 530 535 540 Va] Gin Glu Leu Ala Thr lie Arg val Giu Asn Leu Pro Va] Lys Va] 545 550 555 560 Leu Leu Leu Asn Asn Gin His Leu Giy Met Va] Met Gin Trp Glu Asp 565 570 575 Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Phe Leu Giy Asp Pro Ala Page 14 i.

pbpsmml92a. st25.txt 580 585 590 Gin Glu Asp Glu lie Phe Pro Asn Met Leu Leu Phe Ala Ala Ala Cys 595 600 605 Gly lie Pro Ala Ala Arg Val Thr Lys Lys Ala Asp Leu Arg Glu Ala 610 615 620 lie Gin Thr Met Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val lie 625 630 635 640 Cys Pro His Gin Glu His vai Leu Pro Met lie Pro Asn Gly Gly Thr 645 650 655 Phe Asn Asp Val lie Thr Glu Gly Asp Gly Arg lie Lys Tyr 660 665 670 <210> 3 <211> 139 <212> PRT <213> Artificial <220> <223> Synthetic Construct <400> 3 Met Leu Arg Pro Vai Glu Thr Pro Thr Arg Giu lie Lys Lys Leu Asp 1 5 10 15 Giy Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly lie Asp Gin 25 30 Arg Trp Trp Glu Ser Ala Leu Gin Giu Ser Arg Ala lie Ala Vai Pro 40 45 Giy Ser Phe Asn Asp Gin Phe Ala Asp Ala Asp lie Arg Asn Tyr Ala 55 60 Giy Asn vai Trp Tyr Gin Arg Giu Val Phe lie Pro Lys Gly Trp Ala 70 75 80 Giy Gin Arg lie Vai Leu Arg Phe Asp Ala Vai Thr His Tyr Gly Lys 90 95 vai Trp vai Asn Asn Gin Giu vai Met Giu His Gin Gly Giy Tyr Thr 105 110 Pro Phe Giu Ala Asp Vai Thr Pro Tyr val lie Ala Gly Lys Ser Vai 120 125 Arg Lys Phe Leu Leu Leu Pro Leu lie Tyr lie 135 Page 15 pbpsmmlg2a. st25.txt <210> 4 <211> 16 <212> PRT <213> Artificial <220> <223> Synthetic Construct <400> 4 Leu Ser Leu lie Ser Ser ASn lie lie Phe Gin lie Phe Phe Ser Lys 1 5 10 15 <210> 5 <211> 20 <212> PRT <213> Artificial <220> <223> Synthetic Construct <400> 5 Lys Asn Vai Val Tyr Ser Asn Cys Phe Ser Val Val Tyr Lys Cys Val 1 5 10 15 Tyr Phe Asn Leu <210> 6 <211> 6 <212> PRT <213> Artificial <220> <223> Synthetic Construct <400> 6 Tyr Met Thr Lys lie Cys 1 5 <210> 7 <211> 476 <212> PRT <213> Artificial <220> <223> Synthetic Construct <400> 7 Cys Ala Gly lie Thr Vai CyS Vai Asn Asn Glu Leu Asn Trp Gin Thr 1 5 10 15 lie Pro Pro Giy Met Vai lie Thr Asp Giu Asn Giy Lys Lys Lys Gin 25 30 Ser Tyr Phe His Asp Phe Phe Asn Tyr Ala Gly lie His Arg Ser Va] 40 45 Met Leu Tyr Thr Thr Pro Asn Thr Trp Vai Asp Asp lie Thr vai Vai Page 16 pbpsmmlg2a. st25.txt 55 60 Thr HiS Va] Ala Gin Asp Cys Asn His Ala Ser Val Asp Trp Gin Va] 70 75 80 Va] Ala Asn Gly Asp Vai Ser Va] Glu Leu Arg Asp Ala Asp Gin Gin 90 95 Va] Val Ala Thr Gly Gin Gly Thr Ser Gly Thr Leu Gin Va] Val Asn 105 110 Pro His Leu Trp Gin Pro Gly Giu Gly Tyr Leu Tyr Giu Leu Cys Va] 120 125 Thr Ala Lys Ser Gin Thr Glu Cys Asp lie Tyr Pro Leu Arg Va] Giy 135 140 lie Arg Ser Vai Aia Val Lys Giy Gin Gin Phe Leu lie Asn His Lys 150 155 160 Pro Plie Tyr Phe Thr Giy Phe Gly Arg His Giu Asp Ala Asp Leu Arg 170 175 Giy Lys Giy Phe Asp Asn Val Leu Met Vai HisAsp His Ala Leu Met 185 190 Asp Trp lie Giy Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr Aia 200 205 Giu Giu Met Leu Asp Trp Ala Asp Giu His Giy lie Val Val lie Asp 210 215 220 Glu Thr Ala Ala Val Giy Phe Asn Leu Ser Leu Giy lie Giy Phe Giu 225 230 235 240 Ala Gly Asn Lys Pro Lys Giu Leu Tyr Ser Glu Glu Ala Va] Asn Gly 245 250 255 G]u Thr Gin Gin Ala His Leii Gin Aia lie Lys Glu Leu lie Ala Arg 260 265 270 Asp Lys Asn His Pro Ser Vai Vai Met Trp Ser lie Ala Asn Glu Pro 275 280 285 Asp Thr Arg Pro Gin Va] His Gly Asn lie Ser Pro Leu Ala Glu Aia 290 295 300 Thr Arg Lys Leu Asp Pro Thr Arg Pro lie Thr Cys Va] Asn Va] Met 305 310 315 320 Phe Cys Asp Ala His Thr Asp Thr lie Ser Asp Leu Phe Asp Va] Leu 325 330 335 Page 17 pbpsmml92a. st25.txt Cys Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gin Ser Gly Asp Leu Glu 340 345 350 Thr Ala Giu Lys val Leu Glu Lys Giu Leu Leu Ala Trp Gin Glu Lys 355 360 365 Leu His Gin Pro lie lie lie Thr Giu Tyr Gly Vai Asp Thr Leu Ala 370 375 380 Gly Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu Giu Tyr Gin Cys 385 390 395 400 Ala Trp Leu Asp Met Tyr His Arg Vai Phe Asp Arg Va] Ser Ala Val 405 410 415 val Gly Glu Gin Val Trp Asn Phe Ala Asp Phe Ala Thr Ser Gin Gly 420 425 430 lie Leu Arg val Gly Gly Asn Lys Lys Gly lie Phe Thr Arg Asp Arg 435 440 445 Lys Pro Lys Ser Ala Ala Phe Leu Leu Gin Lys Arg Trp Thr Giy Met 450 455 460 Asn Phe Gly Glu Lys Pro Gin Gin Giy Gly Lys Gin 465 470 475 <210> 8 <211> 1024 <212> DNA <213> Arabidopsis thaliana <220> <221> promoter <222> (1).. (1024) <223> promoter region of acetohydroxyacid synthase large subunit from Arabi dopsi s putative TATA box: 89-7-911 <400> 8 atahasrmtr tttctttacg aggtaattga tctcgcatta tatatctaca ttttggttat 60 gttacttgac atatagtcat tgattcaata gttctgttaa ttcctttaaa gatcattttg 120 actagaccac attcttggtt cattcctcaa taatttgtaa tcatattggt ggatatagaa 180 gtagattggt tatagatcag atagtggaag actttaggat gaatttcagc tagttttttt 240 ttttggctta ttgtctcaaa agattagtgc tttgctgtct ccattgcttc tgctatcgac 300 acgcttctgt ctccttgtat ctttattata tctattcgtc ccatgagttt tgtttgttct 360 gtattcgttc gctctggtgt catggatgga gtctctgttc catgtttctg taatgcatgt 420 tgggttgttt catgcaagaa atgctgagat aaacactcat ttgtgaaagt ttctaaactc 480 Page 18 3c, pbpsmmlg2a. st25.txt tgaatcgcgc tacaggcaat gctccgagga gtaggaggag aagaacgaac caaacgacat 540 tatcagccct ttgaggaagc tcttagtttt gttattgttt ttgtagccaa attctccatt 600 cttattccat tttcacttat ctcttgttcc ttatagacct tataagtttt ttattcatgt 660 atacaaatta tattgtcatc aagaagtatc. tttaaaatct aaatctcaaa tcaccaggac 720 tatgtttttg tccaattcgt ggaaccaact tgcagcttgt atccattctc ttaaccaata 780 aaaaaagaaa gaaagatcaa tttgataaat ttctcagcca caaattctac atttaggttt 840 tagcatatcg aaggctcaat cacaaataca atagatagac tagagattcc agcgtcacgt 900 gagttttatc tataaataaa ggaccaaaaa tcaaatcccg agggcatttt cgtaatccaa 960 cataaaaccc ttaaacttca agtctcattt ttaaacaaat catgttcaca agtctcttct 1020 tctt 1024 <210> 9 <211> 2022 <212> DNA <213> Arabidopsis thaliana <220> <221> CDS <222> (1).. (2022) <223> acetohydroxyacid synthase large subunit gene <400> 9 ata has cds atg gcg gcg gca aca aca aca aca aca aca tct tct tcg 48 lie Xaa Xaa Met Ala Ala Ala Thr Thr Thr Thr Thr Thr ser Ser Ser 1 5 10 15 atc tcc ttc tcc acc aaa cca tct cct tcc tcc tcc aaa tca cca tta 96 lie Ser Phe Ser Thr Lys Pro Ser Pro Ser Ser Ser Lys Ser Pro Leu 25 30 cca atc tcc aga ttc tcc ctc cca ttc tcc cta aac ccc aac aaa tca 144 Pro lie Ser Arg Phe Ser Leu Pro Phe Ser Leu Asn Pro Asn Lys Ser 40 45 tcc tcc tcc tcc cgc cgc cgc ggt atc aaa tcc agc tct ccc tcc tcc 192 Ser Ser Ser Ser Arg Arg Arg Giy lie Lys Ser Ser Ser Pro Ser Ser 55 60 atc tcc gcc gtg ctc aac aca acc acc aat gtc aca acc act ccc tct 240 lie Ser Ala Val Leu Asn Thr Thr Thr Asn Val Thr Thr Thr Pro Ser 70 75 80 cca acc aaa cct acc aaa ccc gaa aca ttc atc tcc cga ttc gct cca 288 Pro Thr Lys Pro Thr Lys Pro Glu Thr Phe lie Ser Arg Phe Ala Pro 90 95 gat caa ccc cgc aaa ggc gct gat atc ctc gtc gaa gct tta gaa cgt 336 Asp Gin Pro Arg Lys Giy Ala Asp lie Leu Val Giu Aia Leu Giu Arg 105 110 caa ggc gta gaa acc gta ttc gct tac cct gga ggt gca tca atg gag 384 Gin Gly Vai Giu Thr vai Phe Ala Tyr Pro Giy Giy Ala Ser Met Giu 120 125 att cac caa gcc tta acc cgc tct tcc tca atc cgt aac gtc ctt cct 432 lie His Gin Ala Leu Thr Arg Ser Ser Ser lie Arg Asn Va] Leu Pro 135 140 Page 19 pbpsmml92a. st25.txt cgt cac gaa caa gga ggt gta ttc gca gca gaa gga tac gct cga tcc 480 Arg His Glu Gin Gly Gly val Phe Ala Ala Glu Giy Tyr Ala Arg Ser 150 155 160 tca ggt aaa cca ggt atc tgt ata gcc act tca gyt ccc gga gct aca 528 Ser Gly Lys Pro Gly lie cys lie Ala Thr Ser Gly Pro Gly Ala Thr 170 175 aat ctc gtt agc gga tta gcc gat gcg ttg tta gat agt gtt cct ctt 576 Asn Leu Val Ser Gly Leu Ala Asp Ala Leu Leu Asp Ser Val Pro Leu 185 190 gta gca atc aca gga caa gtc cct cgt cgt atg att ggt aca gat gcg 624 Val Ala Tie Thr Gly Gin Val Pro Arg Arg Met lie Gly Thr Asp Ala 200 205 ttt caa gag act ccg att gtt gag gta acg cgt tcg att acg aag cat 672 Phe Gin Glu Thr Pro lie Val Glu Val Thr Arg Ser lie Thr Lys His 210 215 220 aac tat ctt gtg atg gat gtt gaa gat atc cct agg att att gag gaa 720 Asn Tyr Leu Val Met Asp Val Glu Asp lie Pro Arg lie lie Glu Glu 225 230 235 240 gct ttc ttt tta gct act tct ggt aga cct gga cct gtt ttg gtt gat 768 Ala Phe Phe Leu Ala Thr Ser Gly Arg Pro Gly Pro val Leu Val Asp 245 250 255 gtt cct aaa gat att caa caa cag ctt gcg att cct aat tgg gaa cag 816 val Pro Lys Asp lie Gin Gin Gin Leu Ala lie Pro Asn Trp Glu Gin 260 265 270 gct atg aga tta cct ggt tat atg tct agg atg cct aaa cct ccg gaa 864 Ala Met Arg Leu Pro Gly Tyr Met Ser Arg Met Pro Lys Pro Pro Glu 275 280 285 gat tct cat ttg gag cag att gtt agg ttg att tct gag tct aag aag 912 Asp Ser His Leu Glu Gin lie Val Arg Leu lie Ser Glu Ser Lys Lys 290 295 300 cct gtg ttg tat gtt ggt ggt ggt tgt ttg aat tct agc gat gaa ttg 960 Pro Val Leu Tyr Val Gly Gly Gly Cys Leu Asn Ser Ser Asp Glu Leu 305 310 315 320 ggt agg ttt gtt gag ctt acg ggg atc cct gtt gcg agt acg ttg atg 1008 Gly Arg Phe Val Glu Leu Thr Gly lie Pro Val Ala Ser Thr Leu Met 325 330 335 ggg ctg gga tct tat cct tgt gat gat gag ttg tcg tta cat atg ctt 1056 Gly Leu Gly Ser Tyr Pro Cys Asp Asp Glu Leu Ser Leu His Met Leu 340 345 350 gga atg cat ggg act gtg tat gca aat tac gct gtg gag cat agt gat 1104 Gly Met His Giy Thr Va] Tyr Ala Asn Tyr Ala val Glu His Ser Asp 355 360 365 ttg ttg ttg gcg ttt ggg gta agg ttt gat gat cgt gtc acg ggt aag 1152 Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys 370 375 380 ctt gag gct ttt gct agt agg gct aag att gtt cat att gat att gac 1200 Leu Glu Ala Phe Ala Ser Arg Ala Lys lie Val His Tie Asp lie Asp 385 390 395 400 tcg gct gag att ggg aag aat aag act cct cat gtg tct gtg tgt ggt 1248 Ser Ala Glu lie Gly Lys Asn Lys Thr Pro His Va] Ser Val Cys Gly 405 410 415 gat gtt aag ctg gct ttg caa ggg atg aat aag gtt ctt gag aac cga 1296 Page 20 pbpsmmlg2a. st25.txt Asp Va] Lys Leu Ala Leu Gin Gly Met Asn Lys Va] Leu Glu Asn Arg 420 425 430 gcg gag gag ctt aag ctt gat ttt gga gtt tgg agg aat gag ttg aac 1344 Ala Glu Glu Leu Lys Leu Asp Phe Gly Val Trp Arg Asn Glu Leu Asn 435 440 445 gta cag aaa cag aag ttt ccg ttg agc ttt aag acg ttt ggg gaa gct 1392 Val Gin Lys Gin Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala 450 455 460 att cct cca cag tat gcg att aag gtc ctt gat gag ttg act gat gga 1440 lie Pro Pro Gin Tyr Ala lie Lys Va] Leu Asp Glu Leu Thr Asp Gly 465 470 475 480 aaa gcc ata ata agt act ggt gtc ggg caa cat caa atg tgg gcg gcg 1488 Lys Aia lie lie Ser Thr Gly Val Giy Gln His Gin Met Trp Ala Ala 485 490 495 cag ttc tac aat tac aag aaa cca agg cag tgg cta tca tca gga ggc 1536 Gin Phe Tyr Asn Tyr Lys Lys Pro Arg Gin Trp Leu Ser Ser Gly Gly 500 505 510 ctt gga gct atg gga ttt gga ctt cct gct gcg att gga gcg tct gtt 1584 Leu Gly Ala Met Gly Phe Giy Leu Pro Ala Ala lie Giy Ala Ser Va] 515 520 525 gct aac cct gat gcg ata gtt gtg gat att gac gga gat gga agc ttt 1632 Ala Asn Pro Asp Ala lie Val Val Asp lie Asp Gly Asp G]y Ser Phe 530 535 540 ata atg aat gtg caa gag cta gcc act att cgt gta gag aat ctt cca 1680 lie Met Asn Vai Gin Glu Leu Ala Thr lie Arg Va] Glu Asn Leu Pro 545 550 555 560 gtg aag gta ctt tta tta aac aac cag cat ctt ggc atg gtt atg caa 1728 Va] Lys vai Leu Leu Leu Asn Asn Gin His Leu Gly Met Va] Met Gin 565 570 575 tgg gaa gat cgg ttc tac aaa gct aac cga gct cac aca ttt ctc ggg 1776 Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Phe Leu G]y 580 585 590 gat ccg gct cag gag gac gag ata ttc ccg aac atg ttg ctg ttt gca 1824 Asp Pro Ala Gin Glu Asp Glu lie Phe Pro Asn Met Leu Leu Phe Ala 595 600 605 gca gct tgc ggg att cca gcg gcg agg gtg aca aag aaa gca gat ctc 1872 Ala Ala Cys Gly lie Pro Ala Ala Arg Va] Thr Lys Lys Ala Asp Leu 610 615 620 cga gaa gct att cag aca atg ctg gat aca cca gga cct tac ctg ttg 1920 Arg Glu Ala lie Gin Thr Met Leu Asp ihr Pro Giy Pro Tyr Leu Leu 625 630 635 640 gat gtg att tgt ccg cac caa gaa cat gtg ttg ccg atg atc ccg aat 1968 Asp Val lie Cys Pro His Gln Glu His Val Leu Pro Met lie Pro Asn 645 650 655 ggt ggc act ttc aac gat gtc ata acg gaa gga gat ggc cgg att aaa 2016 Gly Gly Thr Phe Asn Asp Va] lie Thr Giu Gly Asp Gly Arg lie Lys 660 665 670 tac tga 2022 Ty r <210> 10 Page 21 pbpsmml92a. st25.txt <211> 673 <212> PRT <213> Arabidopsis thaliana <220> <221> misc_feature <222> (2).. (2) <223> The Xaa' at location 2 stands for Lys, Asn, Gin, His, or Tyr. <220>

<221> misc_feature <222> (3).. (3) <223> The Xaa' at location 3 stands for Gln, His, Arg, or Leu.

<400> 10 lie Xaa Xaa Met Ala Ala Ala Thr Thr Thr Thr Thr Thr ser Ser Ser 1 5 10 15 lie Ser Phe Ser Thr Lys Pro Ser Pro Ser Ser Ser Lys Ser Pro Leu 25 30 Pro lie Ser Arg Phe Ser Leu Pro Phe Ser Leu Asn Pro Asn Lys Ser 40 45 Ser Sr Ser Ser Arg Arg Arg Gly lie Lys Ser Ser Ser Pro Ser Ser 55 60 lie Ser Ala Val Leu Asn Thr Thr Thr Asn Vai Thr Thr Thr Pro Ser 70 75 80 Pro Thr Lys Pro Thr Lys Pro Glu Thr Phe lie Ser Arg Phe Ala Pro 90 95 Asp Gin Pro Arg Lys Gly Ala Asp lie Leu Val Glu Ala Leu Glu Arg 105 110 Gin Gly Val Glu Thr val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu 120 125 lie His Gin Ala Leu Thr Arg Ser Ser Ser lie Arg Asn val Leu Pro 135 140 Arg His Glu Gin Gly Gly Val Phe Ala Ala Glu Giy Tyr Ala Arg Ser 150 155 160 Ser Gly Lys Pro Gly lie Cys lie Ala Thr Ser Gly Pro Gly Ala Thr 170 175 Asn Leu Vai Ser Gly Leu Ala Asp Ala Leu Leu Asp Ser Vai Pro Leu 185 190 val Ala lie Thr Gly Gin Val Pro Arg Arg Met lie Gly Thr Asp Ala 200 205 Phe Gin Giu Thr Pro lie Va] Glu Val Thr Arg Ser lie Thr Lys His Page 22 pbpsmml92a.st25.txt 210 215 220 Asn Tyr Leu Val Met Asp Val Glu Asp lie Pro Arg lie lie Glu Glu 225 230 235 240 Ala Phe Phe Leu Ala Thr Ser Gly Arg Pro Gly Pro Val Leu Val Asp 245 250 255 Val Pro Lys Asp lie Gin Gin Gin Leu Ala lie Pro Asn Trp Giu Gin 260 265 270 Ala Met Arg Leu Pro Gly Tyr Met Ser Arg Met Pro Lys Pro Pro Glu 275 280 285 Asp Ser His Leu Glu Gin lie Va] Arg Leu lie Ser Giu Ser Lys Lys 290 295 300 Pro val Leu Tyr Val Gly Giy Gly Cys Leu Asn Ser Ser Asp Glu Leu 305 310 315 320 Gly Arg Phe Val Glu Leu Thr Gly lie Pro Val Ala Ser Thr Leu Met 325 330 335 Gly Leu Gly Ser Tyr Pro Cys Asp Asp Glu Leu Ser Leu His Met Leu 340 345 350 Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Glu His Ser Asp 355 360 365 Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Vai Thr Giy Lys 370 375 380 Leu Glu Ala Phe Ala Ser Arg Ala Lys lie Val His lie Asp lie Asp 385 390 395 400 Ser Ala Glu Ile Gly Lys Asn Lys Thr Pro His Val Ser Val Cys Giy 405 410 415 Asp Val Lys Leu Ala Leu Gin Gly Met Asn Lys Val Leu Glu Asn Arg 420 425 430 Ala Glu Glu Leu Lys Leu Asp Phe Gly Val Trp Arg Asn Glu Leu Asn 435 440 445 Val Gin Lys Gin Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala 450 455 460 lie Pro Pro Gln Tyr Ala lie Lys Vai Leu Asp Glu Leu Thr Asp Giy 465 470 475 480 Lys Ala Ile lie Ser Thr Gly Vai Gly Gin His Gin Met Trp Ala Ala 485 490 495 Page 23 pbpsmmlg2a. st25.txt Gin Phe Tyr Asn Tyr Lys Lys Pro Arg Gin Trp Leu Ser Ser Gly Giy 500 505 510 Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala lie Gly Ala Ser Val 515 520 525 Ala Asn Pro Asp Ala lie val Val Asp lie Asp Gly Asp Gly Ser Phe 530 535 540 lie Met Asn Vai Gin Glu Leu Ala Thr lie Arg Val Glu Asn Leu Pro 545 550 555 560 vai Lys Val Leu Leu Leu Asn Asn Gin His Leu Giy Met Val Met Gin 565 570 575 Trp Giu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Phe Leu Gly 580 585 590 Asp Pro Ala Gin Glu Asp Giu lie Phe Pro Asn Met Leu Leu Phe Ala 595 600 605 Ala Ala Cys Gly lie Pro Ala Ala Arg Val Thr Lys Lys Ala Asp Leu 610 615 620 Arg Glu Ala lie Gin Thr Met Leu Asp Thr Pro Gly Pro Tyr Leu Leu 625 630 635 640 Asp val lie Cys Pro His Gin Glu His Val Leu Pro Met lie Pro Asn 645 650 655 Gly Gly Thr Phe Asn Asp Val lie Thr Giu Gly Asp Gly Arg lie Lys 660 665 670 Ty r Page 24

Claims

J-,u,2-Claims 1. A method for generating a transgenic plant of the

genus Linum comprising the following steps a. introducing into a cell of a plant of the genus linum a construct comprising i) a first nucleic acid sequence encoding an acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide operably linked to a promoter active in said cell and ii) optionally a second nucleic acid sequence operably linked to a promoter active in said plant, and b. incubating the cell of step a) on a selection medium comprising an acetohydroxyacid synthase inhibiting herbicide in a concentration of 0.1 pM or more for a time period of 5 days or more, and c. transferring the cell of step b) to a regeneration medium and regenerating and optionally selecting the plant comprising said construct.

2. The method of claim 1 comprising the steps of a. isolating a hypocotyl segment of a plant of the genus linum and b. introducing the construct of step a) of claim 1 with the aid of a bacterium of the genus Rhizobiaceae into said hypocotyl segment by co-cultivating said hypocotyl segment with said bacterium and, c. optionally transferring the co-cultivated hypocotyl segment to a regeneration medium, said regeneration medium lacking a phytotoxic effective amount of an acetohydroxyacid synthase inhibiting herbicide prior to selection, d. inducing formation of a callus and selecting transgenic callus on a medium comprising, i. an effective amount of at least one auxin compound, and ii. an acetohydroxyacid synthase inhibiting herbicide in a concentration of 0.1 pM or more, and e. regenerating and optionally selecting plants containing the construct from the said transgenic callus.

3. The method of claim 1 or 2, wherein the construct of step a of claim 1 is part of a T-DNA.

4. The method of any of claims 1 to 3, wherein the acetohydroxyacid synthase inhibiting herbicide is selected from the group consisting of imidazolinOnes, sulfonylureas, triazolopyrimidine sulfonamides, pyrimidyloxy-benzoic acids, sulfamoylureas, sulfonylcarboxim ides and corn bi nations thereof.

5. The method of any of claims 1 to 4, wherein the acetohydroxyacid synthase is selected from the group consisting of i) an acetohydroxyacid synthase with an amino acid sequence as described by SEQ ID NO: 10, ii) an acetohydroxyacid synthase having an amino acid sequence identity of 80% or more to the amino acid sequence as described by SEQ ID NO: 10, and iii) an acetohydroxyacid synthase encoded by a nucleic acid sequence capable to hybridize to the nucleic acid sequence as described by SEQ ID NO: 9 or its corn plement.

6. The method of any of claims 1 to 5, wherein the selection of step b) of claim 1 or step d) of claim 2 is done in the presence of 1 to 1,5 pM imidazolinone.

7. The method of any of claims 1 to 6, wherein the total selection time period is from 6to20 weeks.

8. The method of any of claims 1 to 7, wherein the selection of step b) of claim 1 or step d) of claim 2 is done in a two step process, using a first selection step for a time period of 1 to 20 weeks then transferring the surviving cells or tissue to a second selection medium for additional 2 to 7 weeks.

9. The method of any of claim 1 to 8, wherein said plant of the genus linum is mum usitatissimum.

10. The method of any of claims 1 to 9, wherein said promoter active in said cell of a plant of the genus linum is an Arabidopsis thaliana acetohydroxyacid synthase promoter.

11. The method of any of claims 1 to 10, wherein the acetohydroxyacid synthase promoter is selected from the group consisting of a) a nucleic acid sequence comprising the nucleic acid sequence as described by SEQ ID NO: 8, and b) a nucleic acid sequence having promoter activity in a cell of a plant of the genus linum comprising at least one fragment of at least 50 consecutive base pairs of the nucleic acid sequence as described by SEQ ID NO: 8, and, c) a nucleic acid sequence having promoter activity in a cell of a plant of the genus linum comprising a nucleic acid sequence having at least 60% identity to the nucleic acid sequence as described by SEQ ID NO: 8, and, d) a nucleic acid sequence having promoter activity in a cell of a plant of the genus linum comprising a nucleic acid sequence hybridizing to the nucleic acid sequence as described by SEQ ID NO: 8 or its complement.

12. Construct comprising a) a first nucleic acid sequence encoding an acetohydroxyacid synthase as defined in claim 5 linked to the promoter as defined in claim 10 or 11 and b) optionally a second nucleic acid sequence conferring to a plant of the genus linum an agronomically valuable trait operably linked to a promoter active in a plant of the genus linum.

13. A plant or a part of a plant of the genus linum comprising a) a nucleic acid sequence encoding an acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide operably linked to a promoter active in said plant and optionally b) a second nucleic acid sequence conferring to said plant of the genus linum an agronomically valuable trait operably linked a promoter active in said plant.

14. A method for subsequently introducing at least two constructs into a cell of a plant of the genus linum comprising the steps of a) introducing a first construct comprising at least one nucleic acid encoding an acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide operably linked to a promoter active in said cell, and b) introducing a second construct comprising a second nucleic acid sequence encoding a marker protein which is not conferring resistance against imidazolinone operably linked to a promoter active in said cell.

15. A plant or a part of a plant of the genus linum comprising a) a first nucleic acid sequence encoding an acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide operably linked to a promoter active in said plant, and b) a second nucleic acid sequence encoding a selection marker protein, which is not conferring resistance against imidazolinone.

16. The plant or the part of a plant of claim 15, wherein the acetohydroxyacid synthase resistant to an acetohydroxyacid synthase inhibiting herbicide is defined by claim 5.

17. A plant or a part of a plant comprising the construct of claim 12.