CN1351667A

CN1351667A - Transgenic plants that are resistant to a broad specrum of pathogens

Info

Publication number: CN1351667A
Application number: CN 00805132
Authority: CN
Inventors: 桑托什·米斯拉; 威廉·W·凯
Original assignee: OF VCTORIA INNOVATION AND DEVELOPMENT CORP, University of
Current assignee: OF VCTORIA INNOVATION AND DEVELOPMENT CORP, University of
Priority date: 1999-03-17
Filing date: 2000-03-16
Publication date: 2002-05-29
Also published as: WO2000055337A1; AU772964C; AU772964B2; AU3267900A; CA2365099A1; CA2365099C; EP1163352A1; JP2002538828A

Abstract

Transgenic plants that express dermaseptin and/or temporin peptides are disclosed. In certain embodiments, these plants have enhanced, broad-spectrum pathogen resistance and are useful as agricultural or horticultural crops. In other embodiments, the plants are used to produce large quantities of the dermaseptin and/or temporin peptides.

Description

The transgenic plant of anti-broad spectrum of pathogens

Invention field

The present invention relates to engineered plant, it is transformed in order to express the peptide that one or more belong to temporin and/or dermaseptin family.

Background technology

Plant is the host by the thousands of kind communicate illnesss that caused by a large amount of plant pathogen epiphytes, bacterium, virus and nematode.These pathogenic agent are to cause global significant crop loss, cause the major cause of the destruction of the infection of growing plant and harvesting crops.The method that reduces the broad practice of the infringement that caused by these pathogenic agent comprises various chemical reagent.Unfortunately, many pathogenic agent produce the resistance to these chemical agents, and some pathogenic agent (particularly virus) also are not easy to control by chemical means.In addition, many in the chemical reagent of employing have the wide spectrum toxin, manyly cause the serious environmental infringement, and the intravital toxicity of people.

Recently, plant breeding and genetic engineering technique have been used to resist phytopathogen.Under certain situation, breeder and molecular biologist have successfully been transformed the resistance to some pathogenic agent.In some years in past, many plant R (resistance) gene is separated from plant.When introducing other easily catch an illness during crop, these R genes produce the enhanced resistance to some pathogenic agent.For example, United States Patent (USP) 5,571,706 have introduced the separation of tobacco N gene, and it gives the resistance to tobacco mosaic virus (TMV).Yet although the breeding of the routine of hitherto reported and gene engineering method can successfully strengthen the resistance of pathogenic agent, they generally solve only by a kind of pathogenic agent, perhaps the problem that causes of the pathogenic agent that is closely connected in a small amount.As a result, although the crop of adopting these methods to produce has the protection of the anti-a kind of pathogenic agent of enhanced, still must adopt conventional chemical reagent to control other pathogenic agent.

Production comprises bacterium and fungal pathogens to broad spectrum of pathogens, has the plant of enhanced resistance, will have great agriculture interests.The present invention relates to such plant.

Summary of the invention

The present inventor has been found that the broad spectrum of pathogens resistance is given in the expression of some peptide in the transgenic plant, comprises fungi and bacterium enhanced resistance.These peptides are peptides a spot of, that positively charged (cationic) belongs to temporin and dermaseptin family, their natural existing in some skin of planting frog.Transgenic plant provided by the invention can be used for conventional agricultural use, for example food crop.Also can gather in and handle these plants extracting temporin and/or the dermaseptin peptide expressed, it then can purifying to be used for medical treatment and other purposes.

Thereby the present invention includes the transgenic plant of expressing at least a dermaseptin or temporin peptide, and the method for producing these plants.The part of these plants comprises seed, fruit, stem, Ye Hegen, can utilize in a usual manner, as foodstuff raw material, perhaps as the source of dermaseptin or temporin peptide.Because all one or more phytopathogens of floristics easy infection can be used the present invention effectively to produce resistance of wide spectrum in any floristics.Thereby, the present invention can be applied to monocotyledons, dicotyledons and gymnosperm, comprise but be not limited to, beans, rape/vegetable seed (canola), alfalfa, flax, Sunflower Receptacle, safflower, rape, cotton, flax, peanut, the trifolium of corn, wheat, paddy rice, barley, soybean, cotton, general meaning; Vegetables, for example lettuce, tomato, cucurbit, cassava, potato, Radix Dauci Sativae, radish, pea, root of Szemao crotalaria, wild cabbage, Cauliflower, cabbage, Brussels sprouts, pepper; The tree fruit is oranges and tangerines, apple, pear, peach, apricot, English walnut for example; And flower, for example orchid, carnation and rose.

In its basic form, the invention provides the transgenic plant of expressing one or more dermaseptin and/or temporin peptide.The member of dermaseptin and temporin peptide family is known in the art.The example of the adoptable dermaseptin of the present invention comprises, but be not limited to, by people such as Mor., Biochemistry, 30:8824-8830,1991, Strahilevitz, Biochemistry, 33:10951-10960,1994 and Wechselberger, Biochim.Biophys.Acta 1388:279-283,1998. dermaseptin that introduce.Adoptable emporin example comprises, but is not limited to, by people such as Simmaco, and Eur.J.Biochem., 242:788-92,1996 temporin that introduce.In their native state (that is, expressing in the frog cell), dermaseptin and temporin peptide produce with precursor forms, shear processing to form mature protein by proteolysis subsequently.The mature form length of Dermaseptin is generally about 27-34 amino acid, and the mature form length of temporin is generally about 10-13 amino acid.The present invention expects naturally occurring total length (undressed mistake) form of these peptides, and the purposes of the maturation of peptide (processing) form and intermediate form.In addition, also can adopt the synthesized form of these peptides.It is not naturally occurring form that the synthesized form of peptide comprises any, and is included in and is different from the natural peptide that exists on the aminoacid sequence, but still keeps dermaseptin or the bioactive peptide of temporin.This sequence variant usually keeps at least 40% amino acid sequence identity with at least a naturally occurring dermaseptin or temporin peptide.

The synthesized form of other adoptable dermaseptin or temporin comprises having the form that the N-terminal peptide is extended.Such peptide extends the part of the precursor forms that can comprise common dermaseptin that removes or temporin in the protein course of processing, perhaps can be composition sequence.These N-terminal peptide are extended and can be sheared generation enhanced resistibility to proteolysis, and also can strengthen the anti-microbial activity of peptide.Normally, the terminal development length of these N-is between 2 and 25 amino acid, although also can adopt longer extension.The example of the terminal extension sequence of the N-that adopts comprises peptide sequence MAMWK and MASRH in certain embodiments.The AMWK sequence is that naturally occurring peptide extends; It is the part of the common total length dermaseptin-b peptide sequence of shearing in the course of processing.ASRH is a kind of synthetic extension sequence.Under every kind of situation, the N-terminal methionine adds extension peptide to guarantee the suitable expression of peptide.

Basic sides of the present invention is based on the expression of temporin in transgenic plant and dermaseptin peptide, and other aminoacid sequence also can connect in these peptides, so that the preparation fusogenic peptide.The expression of this fusogenic peptide can provide even than the more effective broad spectrum of pathogens resistance of the expression of independent temporin or dermaseptin peptide in the transgenic plant, the stability that perhaps can strengthen the temporin/dermaseptin molecule of having expressed is to provide higher expression level, and therefore promotion comes from the purifying of the peptide of plant tissue.Thereby, in another embodiment, the invention provides the transgenic plant of expressing fusogenic peptide, it comprises:

(1) is first peptide sequence of temporin or dermaseptin; With

(2) second peptide sequence that is operably connected with first peptide sequence.

Second peptide sequence but is not necessarily normally, is connected with the amino (N-) of first peptide sequence is terminal.

In certain embodiments, second peptide sequence comprises anionic (electronegative) " proparea " peptide sequence.During this proparea peptide is used for and the positively charged ion essence of dermaseptin or temporin, thereby in cellular environment, can provide enhanced stability.Therefore, these propareas generally include a large amount of electronegative amino acid, for example L-glutamic acid (Glu or E) and aspartic acid (Asp or D).The proparea that is fit to comprises that discovery is at (total length) dermaseptin of naturally occurring undressed mistake and in the temporin peptide those, and the anionic proparea that is derived from other peptide, comprise those that Mammals originates from, for example be derived from the proteinic proparea of sheep cathelin.The fusogenic peptide that comprises such proparea can represent that wherein P is the proparea peptide with P-D or P-T, and T is the temporin peptide, and D is the dermaseptin peptide.

The proparea peptide of even now can directly connect into the N-end of dermaseptin or temporin peptide, and adopting spacer peptide is useful to connect these two kinds of peptides.The application that connects the spacer peptide of two kinds of peptides in this area is known; This spacer peptide normal length and provides the hinge flexibly of a kind of connection first peptide sequence to the second peptide between 2 and 25 amino acid.Be used to provide the intervening sequence of the hinge flexibly that connects two kinds of peptides to comprise glycine (4) Serine (GGGGS x3) at interval, people such as Chaudhary introduce, Nature 339:394-397,1989.The N-terminal peptide extension of as above introducing also can be used for providing the spacer peptide function.The fusogenic peptide that comprises proparea peptide, spacer peptide and dermaseptin or temporin peptide can be expressed as P-S-D or P-S-T, and wherein S represents spacer peptide.

Intervening sequence also can comprise shearing site, for example the peptide sequence of being discerned and being sheared by proteolytic enzyme.The purifying that such site helps to follow plant tissue is removed the proparea afterwards from dermaseptin or temporin peptide.

Following chapters and sections introduce in more detail of the present invention these and other aspect.Sequence table

Nucleic acid and the aminoacid sequence of display column in subsidiary sequence table adopts the standard alphabet abbreviation for nucleotide base, adopts three-letter code for amino acid.Each nucleotide sequence only shows a chain, but comprises complementary strand by thinking with reference to the chain that shows.

SEQ ID:1 shows dermaseptin b cDNA sequence.

SEQ ID:2 shows the aminoacid sequence of precursor dermaseptin b peptide (undressed mistake).

SEQ ID:3 shows 27 amino acid whose sequences of ripe dermaseptin b peptide.

SEQ ID:4 shows 31 amino acid whose sequences of ripe dermaseptin B peptide.

SEQ ID:5-14 shows the aminoacid sequence of various maturations (processing) dermaseptin peptide.

SEQ ID:15 shows the cDNA sequence of a kind of temporin G that encodes.

SEQ ID:16 shows the aminoacid sequence of temporin G precursor (undressed mistake) form.

SEQ ID:17 shows 13 amino acid whose sequences of ripe temporin G peptide.

SEQ IDs:18-26 shows the aminoacid sequence of various maturations (processing) temporin peptide.

SEQ ID:27 code displaying MSRA ₂Nucleotide sequence.

SEQ ID:28 shows MSRA ₂Aminoacid sequence.

SEQ ID:29-32 demonstration is used for producing coding MSRA ₂The oligonucleotide of nucleotide sequence.

SEQ ID:33 code displaying MSRA ₃Nucleotide sequence.

SEQ ID:34 shows MSRA ₃Aminoacid sequence.

SEQ ID:35-38 demonstration is used for producing coding MSRA ₃The oligonucleotide of nucleotide sequence.

SEQ ID:39-41 shows the aminoacid sequence of the terminal extension sequence of various N-.

Brief Description Of Drawings

Fig. 1 shows the test-results figure of the anti-soft rot of test transgenosis Marxism-Leninism bell potato stem tuber.By Desiree contrast and express Demaseptin B (sample number into spectrum D1, D2, D6, D10) or Temporin A (dish of the stem tuber preparation of transgenic plant T3) is infected by the Radix Dauci Sativae owen bacteria for sample number into spectrum T1, T2.Place under the room temperature after 6 days, from dish, remove septic tissue lightly, the susceptibility/resistance of Radix Dauci Sativae owen bacteria is represented with the weight loss of stem tuber tissue.

Fig. 2 is show peptide MSRA ₂(Dermaseptin B) and MSRA ₃(Temporin A) is to the figure of colibacillary anti-microbial effect.This cell culture is at room temperature at the Dermaseptin of prescribed concentration B (DSB; 7 μ g/ml, 30 μ g/ml and 75 μ g/ml), Temporin A (TA; 75 μ g/ml, 133 μ g/ml, 200 μ g/ml) and the combination (133 μ g/ml Temporin A and 30 μ g/ml Dermaseptin B) of Temporin A and Dermaseptin B cultivated 4 hours when existing, dilute and be coated on the LB plate.Be after 37 ℃ of overnight incubation, to enumeration and write down the bacterium of survival.

Fig. 3 is show peptide MSRA ₂(Dermaseptin B) and MSRA ₃(Temporin A) is to the figure of the anti-microbial effect of Radix Dauci Sativae owen bacteria.This cell culture is under room temperature, at the Dermaseptin of prescribed concentration B (DSB; 23 μ g/ml, 45 μ g/ml) or Temporin A (TA; 67 μ g/ml, 133 μ g/ml) cultivated 4 hours when existing, dilute and be coated on the LB plate.Be after 28 ℃ of overnight incubation, to enumeration and write down the bacterium of survival.

I. definition

Dermaseptin: adopt such as this paper, term " dermaseptin " refers to naturally occurring title Be any composition of the cationic peptide family of dermaseptins, (Strahilevitz, Biochemistry, 33:10951-960,1994), and these naturally occurring peptides show below definition The bioactive fragment of dermaseptin and variant.

Dermaseptin is at first in the skin extraction of state, South America tree frog Phyllomedusa sauvagii Identify (people such as Mor, J.Biol.Chem., 269:31635-31641,1994) in the thing. They are The microbicidal peptide of wide spectrum can suppress filament shape fungi and bacterium, yeast and protozoic Growth (Strahilevitz, Biochemistry, 33:10951-10960,1994). Since identifying A dermaseptin, i.e. dermaseptin S, other a large amount of composition industry of this peptide family Characterized and cloned, being comprised: dermaseptin-b, separate from Phyllomedusa bicolor Skin (people such as Mor, J.Biol.Chem., 269:31635-31641,1994), (SEQ ID: 2); Isolate two kinds of dermaseptin from Pachymedusa dacnicolor, it is by clone PD-3-3 and PD-2-2 coding are introduced Biochim.Biophys.Acta such as Wechselberger 1388:279-283,1998 (being respectively the peptide sequence shown in the SEQ ID:5-6); From Agalychnis Annae isolates three kinds of dermaseptin, and it is by cloning AA-3-6, AA-3-3, AA-3-1 volume Code is introduced Biochim.Biophys.Acta 1388:279-283,1998 such as Wechselberger (being respectively the peptide sequence shown in the SEQ ID:7-9); And be derived from Phyllomedusa sauvagii Five kinds of dermaseptin peptides, be called dermaseptin 5, dermaseptin 4, dermaseptin 3, dermaseptin 2 and dermaseptin 1 introduce Journal such as Mor and Nicolas Biochemical Chemistry, 269:1934-1939,1994 (are respectively SEQ ID:10-14 institute The peptide sequence that shows). These sequences are easy to obtain from public's database, comprise deriving from GenBank.

The dermaseptin peptide is typically expressed as the precursor forms of about 60-80 amino acid length, And be processed into subsequently the mature form of about 27-34 amino acid length. For example, coding The cDNA of dermaseptin-b (SEQ ID:1; The people such as Amiche, J.Biol.Chem. 269:1747-1852,1994; The people such as Chapentier, Biol.Chem.273:14690-14697, 1998; Be positioned at the GenBank nucleotide sequence database, entering to hide registration number is X72387) coding The precursor peptide of 78 amino acid lengths (SEQ ID:2). Process the front bodily form of this dermaseptin-b Formula is called dermaseptin b and dermaseptin B to produce two kinds of mature forms (Strahilevitz, Biochemistry, 33:10951-10960,1994). Dermaseptin b (SEQ ID:3) length is 27 amino acid and the 49-75 amino acids residue that comprises precursor forms. Dermaseptin B is that another kind of length is 31 amino acid whose shearing products, comprises 4 ammonia Terminal (AMWK) (the SEQ ID:4) that extend of the N of base acid. Dermaseptin B comprises the front bodily form The 45-75 amino acids residue of formula. SEQ ID except demonstration dermaseptin b total length precursor forms: Outside 1 and 2, the dermaseptin peptide that sequence table shows represents finished, the ripe shape of peptide Formula.

If can obtain the nucleotide sequence of dermaseptin peptide sequence on a large scale and these peptides of coding, Those of ordinary skill in the art, the Protocols in Molecular Biology of employing standard can easily be given birth to Produce these peptides and their corresponding nucleotide sequences.

Except the naturally occurring dermaseptin that adopts above introduction, adopt with natural The dermaseptin peptide that exists is slightly different, yet still gives enhancing when expressing in plant The peptide of broad spectrum of pathogens resistance also can be implemented the present invention, and this is for the knack of this area Personnel are obvious. For example, the helical form two of ripe dermaseptin peptide N-end The property molecular moiety, especially front 18 amino acid residues are proved to be for antibacterial activity Important (people such as Mor, J.Biol.Chem., 269:31635-31641,1994; Mor and Nicolas, Journal Biochemical Chemistry, 269:1934-39,1994), and should Fragment can be used to replace total length dermaseptin. Therefore, term " dermaseptin " also comprises The dermaseptin peptide of variation, and the natural fragment that has peptide, they are shared and natural existence The particular sequence homogeneity level of dermaseptin peptide, perhaps conservative by one or more Amino acid substitution is different from naturally occurring dermaseptin peptide.

The peptide of these variations and fragment have kept the dermaseptin biologically active, it can by with The method of lower introduction is measured. The dermaseptin of variation has usually with naturally occurring The amino acid sequence of dermaseptin peptide (what for example SEQ ID:3 showed is the sort of) at least 40% together One property, and the method by following introduction is measured.

The dermaseptin biologically active: dermaseptin peptide bacteria growing inhibiting and/or fungi give birth to Long ability. Can determine easily that by adopting following given scheme dermaseptin is biological alive The property.

The antibacterial activity of given dermaseptin peptide suppresses the pectin bacterium by measuring it Strain is the capability evaluation of carrot owen bacteria or bacillus coli DH 5 for example. By in LB, connecting Dilute in the hole of peptide and 100: 1 aliquot to 96 hole microtiter plates continuously, measure given peptide Activity. Fresh bacterial cultures (～0.3 A550) is then at Luria-Bertani culture medium (LB) Middle growth (1%w/v tryptone and 0.5%w/v yeast extract) also is diluted to 10 in LB^-2To represent about 10⁴-10 ⁵CFU (CFU) ml^-1 10: 1 bacterial cultures connects then Plant to enter to contain in the hole of peptide, sample was cultivated 4 hours at 37 ℃. The composition in hole is then with LB Dilution is coated on the LB agar and 37 ℃ of placements and spends the night. Then count and each dermaseptin The corresponding bacterium colony of dilution (with the contrast that does not add peptide), the antibacterial activity of the peptide under the test By with the comparative measurements of control board.

Under the condition of this test, if it can suppress at least 10% in the concentration of 7:g/ml Bacterial growth (that is, and in this concentration, the quantity of bacterial clump be no more than control board 90%), just Measure the dermaseptin peptide and have biologically active.

The antimycotic biologically active of given dermaseptin peptide is by utilizing the fungal bacterial strain cactus Mould and/or the Fusaium solani assessment of epidemic disease. Selected fungal bacterial strain is at five kinds of cereal agar (Five Cereal Agar contains 20gL^-1Five kinds of cereal pablums make things convenient for thin slice and 8gL^-1Agar The upper growth of 3 FCA people such as (, The Plant Cell 7:573-588,1995) Terras. Under the room temperature After growing 5 days, take out the mycelium plug and be upside down in fresh FCA plate center. The nothing of test peptides Bacterium solution (10: 1) is then introduced in the hole of panel edges 3cm, contains aseptic in same plate foundation The control wells of water. Can test various concentration at (perhaps on other plate) on the same plate Test peptides. Cultivated 5 days under the breadboard room temperature, measure afterwards growth inhibition district on every side, each hole.

Under this experimental condition, if it can be at the control of the concentration conk of 5:g/ml (that is, The hole of containing this concentration peptide has recognizable inhibition zone on every side), just determine the dermaseptin peptide Has biologically active.

Temporin: adopt such as this paper, term " temporin " refers to naturally occurring being called Any composition of the cationic peptide family of temporins (people such as Simmaco, Eur.J. Biochem., 242:788-92.1996), and these naturally occurring peptides show below definition The bioactive fragment of temporin and variant.

Temporin knows from the cDNA storehouse by the preparation of Rana temporaria frog skin at first Other has the small-sized cationic peptide of antibacterial activity. These peptides show and separate from the Vespa snake venom Some sequence similarities of haemolysis peptide, yet the temporin peptide is not (the Simmaco of haemolysis Deng people, Eur.J.Biochem., 242:788-92.1996).

Ten compositions of temporin family, temporin A, B, C, D, E, F, G, H, K and L are introduced Eur.J.Biochem., 242:788-92. by people such as Simmaco 1996. Similar dermaseptin, temporin is typically expressed as precursor forms, subsequently processing To produce mature form. For example, the cDNA molecule of coding temporin G (be shown SEQ ID: 15, be positioned at the GenBank nucleotide sequence database, entering to hide registration number is Y09395) coding one Plant 61 amino acid whose temporin G precursor forms (being shown SEQ ID:16). Amino acid/11-22 Comprise burst, amino acid 23-46 comprises the proparea, that amino acid 47-59 comprises is processed, Ripe temporin G peptide (mature form is shown SEQ ID:17). Usually, the one-tenth of expectation Ripe temporin peptide length is between 10 and 13 amino acid, and some have been found in the C-end Amidatioon (people such as Simmaco, Eur.J.Biochem., 242:788-92.1996). Temporin The mature form of A, B, C, D, E, F, G, H, K and L be shown respectively SEQ ID:18, 19,20,21,22,23,17,24,25 and 26.

If can obtain the nucleotide sequence of temporin peptide sequence on a large scale and these peptides of coding, this The those of ordinary skill in field, the Protocols in Molecular Biology of employing standard can easily be produced These peptides and their corresponding nucleotide sequences.

Except the naturally occurring temporin peptide that adopts above introduction, adopt and natural depositing The temporin peptide slightly different, yet still give the wide spectrum of enhancing when in plant, expressing The peptide of pathogen-resistance also can be implemented the present invention, and this is for those skilled in the art Obvious. Thereby term " temporin " also comprises the temporin peptide of variation, with And the natural fragment that has peptide, they show the particular sequence with naturally occurring temporin peptide The homogeneity level is perhaps by one or more conservative amino acid substitutions and natural temporin The peptide difference.

The peptide of such variation and fragment have kept the biologically active of temporin, this biological living Property can be measured by method described below. Method is measured as described below, a kind of variation Temporin have naturally occurring temporin peptide (what for example SEQ ID:17 represented is the sort of) At least 40% amino acid sequence identity.

Temporin biologically active: the ability of temporin peptide bacteria growing inhibiting.

The antibacterial activity of given temporin peptide suppresses pectin bacterial strain example by measuring it Capability evaluation such as carrot owen bacteria or bacillus coli DH 5 . By in LB continuously Measure the work of given peptide in the hole of dilution peptide and 100: 1 aliquot to 96 hole microtiter plates The property. Fresh bacterial cultures (～0.3 A550) is then in Luria-Bertani culture medium (LB) Growth (1%w/v tryptone and 0.5%w/v yeast extract) also is diluted to 10 in LB^-2To represent about 10⁴-10 ⁵CFU (CFU) ml^-1 10: 1 bacterial cultures is inoculated then Enter to contain in the hole of peptide, sample was cultivated 4 hours at 37 ℃. The composition in hole is then rare with LB Release, be coated on the LB agar and 37 ℃ of overnight incubation. Then number is rare with each temporin Release the corresponding bacterium colony of liquid (with the contrast that does not add peptide), the antibacterial activity of the peptide under the test passes through Comparative measurements with control board.

Under the condition of this test, if it the concentration of 100:g/ml can suppress at least 10% bacterial growth (that is, and in this concentration, the quantity of bacterial clump be no more than control board 90%), just measure the temporin peptide and have biologically active

The antimycotic biologically active of given temporin peptide is by utilizing fungal bacterial strain cactus epidemic disease mould And/or Fusaium solani assessment. Selected fungal bacterial strain (contains 20gL at five kinds of cereal agar^-1Five kinds of cereal pablums make things convenient for thin slice, and 8gL^-1Agar³FCA (people such as Terras, The Plant Cell 7:573-588,1995) upper growth. After growing 5 days under the room temperature, take out bacterium The filament plug also is upside down in fresh FCA plate center. A kind of sterile solution of test peptides (10: 1) then Introducing is set up the control wells that contains sterilized water at same plate in the hole of panel edges 3cm. Same On one plate or on other plate, can test the test peptides of various concentration. Under the breadboard room temperature Cultivated 5 days, and measured afterwards growth inhibition district around each hole.

Under this experimental condition, if it can be at the control of the concentration conk of 5:g/ml (that is, The hole of containing this concentration peptide has recognizable inhibition zone on every side), just determine temporin peptide tool Biologically active is arranged.

Genetically modified plants: adopt such as this paper, this term refers to contain usually not in this kind wild type The plant of the genetic recombination material of finding in the plant. Thereby, put restructuring by being introduced into by conversion The plant of the plant cell growth of DNA is genetically modified plants, contains the genetically modified plant of introducing All offsprings (no matter being sexual or vegetative propagation) also be.

Sequence homogeneity: two nucleotide sequences, perhaps similar between two amino acid sequences The property, represent with the level of the sequence homogeneity shared between the sequence. Sequence homogeneity usually according to Percentage homogeneity represents; Percentage is more high, and two sequences are more similar.

The control methods of more various sequences is being known in the art. Following Introduction of Literatures each The program of kind and contrast algorithm: Smith and Waterman, Adv.Appl.Math, 2:482,1981; Needleman and Wunsch, J.Mol.Biol., 48:443,1970; Pearson and Lipman, Proc.Natl.Acad.Sci.USA, 85:2444,1988; Higgins ﹠ Sharp, Gene, 73:237-244,1988; Higgins ﹠ Sharp, CABIOS, 5:151-153,1989; Corpet etc. The people, Nucleic Acids Research, 16:10881-10890,1988; Huang waits the people, Computer Applications in the Biosciences, 8:155-165,1992; And Pearson Deng the people, Methods in Molecular Biology, 24:307-331,1994. The people such as Altschul, Nature Gene 6:119-129,1994 introduced a kind of about sequence control methods and homology meter The detailed consideration of calculating.

Can obtain from several sources the basic local sequence contrast research tool (BLAST) of NCBI (people such as Altschul, J.Mol.Biol.215:403-410,1990) comprise national biological information Center (NCBI, Bethesda, MD) and on the internet, with relevant sequence analysis program Blastp, blastn, blastx, tblastn and tblastx use together. Http//www.ncbi.nlm.nih.gov/BLAST/ can access it. How https://www.ncbi.nlm.nih.gov/BLAST/blast_help.html can be adopted The introduction of this program determination sequence homogeneity.

The variant one that is used for naturally occurring dermaseptin of the present invention and temporin peptide As be characterised in that, when adopting NCBI Blast 2.0.1 contrast, with naturally occurring temporin Or the contrast of the amino acid sequence total length of dermaseptin, have at least 40% sequence homogeneity (people such as Altschul introduces, Nucleic Acids Res.25:3389-3402,1997). In order to compare Surpass about 30 amino acid whose amino acid sequences, adopt Blast 2 functional nucleotide sequences, default The BLOSUM62 arranged in matrix becomes default parameters (gap existence value 11, each residue gap width 1). When contrast small peptide (less than about 30 amino acid), adopt Blast 2 functional nucleotide sequences, PAM30 Arranged in matrix becomes default parameters (open gap 9 is extended gap 1 punishment), carries out the sequence contrast. When assessing by the method, the protein that has with the higher similitude of reference peptide can show higher Percentage homogeneity, for example at least 45%, at least 50%, at least 70%, at least 80%, At least 85%, at least 90% or at least 95% sequence homogeneity.

Restructuring: recombinant nucleic acid is a kind of sequence that non-natural exists that has, and perhaps has a kind of logical Cross the artificial sequence in conjunction with preparation of two independent sequence fragments. This artificial combination by by Chemical synthesis is finished, more generally, and by the manually-operated to the nucleic acid fragment that separates, for example, Finish by technique for gene engineering.

Oligonucleotides (oligo): a kind of length is the linear multinuclear glycosides of about 100 nucleotide bases at the most Acid sequence.

Probe and primer:, can easily prepare nucleic acid probe and primer based on aminoacid sequence provided by the invention.Probe comprises the isolating nucleic acid that is connected with detectable label or reporter molecule.Conventional mark comprises radio isotope, part, chemoluminescence agent and enzyme.Marking method and the selection guide that is applicable to the mark of various purposes, referring to, for example, people such as Sambrook, MolecularCloning:A Laboratory Manual, Cold Spring Harbor, 1989 and people such as Ausubel, Current Protocals in Molecular Biology, Greene publishing company and Wiley-Intersciences, 1987.

Primer is short nucleic acid, long 15 Nucleotide or more of preferred DNA oligonucleotide.Primer can be annealed into the complementary target dna strand by the hybridization of nucleic acid, to form heterozygote between primer and target dna strand, then extends along target dna strand by archaeal dna polymerase.Primer is to being used for the amplification of nucleotide sequence, for example by polymerase chain reaction (PCR) or other nucleic acid amplification method well known in the art.

Preparation and use probe and the method for primer referring to, for example, people such as Sambrook, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor, 1989; People such as Ausubel, Current Protocals in Molecular Biology, Greene Greene publishing company and Wiley-Interscienes, 1987; And people such as Innis, PCR Protocols, AGuide to Methods and Applications, 1990.The PCR primer be to being derived by known sequence, for example by adopt with for example primer be target computer program (0.5 edition, 1991, Whitehead biomedical research institute, Cambridge, MA).Those skilled in the art will appreciate that the specificity of specific probe or primer increases with its length.Therefore, for example, a kind of primer that comprises 20 continuous nucleotides can with have than the more target annealing of high specific of the primer that 15 Nucleotide are only arranged accordingly.Thereby, in order to obtain higher specificity, can select to comprise 20,25,30,35,40,50 or the probe or the primer of more continuity Nucleotide.

Isolating a kind of " isolating " biotic component (for example nucleic acid or protein or organoid) is to separate basically or purifying with other biology composition from the biological cell of natural this composition of existence, promptly separates with RNA, protein and organoid with extrachromosomal DNA with other karyomit(e) or comes out from purifying wherein." isolating " nucleic acid and protein comprise nucleic acid and the protein by standard purification method purifying.This term also comprises the nucleic acid by the nucleic acid of recombinant expressed preparation in host cell and protein and chemosynthesis.

Carrier: a kind of nucleic acid molecule is introduced host cell, therefore produces a kind of transformed host cell.Carrier can comprise permission its nucleotide sequence in host cell clone, for example replication orgin.Carrier also can comprise one or more selectable marker gene and other gene well known in the art.

Be operably connected: when first nucleotide sequence and second nucleotide sequence were placed with function association, first nucleotide sequence was operably connected with second nucleotide sequence.For example, if promotor influences transcribing or expressing of encoding sequence, promotor is operatively coupled on the encoding sequence.Usually, the dna sequence dna that can be operatively connected is an adjacency, and, two encoding histones must be connected in the mode that meets same frame.By ormal peptide link, perhaps pass through the covalently bound of other, two peptide sequences can be operably connected.

The selection of II.dermaseptin and temporin peptide

The a.dermaseptin peptide

Some exemplary dermaseptin peptides more than are provided.The encode nucleic acid molecule of these dermaseptin polypeptide or derive to this peptide sequence by the genetic code simple application perhaps can adopt naturally occurring cDNA or gene order.For example the encode cDNA sequence shown in SEQ ID:1 (and being disclosed in people such as Amiche, J.Biol.Chem.269:1747-852,1994) of dermaseptin-b.Usually, select the dermaseptin peptide of the mature form be suitable for expressing.Yet, can select any fragment of total length dermaseptin peptide, decide with having the bioactive fragment of dermaseptin, if it is used to produce the antipathogen plant.

Those of ordinary skill in the art can understand, and has the various dermaseptin peptides of anti-microbial activity in various degree, and it has the more effectively effect of anti-some pathogenic agent than other.Therefore, when selecting peptide to be used to produce the transgenic plant with the anti-pathogenic agent of enhanced, in various factors, the kind of the plant of wanting expression of peptides is depended in the selection of concrete dermaseptin, and the kind that infects the sort of floristic pathogenic agent usually.

Choose the desirable dermaseptin that will express, can be by the nucleic acid molecule of this peptide of standard molecular biological technique preparation coding.Because the dermaseptin peptide is short relatively, the synthetic the simplest method of this nucleic acid molecule is likely on commercial available oligonucleotide synthesizer carries out via synthetic overlapping oligonucleotide.Then oligonucleotide in vitro is assembled into full length coding region.This method codon that reflection wherein will introduce the plant of nucleic acid molecule that also allows to select to encode uses the codon of the particular amino acid residue of skewed popularity, thereby strengthens expression efficiency.The detailed example of the generation of the encoding sequence that adopts this method is provided in following embodiment.

The b.temporin peptide

Some exemplary temporin peptides more than are provided.The encode nucleic acid molecule of these temporin polypeptide or derive to this peptide sequence by the genetic code simple application perhaps adopts naturally occurring cDNA or gene order.For example, the cDNA sequence of the coding temporin G shown in SEQ ID:15.Usually, select the temporin peptide of the mature form be suitable for expressing.Yet, can select any fragment of total length temporin peptide, decide with having the bioactive fragment of temporin, if it is used to produce the antipathogen plant.

When selecting the dermaseptin peptide, those of ordinary skill in the art can understand, and has the various dermaseptin peptides of the anti-microbial activity of change degree, and it has the more effectively effect of anti-some pathogenic agent than other.Therefore, when selecting peptide to be used to produce the transgenic plant with the anti-pathogenic agent of enhanced, in various factors, the kind of the plant of wanting expression of peptides is depended in the selection of special temporin, and the kind that causes the pathogenic agent of the sort of floristics loss.

As above introduction to dermaseptin, the synthetic and assembling of overlapping oligonucleotide is a kind of method of simply and easily producing the nucleic acid molecule of coding temporin.

C. the interpolation of other peptide sequence

Temporin and dermaseptin albumen also can be with the formal representations of fusion rotein in transgenic plant.Although the temporin that is suitable for expressing and dermaseptin albumen and any desirable peptide fusion of selecting can be comprised that the Expression of Fusion Protein expection of the N-terminal negatively charged ion of a kind of dermaseptin of being operatively coupled on or temporin proparea peptide is useful especially in plant.For this purpose can adopt any negatively charged ion proparea peptide, be included in the negatively charged ion proparea of finding in naturally occurring total length (that is, undressed) dermaseptin and the temporin peptide.For example, the proparea (being shown as SEQ ID:16) that comprises the 23-46 amino acids of temporin G can be used as a kind of proparea.Such proparea peptide in order in and the positively charged ion essence of dermaseptin or temporin, thereby enhanced stability can be provided in cellular environment.Therefore, these zones generally include a large amount of electronegative amino acid, for example L-glutamic acid (Glu or E) and aspartic acid (Asp or D).

Other the example of naturally occurring proparea peptide well known in the art comprises following proteic proparea peptide: people's neutrophilia defensin albumen (people such as Daher, Proc.Natl.Acad.Sci USA, 85:7327-7331,1988); Ox germ resistance cathelicidin protein B MAP28 (people such as Skerlavaj, J.Biol.Chem.271:28375-381,1996); Sheep germ resistance cathelin family protein (people such as Mahoney., FEBS Lett.377:519-522,1995); Ox indolicidin (people such as DelSal, Biochem.Biophys.Res.Commun.187:467-472,1992); Pig antibacterial peptide prophenin-2 and PR-39 (people such as Zhao, FEBS Lett.367:130-134,1995) and PMAP-37 (people such as Tossi, Eur.J.Biochem, 15:941-946,1995); The antibiotic lipopolysaccharide binding protein CAP18 of people (people such as Larrick, Infect.Immun.63:1291-1297,1995); And mouse albumen E3 (Scott and Collins, Blood 88:2517-2530,1996).

Although negatively charged ion proparea peptide can directly combine with cationic peptide N-is terminal, another embodiment comprises that employing spacer peptide sequence connects the proparea peptide to dermaseptin or temporin peptide.The use that connects the spacer peptide in two peptide territories in this area is known; Such spacer peptide length is normally between 2 and 25 amino acid, and the hinge that connects first peptide sequence to the second peptide flexibly is provided.Be used to provide the intervening sequence of the hinge flexibly that connects two peptide sequences to comprise glycine glycine (4)-Serine (GGGGS x3) at interval, described Nature 339:394-397,1989 by people such as Chaudhary.The N-terminal peptide extension of following introduction also can be used for providing the spacer peptide function.The intervening sequence peptide also can comprise shearing site, for example by proteolytic enzyme identification and the peptide sequence sheared, as factor Xa.Such site helps to remove the proparea from dermaseptin or temporin after purifying from plant tissue.In microorganism system, adopt negatively charged ion proparea peptide and spacer peptide being known in the art to express some cationic peptide, and in the United States Patent (USP) 5,593,866 of authorizing Hancock.

In certain embodiments, the terminal extension peptide sequence of N-can be added into dermaseptin or temporin peptide.Such peptide extends can comprise the part of the precursor forms of the dermaseptin of removing or temporin during albumen processing usually, perhaps can be the synthetic sequence.These N-terminal peptide are extended and are used to provide the anti-proteolysis of enhanced to shear property, strengthen transcriptional level or strengthen the anti-microbial activity of these peptides.Usually, the terminal development length of these N-is between 2 and 25 amino acid, although also can adopt longer extension.The example of the terminal extension sequence of the N-that adopts comprises peptide sequence AMWK, ASRH and ALWK in certain embodiments.AMWK (SEQ ID:39) sequence is that naturally occurring peptide extends; It is the part of total length dermaseptin-b peptide, cuts off in the course of processing usually.Reported to the N-of dermaseptin b peptide end and added the antibacterial activity in vitro (Strahilevitz, Biochemistry, 33:10951-10960,1995) that this sequence (to produce dermaseptin B) can strengthen this peptide.ASRH (SEG ID:41) and ALWK (SEQ ID:41) peptide are the synthetic extension sequences.Under each situation, the methionine(Met) that adds the N-end is to guarantee the suitable extension of this peptide.Those skilled in the art can understand the influence to the biologic activity of the peptide (dermaseptin or temporin) in producing that adds any specific terminal extension peptide of N-, can adopt the biological activity test of above introduction easily to assess.

D.dermaseptin and temporin peptide varient

As described above, a large amount of naturally occurring temporin and dermaseptin peptides are known, by being presented at those illustrations in the sequence table.By introducing amino acid replacement, the amino-acid residue that adds or passing through the deletion amino-acid residue, can select the varient of these naturally occurring peptides.These variation peptides or by chemosynthesis production (for example, this variation peptide reservation function activity for confirmation) are perhaps produced in the biology expression system.Under latter event, the nucleotide sequence that can operate the corresponding naturally occurring peptide of coding is so that its coding variation peptide.This can implement by many methods, for example by adopting site-directed mutagenesis or polymerase chain reaction.Because this peptide is short relatively molecule, the expression vector that is fit to is also introduced in the coding region of variation peptide de novo synthesis simply.

The simplest modification of aminoacid sequence comprises with one or more amino acid replaces the amino acid with similar biological property.These so-called conservative replacements have minimum influence to the protein active of gained probably.Therefore, replace and naturally occurring temporin or the different peptide of dermaseptin, can be used to replace naturally occurring peptide in the present invention by one or more conservative amino acid.Table 1 is presented at replaceable primary amino acid in the protein and is considered to the amino acid of conservative alternative.

Table 1

Original residue is guarded alternative

Ala ser

Arg lys

Asn gln；his

Asp glu

Gln asn

Glu asp

Gly pro

His asn；gln

Ile leu；val

Leu ile；val

Lys arg；gln；glu

Met leu；ile

Phe met；leu；tyr

Ser thr

Thr ser

Trp tyr

Tyr trp；phe

Val ile；leu

By selecting those not conservative alternatives than table 1, can obtain more substantial variation on function or further feature, promptly be chosen in visibly different residue on its following effects: keep (a) to replace district's polypeptide backbone structure, for example, as folding or spiral structure, (b) electric charge of target position molecule or hydrophobicity, the perhaps size of (c) side chain.Wish that generally producing the maximum alternative that changes on protein capability is those groups, (a) a kind of wetting ability residue wherein, for example seryl or Threonyl, replace hydrophobic residue, for example leucyl, isoleucyl, phenylalanyl, valyl or alanyl (or by its replacement); (b) halfcystine or proline(Pro) replace any other residue (or by its replacement); (c) a kind of residue of positively charged side chain, for example lysyl, arginyl, histidyl-replace electronegative residue, for example glutamyl or aspartyl (or by its replacement); Perhaps (d) has the residue of bulky side chain, and phenylalanine for example replaces for example glycine (or by its replacement) of a kind of group that does not have a side chain.The present invention also can adopt has these more variation peptides of one or more in the variation of essence, and condition is to keep temporin or dermaseptin biologic activity.

Amino acid changes also and can carry out at variation dermaseptin or temporin widely.Yet as above explain, when adopting the above contrast program of introducing and their separately natural to have the aminoacid sequence total length contrast of sequence, these peptides that make a variation are characterised in that usually, calculate them and have at least 40% sequence identity.In addition, these variation peptides keep biologic activity.

Adopt the above test system of introducing to confirm that dermaseptin or temporin peptide have biologic activity.Confirm that peptide has after the activity of hope, adopt standard molecular biological technique can easily produce the nucleic acid molecule of this peptide of coding.When suitable, the selection of open reading frame will consider wherein to want the codon of the plant species of expression of peptides to use skewed popularity.III.dermaseptin and/or temporin introduced plant

In case produced the nucleotide sequence of coding dermaseptin and/or temporin peptide, can adopt standard technique in transgenic plant, to express this sequence, so that give the plant disease-resistant substance.Basic skills is that cloning nucleic acid enters conversion carrier, like this so that it operationally be connected with the control sequence (for example promotor) of guiding this nucleic acid to express at vegetable cell.This conversion carrier is then by a kind of (for example, electroporation) the introduced plant cell in many technology, and whole plants is by these cell regeneration, and selection contains the descendant plant of the nucleic acid of these introducings.Preferably, the conversion carrier stable integration of all or part is gone into the vegetable cell genome.That part of recombinant expression cassettes that is called as of conversion carrier that is integrated into vegetable cell and contains calling sequence and be used to control the relevant sequence (" transgenosis " of introducing) of expression.

Containing the selection of introducing genetically modified descendant plant can carry out based on detecting the phenotype that changes.The disease resistance that a kind of like this phenotype can directly be given from calling sequence, perhaps conduct imports to the result that the selectable dominant marker's gene in the conversion carrier is introduced, and is proved to be to have chemical reagent (for example microbiotic) enhanced resistance.

In technology and scientific literature, be full of the example that is characterized as with the success of cloning nucleic acid sequences plant transformed modification.Selectedly be used to illustrate that the example of the described knowledge in present technique field comprises:

United States Patent (USP) 5,571,706 (" plant virus resistance gene and methods ")

United States Patent (USP) 5,677,175 (" phytopathogen inducible proteins ")

United States Patent (USP) 5,510,471 (" mosaic genes that are used for Plant Transformation ")

United States Patent (USP) 5,750,386 (" antipathogen transgenic plant ")

United States Patent (USP) 5,597,945 (" heredity strengthens the plant of disease resistance ")

United States Patent (USP) 5,589,615 (" having the production method of transgenic plants of the nutritive value of improvement ") by the albuminised expression of 25 storages of having modified

United States Patent (USP) 5,750,871 (" conversion of rape kind and exogenous gene expressions ")

United States Patent (USP) 5,268,526 (" overexpressions of plant pigments in the transgenic plant ")

United States Patent (USP) 5,780,708 (" fertile rotaring gene corn plants ")

United States Patent (USP) 5,538,880 (" production methods of fertile rotaring gene corn plant ")

United States Patent (USP) 5,773,269 (" fertile transgenosis oat plants ")

United States Patent (USP) 5,736,369 (" production methods of rotaring gene paddy quasi-plant ")

United States Patent (USP) 5,610,042 (" the stable method for transformation of wheat ").

The structure that these examples comprise selection, the transformation technology of introducing conversion carrier and are designed to the genetically modified construct that overexpression introduces.

A. floristics

The disease that is caused by many pathogenic agent acts on the various plants kind.These plants are monocotyledons, dicotyledons or gymnosperm.Therefore, for example, dermaseptin and/or temporin peptide can the exotic plant kind comprise, but be not limited to beans, rape/vegetable seed (canola), alfalfa, flax, Sunflower Receptacle, safflower, rape, cotton, tobacco, flax, peanut, trifolium, cowpea, the grape of corn, wheat, rice, barley, soybean, cotton, general meaning; Vegetables, for example lettuce, tomato, cucurbit, cassava, potato, Radix Dauci Sativae, radish, pea, root of Szemao crotalaria, wild cabbage, Cauliflower, cabbage, Brussels sprouts, pepper; The tree fruit is oranges and tangerines, apple, pear, peach, apricot, English walnut for example; Fur is set for example Pseudotsuga menziesii (Mirbel) Franco and torch pine; And flower, for example carnation and rose.

B. vector construction and promotor are selected

By the agency of be suitable for the recombinant vectors that vegetable cell stable transfection or transgenic plant are established in a large number, be included in people such as Pouwels, Cloning Vectors:A Laboratory Manual, 1985, supp., 1987; Weissbach and Weissbach, Methods for Plant MolecularBiology, Academic Press, 5:173-184,1989; And people such as Gelvin, PlantMolecular Biology Manual, press of Kluwer institute, those that introduce in 1990.Usually, plant conversion carrier be included in 5 ' and 3 ' regulate one or more sequences of having cloned under sequence and the dominant selectable marker control.Such plant conversion carrier also comprises promotor regulatory region (for example, a kind of control is induced or composing type, through environment or regulatory region that grow to regulate or cell or tissue is specific expressed), transcription initiation site, ribosome bind site, RNA processing signal, Transcription Termination site and/or polyadenylation signal usually.Can be used for expressing a kind of genetically modified constitutive plant promoters comprises: cauliflower mosaic virus (CaMV) 35S promoter, it can in most of plant tissues, give composing type, high-caliber expression (referring to for example, people such as Odel, Nature, 313:810,1985; People such as Dekeyser, Plant Cell, 2:591,1990; Terada and Shimamoto, Mol.Gen.Genet.220:389,1990; And Benfey and Chua, Science, 250:959-966,1990); Nopaline synthase promoter (people such as An, Plant Physiol.88:547,1988); Octopine synthase promoter (people such as Fromm, PlantCell, 1:977,1989) and 2x CaMV/35S promotor (people such as Kay, Science, 236:1299-1302,1987) with transposition enhancement sequences.

Response environment, hormone, chemistry and/or grow signal and the various plant gene promoters that are conditioned also can be used in vegetable cell transfer expression of gene, comprise the promotor of regulating: (a) heat (people such as Callis by following factor, Plant Physiol., 88:965,1988; People such as Ainley., Plant Mol.Biol., 22:13-23,1993; With people such as Gilmartin, The Plant Cell, 4:839-949,1992) (b) light (for example, pea rbcS-3A promotor, people PlantCell such as Kuhlemeier, 1:471,1989 and corn rbcS promotor, Schaffner ﹠amp; Sheen, Plant Cell, 3:997,1991); (c) hormone, for example dormin (people such as Marcotte, Plant Cell, 1:471,1989); (d) wound (for example, potato PinII promotor (people such as Keil, Nucl.Acids.Res.14:5641-5650,1986), Agrobacterium mas promotor (people Bio/Technology 10:305-308 such as Langridge, 1989) and grape vine vstl promotor (people .Plant Mol.Biol. such as Weise, 26:667-677,1994); (e) for example methyl jasmonate or Whitfield's ointment (also referring to people .Plant Mol.Biol.48:89-108 such as Gatz, 1997) of chemical.

Tissue specificity (for example root, leaf, flower and seed) promotor (people such as Carpenter, ThePlant Cell 4:557-571,1992; People such as Denis, Plant Physiol.101:1295-1304,1993; People such as Opperman, Science 263:221-223,1993; People such as Stockhause, ThePlant Cell 9:479-489,1997; People such as Roshal, The EMBO J.6:1155,1987; People such as Schernthaner, EMBO J.7:1249,1988; People such as Yamamoto, Plant Cell3:371-382,1990; People such as and Bustos, Plant Cell 1:839,1989) also can be merged and be gone into encoding sequence to obtain expression specific in each organ.

Plant conversion carrier also can comprise the RNA processing signal, for example, intron, it can be positioned at the upstream or the downstream of this transgenosis ORF sequence.In addition, expression vector also can comprise from 3 of plant gene '-other of non-translational region regulate sequence, for example, a kind of 3 ' terminator that strengthens the mRNA stability of mRNA is as PI-II terminator or octopine or nopaline synthase (NOS) 3 ' terminator of potato.

At last, as above indicate, plant conversion carrier also can comprise the dominant selectable marker gene, to allow easily to select transformant.These groups comprise those of coding antibiotics resistance group (for example, moisture resistance mycin, kantlex, bleomycin, G418, Streptomycin sulphate or Trobicin) and anti-herbicide gene (for example, phosphinothricin acetyltransferase).

C. transform and regeneration techniques

The conversion of the vegetable cell of monocotyledons and dicotyledons and regeneration are conventional now, and the professional can determine appropriate transformation technology.The selection of method changes with vegetation type to be transformed; Those skilled in the art can discern ad hoc approach for given floristic appropriateness.Appropriate means comprises, but is not limited to: the electroporation of plant protoplast; Liposome-mediated conversion; The conversion of polyoxyethylene glycol (PEG) mediation; Adopt the conversion of virus; The vegetable cell microinjection; The vegetable cell microparticle bombardment; Vacuum is infiltrated; And the conversion of agrobacterium tumefaciens (AT) mediation.The patent documentation that these chapters and sections begin column goes out has been described the exemplary steps of conversion and aftergrowth.

D. transformed the selection of plant

After adopting conversion carrier to carry out Plant Transformation and regeneration, adopt the dominant selectable marker that mixes conversion carrier to select plant transformed usually.Normally, such mark can give transformed plant rice shoot with antibiotics resistance, the selection of transformant can be finished by exposing rice shoot to the microbiotic of proper concn.

Select also can finish by the pathogen resistance that utilization is given plant through transgenosis.Introduce as following examples, such screening or transgenic plant regenerate after, finish, perhaps (method for transformation that depends on employing) carries out in afforesting on the transgenic calli before the plant regeneration.IV. the plant that contains the polycation peptide-coding region

Under some situations, the resistance level of giving by the single copy of transgenosis of coding dermaseptin or temporin peptide, can by introduce single cationic peptide gene repeatedly copy or several genes of the different cationic peptides of encoding strengthen.

By adopting genetically engineered, it is possible that the polycation peptide-coding region is introduced single carrier.Normally (although not necessarily), such carrier comprises two or more dermaseptin and/or temporin open reading frame, and each operationally 5 ' and 3 ' is regulated sequence and be connected with himself.When being introduced into plant, such carrier can cause the expression of multiple cationic peptide.

The creation that contains how genetically modified plant also can be finished by adopting the standard breeding technique.The transgenosis of first cationic peptide of encoding can be introduced into first plant, and second transgenosis of second cationic peptide of encoding can be introduced into second plant.The transgenic plant of gained then can hybridize with production and are loaded with two kinds of genetically modified offsprings.The production of V.dermaseptin and temporin with separate

More than composition of Jie Shaoing and method not only can be used to produce have enhanced, the plant of broad spectrum of pathogens resistance, and can be used to be suitable for the extensively dermaseptin of other purposes and the scale operation of temporin.For example, a large amount of temporin that produce and dermaseptin can purifying and be used for medical use in the plant.

The production of biologically active peptides is extensively implemented now in the plant, and large quantities of expression and purification process are known.Promote the construct example that biologically active proteins produces in the plant in people's such as Goodman United States Patent (USP) 4,956,282, to find.These construct examples comprise promoter region usually and encode the additional nucleotide sequence of the aminoacid sequence of utilization in purifying processing afterwards.Be used to promote dermaseptin and/or the isolating aminoacid sequence of temporin peptide can to shear and abandon subsequently.

The selection and the creation of the nucleotide sequence of embodiment 1. coding dermaseptin and temporin peptide

The a.dermaseptin encoding sequence

Nucleic acid molecule is designed to sophisticated 27 amino acid length forms (SEQ ID:3) of dermaseptin b, has 5 terminal extension sequences of amino acid whose N-, MAMWK.This nucleic acid construct called after MSRA ₂And in single PCR reaction, adopt four overlapping oligonucleotide synthetic.The oligonucleotide that adopts is as shown in table 2.Two oligonucleotide (oligonucleotide #1 and oligonucleotide #2) comprise the nucleotide sequence of the terminal and C-terminal portions of the N-of encoded peptide respectively.These oligonucleotide concentration that adopt in the PCR reaction are 20nM.Latter two oligonucleotide comprises the sequence by various restriction enzyme identifications.Particularly, oligonucleotide #3 comprises the restriction site of XbaI, KpnI and NcoI, and oligonucleotide #4 comprises the restriction site of SstI, PstI and HindIII.These oligonucleotide concentration that adopt in the PRC reaction are 200nM.After the product amplification, adopt the embedding restriction site that it is cloned into conventional cloning vector.MSRA ₂The nucleotide sequence of coding region is shown as SEQ ID:27, and the peptide that is encoded is shown as SEQ ID:28.Oligonucleotide #1-4 is shown as SEQ ID:29-32.

Table 2 oligonucleotides #1:5 '-ATG GCC ATG TGG AAA GAC GTT CTG AAA AAG (SEQ ID:29) ATC GGT ACT GTC GCC CTC CAT GCA GGG-3 ' oligonucleotides #2:3 '-TGA CAG CGG GAG GTA CGT CCC TTC CGG CGC (SEQ ID:30) GAA CCT CGT CAT CGG CTG TGG TAG AGC GTC ATT-5 ' oligonucleotides #3:5 ' TCT AGA GGT ACC ATG GCC ATG TGG AAA GAC G-(SEQ ID:31) 3 ' oligonucleotides #4:3 '-GGC TGT GGT AGA GCG TCA TTC TCG AGA CGT CTT (SEQ ID:32) CGA AC-5 '

Runic Nucleotide is represented the complementary zone between oligonucleotide #1 and the oligonucleotide #2.The underscore of oligonucleotide #1 part is the same with the underscore part of oligonucleotide #3, so allows oligonucleotide #3 and be derived from the PCR product that oligonucleotide #1 and #2 begin to prolong and combine.Similarly, the underscore of oligonucleotide #4 part is the same with the following underscore part of oligonucleotide #2.This allows oligonucleotide #4 to combine with the PCR product of being created by the prolongation of oligonucleotide #1 and #2.

The b.temporin encoding sequence

Nucleic acid molecule is designed to sophisticated 13 amino acid length forms of temporin A, and it has 6 terminal extension sequences of amino acid whose N-, MASRHM (SEQ ID:33).This nucleic acid construct called after MSRA ₃And in single PCR reaction, adopt four overlapping oligonucleotide synthetic.The oligonucleotide that adopts is as shown in table 3.Two oligonucleotide (oligonucleotide #1 and oligonucleotide #2) comprise the nucleotide sequence of coding prototype peptide respectively.Yet different with the oligonucleotide of the prototype dermaseptin peptide that is used to encode, these oligonucleotide are complete complementary, thus eliminated oligonucleotide #3 and #4 in conjunction with before to initial extension round-robin needs.Oligonucleotide #1 that adopts in the PCR reaction and the concentration of oligonucleotide #2 are 20nM.Latter two oligonucleotide comprises the sequence by various restriction enzyme identifications.Particularly, oligonucleotide #3 comprises the restriction site of XbaI, KpnI and NdeI, and oligonucleotide #4 comprises the restriction site of SstI and PstI.These oligonucleotide concentration that adopt in the PCR reaction are 200nM.

After the product amplification, adopt the embedding restriction site that it is cloned into conventional cloning vector.MSRA ₃The nucleotide sequence of coding region is shown as SEQ ID:33, and the peptide of having encoded is shown as SEQID:34.Oligonucleotide #1-4 is shown as SEQ ID:35-38.

Table 3 oligonucleotide #1:5 '-ATG TIT CTG CCC CTA ATC GGG AGG GTT CTC, (SEQ ID:35) TCG GGA ATC CTG TAA-3 ' oligonucleotide #2:3 '-TAC AAA GAC GGG GAT TAG CCC TCC CAA GAG, (SEQ ID:36) AGC CCT TAG GAC ATT-5 ' oligonucleotide #3:5 '-GGT ACC TCT AGA CAT ATG TTT CTG CCC CTA-3 ', (SEQ ID:37) oligonucleotide #4:3 '-GAG AGC CCT TAG GAC ATT CTC GAG ACG TC-5 ', (SEQ ID:38)

Runic Nucleotide is represented the complementary zone between oligonucleotide #1 and the oligonucleotide #2.These sequences as description in the table are complete complementary.Oligonucleotide #2 underscore part is complementary with oligonucleotide #3 underscore part, and oligonucleotide #1 underscore part is complementary with oligonucleotide #4 underscore part.

The double-stranded DNA of gained then clone advances one or more carriers described below.2. the carrier that contains various promoter sequences

Coding MRSA ₂And MRSA ₃Nucleotide sequence be fitted into various plant conversion carriers, thereby be placed on the control of various promotor down.

Clone MRSA ₂And MRSA ₃Sequence enters a kind of such carrier, different sequences is placed under the control of the promotor that two copies comprising the CaMV 35S promoter and AMV RNA4 translation strengthen element people such as (, Science 236:1299-1302,1987) Kay.By the clone who connects into this carrier gained with prefix pD as title.Therefore, pDMSRA ₂Expression is included in the MSRA that contains under two CaMV 35S promoters and the control of AMV RNA4 translational enhancer ₂The carrier of construct.Similarly, pDMSRA ₃Expression is included in the MSRA that contains under two CaMV 35S promoters and the control of AMV RNA4 translational enhancer ₃The carrier of construct.

Designed another kind of carrier, so that MSRA ₂Construct is subjected to rebuild the control of " super promotor ".This promotor is included in mas (mannopine synthase) promotor/region of activation after ocs (octopine synthase) upstream activation sequences (oppositely) triplet people such as (, Bio/Technology 10:305-308,1989) Langridge.This super promotor is introduced in more detail referring to people such as Ni, The Plant J., 7:661-676,1995.This special construct also comprises the coding region of 6 * His mark, is positioned at MSRA ₂Upstream of coding region also operationally is connected with it.Therefore, this 6 * His marker amino acid sequence is expressed as the N-end that is attached to the dermaseptin/MAMWK peptide that is encoded.The carrier of this peptide of encoding is named as pRSHMSRA ₂3. the conversion of potato and tobacco

The potato kind, Russert Burbank and Desiree, and tobacco is used as the representative plant variety that is suitable for transforming.According to DeBlock, Theoret.Appl.Genet.76:767-774,1988, in addition some change, and implement the conversion of plant.

Briefly, by from 4 age in week branch separate leaf (5mm) and stem is implemented the conversion of tobacco and potato.Downcut these leaves with branch and by the further damage of swiping with the glass pipet point.Injured leaf and stem are then put upside down floating in the petri diss of the S2 substratum that contains 15ml.This substratum comprises as Murashige and Skoog, Physiol.Plant.15:473-479, and 1962 listed compositions are supplemented with 0.5g/L MES and the 20g/L N.F,USP MANNITOL of 30g/L sucrose, pH 5.5.

The LB substratum that contains the 60 μ l of Agrobacterium (transforming with interested carrier) adds each culture dish at the back logarithmic phase, and these culture dish were then cultivated 3 days in the presence of Agrobacterium under low light intensity (500lux).Then, blot, put upside down and insert the S4 substratum with filter paper to contain the S2 substratum washing plant tissue of 1g/L Pyocianil.The S4 substratum comprises Murashige and Skoog, Physiol.Plant.15:473-479,1962 compositions of listing deduct sucrose and replenish 200mg/L L-glutamic acid, 0.5g/L MES, the 0.5g/L PVP of pH 5.7,20g/L N.F,USP MANNITOL, 20g/L glucose, 40mg/L gland fat purine-SO ₄, 0.5% agarose, the trans zeatin of 1mg/L, 0.1mg/L NAA, 1g/L Pyocianil and 50 μ g/ml kantlex, 10mg/L AgNO ₃).Placing the plant tissue sample under the room temperature (RT) under 3000lux forms to allow callus.After two weeks, many little callus (Calli) have been formed at the edge of wound of leaf and stem.Take out little callus and be transferred to fresh S6 substratum (S4 that does not contain NAA).2-3 is after week, and these callus are transferred to the S8 substratum and (have replenished 0.1mg/L GA ₃S6) allow to form bud.

After the S8 substratum put for two weeks, first bud (0.5cm) was transferred to the S1 substratum.This substratum comprises the composition (Exp.Cell Res.50:151-158,1968) that people such as Gamborg describes, and also adds 20g/L sucrose, 150mg/L CaCl ₂, pH 5.8 0.4% agarose, 1g/L Pyocianil and 50 μ g/ml kantlex.S1 substratum and bud are put into the Magenta bottle and are formed to allow root.Bud is taken root after one week.For fear of selecting identical root, avoid shifting bud from same or close-connected callus.

The transgenic plant that regenerated is inferred change over to contain 1g/L Pyocianil and 50 μ g/ml kantlex the MS substratum with further analysis.4. the screening of disease resistance callus

Developed the simplification early detection method that is suitable for disease-resistant test.Contrast and transgenosis callus are grown in the S4 substratum, and (the MS substratum does not wherein have sucrose and replenishes 200mg/L L-glutamic acid, 0.5g/L MES, the 0.5g/L PVP of pH 5.7,20g/L N.F,USP MANNITOL, 20g/L glucose, 40mg/L VITAMIN B4-SO ₄, 0.5% agarose, the trans zeatin of 1mg/L, 0.1mg/LNAA, 1g/L Pyocianil and 50 μ g/ml kantlex, 10mg/L AgNO ₃).Sample is then placed under RT and 3000lux to allow callus to form.After two weeks, many little callus have been formed at the edge of wound of leaf and stem.Take out little callus and be transferred to fresh S6 substratum (S4 of no NAA).2-3 is after week, and callus is transferred to fresh culture and at phytopathogen, grows under the condition that Fusarium or phytophthora exist.The experiment ending is write down survival and is kept bright green callus.In control sample, do not find the antifungal property callus, find that the callus of resistant to fungal pathogens transforms.5. the Molecular Identification of transgenic plant

Adopt method described below DNA isolation from transgenic Rhizoma Solani tuber osi and tobacco plant.Carry out purifying, relate to a kind of stricter scheme in some cases, under other situation, relate to a kind of simple thick extraction step.Fresh leaf tissues and freezing sample in liquid nitrogen immediately by obtaining 10 grams begin stricter extraction step.The tissue of freezing mistake then is ground into fine powder and extracts damping fluid (50mM Tris-HCl damping fluid, 5mM EDTA, 0.35M sorbyl alcohol, 0.1%BSA, 0.1% β mercaptoethanol, 10%PEG 4000) with 20ml and extract.Filter homogenate by which floor cheese cloth and one deck micropore cloth.Follow according to people such as Wagner, Proc.Natl.Acad.Sci.U.S.A.84:2097-5100,1987, implement last purification step.

When separating, mainly adopt thick extraction step for the pcr analysis purpose.For this step, collect the fresh leaf of about 200mg and in liquid nitrogen, be ground into powdery.The 0.5NNaOH of 100 μ l adds in the powder and mixes (rotation) 30 seconds.Centrifugal 5 minutes of this suspension, 5 μ l supernatant liquors add in the Tris damping fluid (pH 8.0) of the 100mM of 45 μ l.This thick extracting genome DNA thing is used as the template of pcr amplification.

Implement the PCR reaction by oligonucleotide #3 and #4, carry out MSRA with genomic dna that extracts and above table 2 ₂The detection whether construct exists.The product size of the expectation of reacting since then is 129bp.This method allows to identify uses pDMSRA ₂Or pRSHMSRA ₂Transgene tobacco and potato that construct transforms.

By using the oligonucleotide #3 and the #4 of table 3, perhaps with the combination of specificity, implement the PCR reaction at the oligonucleotide #4 of the primer of 2 * 35SCaMV promotor and last table 3, carry out MSRA ₃The detection of construct.Identify and contain pDMSRA ₃The rotaring gene tobacco plant of construct.

Under the control of the super promotor that contains mas (mannopine synthase) promotor/region of activation after the triplet of ocs (octopine synthase) upstream activation sequences (oppositely), also control plant is transformed into and comprises gus gene (people such as Ni, The Plant J.7:661-676.1995).Confirm that through round pcr this transgenosis inserted the tobacco plant genome.

Under some situation, confirm genetically modified activity expression by the Northern engram analysis.From transgene tobacco and potato plants, separate and be purified into the RNA substrate that is used for these experiments.According to people such as Verwoerd, Nucl.Acids Res.17:2362,1989, implement to be used for this isolating scheme.6. antibacterium pathogenic agent

In order to detect this transgenic Rhizoma Solani tuber osi and tobacco plant resistance, allow this bacterium under the room temperature at LB substratum growth (A600=2.9) whole night to bacterial pathogens Radix Dauci Sativae owen bacteria.The owen bacteria culture of 2ml aliquot is at dH ₂Dilution is 5 times among the O, and the culture of 1ml dilution adds in the 2ml MS liquid nutrient medium and is used for this antibacterial tests.The branch of on transgenic plant or control plant, newly cutting (～3.5cm) insert the test tube bottom immersion bacterial cultures that consequently this plant is cut edge that contains the owen bacteria culture that has diluted.Under cultivating, also observes off and on 3000lux under the developmental tube room temperature.

Contain pDMSRA ₂The transgenic Rhizoma Solani tuber osi plant and contain pDMSRA ₂Or pRSHMSRA ₂Rotaring gene tobacco plant as above introduce and test, and show antipathogen: after one week of growth, this transgenic plant uninfection (determining by visual inspection) also continues to grow in the presence of bacterial cultures.The ground that differs widely, with control plant severe infections after cultivating a week of bacterial cultures struggle, growth is suppressed, plant death after being exposed to fungal pathogens 2-3 week.

In order to detect the resistance of transgenic Rhizoma Solani tuber osi stem tuber, on the stem tuber disk, open an aperture (diameter 2cm, thick 3cm) to bacterial pathogens Erwinia carotovora cvcarotovora.The bacterial cultures overnight (about 2 * 10 of the 100x dilution of 20 microlitres ⁷CFU) move in the discharge hole, dish was at room temperature cultivated 6 days.From dish, remove septic tissue lightly, measure the weight loss of stem tuber tissue.Test the result who obtains thus and show, compare the anti-soft rot of genetically modified potato tissue (Fig. 1) with the non-transgenic control group.

In microtiter plate, to contain about 1 * 10 ⁵The last volume of 220 μ l of bacterium/ml and prescribed concentration antibacterial peptide is measured antibacterial peptide (SEQ ID NO:34 (MRSA ₃) and SEQ ID NO:28 (MRSA ₂)) anti-microbial effect of antagonism intestinal bacteria (Fig. 2) and Radix Dauci Sativae owen bacteria (Fig. 3).Cultivated under the cell culture room temperature 4 hours, and diluted and be coated on the LB plate.After 37 ℃ (intestinal bacteria) or 28 ℃ of (Radix Dauci Sativae owen bacteria) incubated overnight, the bacterium of enumeration and record survival.Two test-results show that this peptide has significant anti-microbial activity.7. antifungal property

Adopt following scheme to measure the resistance of maturation plant to various fungies.Cut 1cm ²* 0.5cm comprises V8 nutrient agar (the 250ml/L V8 juice that Fusarium or phytophthora (Phytophthora sp.) are cultivated substrate and put into the culture dish of 9cm, 7 gram/L agar) center of fresh plate, and at room temperature grew about one month, perhaps thoroughly cover this culture dish up to radicula byssoidea.Downcut transgenic plant bud (～10cm) and change the MS substratum over to further growth.According to various processing, allow plant-growth 3 days or 2 weeks up to the blastogenesis root.Do not injure plant, then with two 1cm ²The mycobiotic agar section of * 0.5cm is applied to the both sides of plant sprout.Vision is determined the gradient of infection of gained then.

In typical test, with pDMSRA ₂The transgenic Rhizoma Solani tuber osi plant that transforms is with pRSHMRSA ₂Or pDMSRA ₂The tobacco plant that transforms, and the potato of contrast and tobacco plant to be exposed to the Root and stem of Cholla epidemic disease mould down.After 7 days, the Root and stem of Cholla epidemic disease is mould in the whole surface growth of MS substratum, and penetrates the root and the stem of control plant, causes the infringement to important plant function.The velamen grievous injury of control plant, this is tangible.Interact between plant and the fungi and cause the secretion of the Huang-brown pigmentation of indicating decay process.Subsequently, the plant dehydration, leaf curls, and the bottom of stem limbers up, root death.Yet transgenic plant still health there is no disease symptoms, even radicula byssoidea covers the MS substratum fully.

More than the enforcement of Jie Shaoing also is used to test the resistance of transgenic plant to phytophthora infestan.These test-results show that transgenic plant also have resistance to phytophthora infestan.

Use fusariun solani bacterium attack pDMSRA ₂Another enforcement of transgenic Rhizoma Solani tuber osi plant.After 6 days, sickle spore bacteria growing spreads all over the MS media surface, and the infringement to root in control plant is serious, and sickle spore bacterium penetrates the stem foot bottom, and stem is softening, and the vein of the leaf of infection demonstrates brown stain clearly and necrosis.After several days, the control plant atrophy is also dead.Yet transgenic plant continue growth, even under the bad attack of fusariun solani bacterium.

Having illustrated and introduced principle of the present invention in a plurality of embodiments and embodiment, do not deviated from these principles and change the present invention on arrangement and details, is tangible to those skilled in the art.Our claimed all changes in the spirit and scope in the claim subsequently.

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Sequence table＜110〉the Sang Tuoshi Meath draws

Bill is triumphant＜120〉anti-broad spectrum of pathogens genetically modified plants＜130〉54447＜140〉＜141＜150〉60/125,072＜151〉1999-03-17＜160〉41＜170〉PatentIn Ver.2.0＜210〉1＜211〉443＜212〉DNA＜213〉Phyllomedusa bicolor＜220〉＜221〉CDS＜222〉(58) .. (294)＜400〉1ccacgcgtcc gattctgtcc tccagtactc aacacattct gaattgtaag aacaaac 57atg gat atc ctg aag aaa tct ctt ttc ctt gta tta ttc ctt gga ttg 105Met Asp Ile Leu Lys Lys Ser Leu Phe Leu Val Leu Phe Leu Gly Leu 15 10 15gtt tcc ctt tcc atc tgt gaa gaa gag aaa aga gaa aat gaa gat gag 153Val Ser Leu Ser Ile Cys Glu Glu Glu Lys Arg Glu Asn Glu Asp Glu

20 25 30gag?aaa?caa?gat?gac?gag?caa?agt?gaa?atg?aag?aga?gct?atg?tgg?aaa 201Glu?Lys?Gln?Asp?Asp?Glu?Gln?Ser?Glu?Met?Lys?Arg?Ala?Met?Trp?Lys

35 40 45gat?gtg?tta?aaa?aaa?ata?gga?aca?gtg?gcc?tta?cat?gca?gga?aaa?gcg 249Asp?Val?Leu?Lys?Lys?Ile?Gly?Thr?Val?Ala?Leu?His?Ala?Gly?Lys?Ala

50 55 60gct?tta?ggt?gca?gtt?gct?gat?aca?ata?agt?caa?gga?gag?caa?taa 294Ala?Leu?Gly?Ala?Val?Ala?Asp?Thr?Ile?Ser?Gln?Gly?Glu?Gln?65 70 75agtgaaaaaa?atttaaaatt?gaattactct?aaatagaaca?attagcaata?attgtgtcaa?354acctacatta?aagcatactg?aaccaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?414aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaa 443<210>2<211>78<212>PRT<213>Phyllomedusa?bicolor<400>2Met?Asp?Ile?Leu?Lys?Lys?Ser?Leu?Phe?Leu?Val?Leu?Phe?Leu?Gly?Leu 1 5 10 15Val?Ser?Leu?Ser?Ile?Cys?Glu?Glu?Glu?Lys?Arg?Glu?Asn?Glu?Asp?Glu

20 25 30Glu?Lys?Gln?Asp?Asp?Glu?Gln?Ser?Glu?Met?Lys?Arg?Ala?Met?Trp?Lys

35 40 45Asp?Val?Leu?Lys?Lys?Ile?Gly?Thr?Val?Ala?Leu?His?Ala?Gly?Lys?Ala

50 55 60Ala?Leu?Gly?Ala?Val?Ala?Asp?Thr?Ile?Ser?Gln?Gly?Glu?Gln?65 70 75<210>3<211>27<212>PRT<213>Phyllomedusa?bicolor<400>3Asp?Val?Leu?Lys?Lys?Ile?Gly?Thr?Val?Ala?Leu?His?Ala?Gly?Lys?Ala 1 5 10 15Ala?Leu?Gly?Ala?Val?Ala?Asp?Thr?Ile?Ser?Gln

20 25<210>4<211>31<212>PRT<213>Phyllomedusa?bicolor<400>4Ala?Met?Trp?Lys?Asp?Val?Leu?Lys?Lys?Ile?Gly?Thr?Val?Ala?Leu?His 1 5 10 15Ala?Gly?Lys?Ala?Ala?Leu?Gly?Ala?Val?Ala?Asp?Thr?Ile?Ser?Gln

20 25 30<210>5<211>36<212>PRT<213>Pachymedusa?dacnicolor<400>5Gly?Met?Trp?Ser?Lys?Ile?Lys?Asn?Ala?Gly?Lys?Ala?Ala?Ala?Lys?Ala 1 5 10 15Ser?Lys?Lys?Ala?Ala?Gly?Lys?Ala?Ala?Leu?Gly?Ala?Val?Ser?Glu?Ala

20 25 30Leu?Gly?Glu?Gln

35<210>6<211>31<212>PRT<213>Pachymedusa?dacnicolor<400>6Ala?Leu?Trp?Lys?Thr?Leu?Leu?Lys?Lys?Val?Gly?Lys?Val?Ala?Gly?Lys 1 5 10 15Ala?Val?Leu?Asn?Ala?Val?Thr?Asn?Met?Ala?Asn?Gln?Asn?Glu?Gln

20 25 30<210>7<211>35<212>PRT<213>Agalychnis?annae<400>7Gly?Met?Trp?Ser?Thr?Ile?Arg?Asn?Val?Gly?Lys?Ser?Ala?Ala?Lys?Ala 1 5 10 15Ala?Asn?Leu?Pro?Ala?Lys?Ala?Ala?Leu?Gly?Ala?Ile?Ser?Glu?Ala?Val

20 25 30Gly?Glu?Gln

35<210>8<211>29<212>PRT<213>Agalychnis?annae<400>8Gly?Met?Phe?Thr?Asn?Met?Leu?Lys?Gly?Ile?Gly?Lys?Leu?Ala?Gly?Gln 1 5 10 15Ala?Ala?Leu?Gly?Ala?Val?Lys?Thr?Leu?Ala?Gly?Glu?Gln

20 25<210>9<211>30<212>PRT<213>Agalychnis?annae<400>9Ser?Leu?Trp?Ser?Lys?Ile?Lys?Glu?Met?Ala?Ala?Thr?Ala?Gly?Lys?Ala 1 5 10 15Ala?Leu?Asn?Ala?Val?Thr?Gly?Met?Val?Asn?Gln?Gly?Glu?Gln

20 25 30<210>10<211>34<212>PRT<213>Phyllomedusa?sauvagei<400>10Ala?Leu?Trp?Lys?Thr?Met?Leu?Lys?Lys?Leu?Gly?Thr?Met?Ala?Leu?His 1 5 10 15Ala?Gly?Lys?Ala?Ala?Leu?Gly?Ala?Ala?Ala?Asp?Thr?Ile?Ser?Gln?Gly

20 25 30Thr?Gln<210>11<211>34<212>PRT<213>Phyllomedusa?sauvagei<400>11Ala?Leu?Trp?Phe?Thr?Met?Leu?Lys?Lys?Leu?Gly?Thr?Met?Ala?Leu?His 1 5 10 15Ala?Gly?Lys?Ala?Ala?Leu?Gly?Ala?Ala?Ala?Asn?Thr?Ile?Ser?Gln?Gly

20 25 30Thr?Gln<210>12<211>30<212>PRT<213>Phyllomedusa?sauvagei<400>12Ala?Leu?Trp?Lys?Asn?Met?Leu?Lys?Gly?Ile?Gly?Lys?Leu?Ala?Gly?Lys 1 5 10 15Ala?Ala?Leu?Gly?Ala?Val?Lys?Lys?Leu?Val?Gly?Ala?Glu?Ser

20 25 30<210>13<211>27<212>PRT<213>Phyllomedusa?sauvagei<400>13Ala?Leu?Trp?Met?Thr?Leu?Leu?Lys?Lys?Val?Leu?Lys?Ala?Ala?Ala?Lys 1 5 10 15Ala?Leu?Asn?Ala?Val?Leu?Val?Gly?Ala?Asn?Ala

20 25<210>14<211>29<212>PRT<213>Phyllomedusa?sauvagei<400>14Gly?Leu?Trp?Ser?Lys?Ile?Lys?Thr?Ala?Gly?Lys?Ser?Val?Ala?Lys?Ala 1 5 10 15Ala?Ala?Lys?Ala?Ala?Val?Lys?Ala?Val?Thr?Asn?Ala?Val

20 25<210>15<211>329<212>DNA<213>Rana?temporaria<220><221>CDS<222>(53)..(238)<400>15cccctccagc?tgtctacatt?ctcataacca?actgaaccac?ccgagcccaa?ag?atg?ttc?58

Met?Phe

1acc?ttg?aag?aaa?tcc?ctc?tta?ctc?ctt?ttc?ttc?ctt?ggg?acc?atc?aac 106Thr?Leu?Lys?Lys?Ser?Leu?Leu?Leu?Leu?Phe?Phe?Leu?Gly?Thr?Ile?Asn

5 10 15tta?tct?ctc?tgt?gag?gaa?gag?aga?gat?gcc?gat?gaa?gaa?aga?aga?gat 154Leu?Ser?Leu?Cys?Glu?Glu?Glu?Arg?Asp?Ala?Asp?Glu?Glu?Arg?Arg?Asp

20 25 30gat?ctc?gaa?gaa?agg?gat?gtt?gaa?gtg?gaa?aag?cga?ttt?ttt?cca?gtg 202Asp?Leu?Glu?Glu?Arg?Asp?Val?Glu?Val?Glu?Lys?Arg?Phe?Phe?Pro?Val?35 40 45 50att?gga?agg?ata?ctc?aat?ggt?att?ttg?gga?aaa?taa?ccaaaaaaag 248Ile?Gly?Arg?Ile?Leu?Asn?Gly?Ile?Leu?Gly?Lys

55 60ttaaaacttt?ggaaatggaa?ttggaaatca?tctaatgtgg?aatgtcattt?agctaaatgc?308acatcaaatg?tcttataaaa?a 329<210>16<211>61<212>PRT<213>Rana?temporaria<400>16Met?Phe?Thr?Leu?Lys?Lys?Ser?Leu?Leu?Leu?Leu?Phe?Phe?Leu?Gly?Thr 1 5 10 15Ile?Asn?Leu?Ser?Leu?Cys?Glu?Glu?Glu?Arg?Asp?Ala?Asp?Glu?Glu?Arg

20 25 30Arg?Asp?Asp?Leu?Glu?Glu?Arg?Asp?Val?Glu?Val?Glu?Lys?Arg?Phe?Phe

35 40 45Pro?Val?Ile?Gly?Arg?Ile?Leu?Asn?Gly?Ile?Leu?Gly?Lys

50 55 60<210>17<211>13<212>PRT<213>Rana?temporaria<400>17Phe?Phe?Pro?Val?Ile?Gly?Arg?Ile?Leu?Asn?Gly?Ile?Leu 1 5 10<210>18<211>13<212>PRT<213>Rana?temporaria<400>18Phe?Leu?Pro?Leu?Ile?Gly?Arg?Val?Leu?Ser?Gly?Ile?Leu 1 5 10<210>19<211>13<212>PRT<213>Rana?temporaria<400>19Leu?Leu?Pro?Ile?Val?Gly?Asn?Leu?Leu?Lys?Ser?Leu?Leu 1 5 10<210>20<211>13<212>PRT<213>Rana?temporaria<400>20Leu?Leu?Pro?Ile?Leu?Gly?Asn?Leu?Leu?Asn?Gly?Leu?Leu 1 5 10<210>21<211>13<212>PRT<213>Rana?temporaria<400>21Leu?Leu?Pro?Ile?Val?Gly?Asn?Leu?Leu?Asn?Ser?Leu?Leu 1 5 10<210>22<211>13<212>PRT<213>Rana?temporaria<400>22Val?Leu?Pro?Ile?Ile?Gly?Asn?Leu?Leu?Asn?Ser?Leu?Leu 1 5 10<210>23<211>13<212>PRT<213>Rana?temporaria<400>23Phe?Leu?Pro?Leu?Ile?Gly?Lys?Val?Leu?Ser?Gly?Ile?Leu 1 5 10<210>24<211>12<212>PRT<213>Rana?temporaria<400>24Leu?Ser?Pro?Asn?Leu?Leu?Lys?Ser?Leu?Leu?Gly?Lys 1 5 10<210>25<211>10<212>PRT<213>Rana?temporaria<400>25Leu?Leu?Pro?Asn?Leu?Leu?Lys?Ser?Leu?Leu 1 5 10<210>26<211>13<212>PRT<213>Rana?temporaria<400>26Phe?Val?Gln?Trp?Phe?Ser?Lys?Phe?Leu?Gly?Arg?Ile?Leu 1 5 10<210>27<211>99<212>DNA<213>Phyllomedusa?bicolor<220><221>CDS<222>(1)..(99)<400>27atg?gcc?atg?tgg?aaa?gac?gtt?ctg?aaa?aag?atc?ggt?act?gtc?gcc?ctc 48Met?Ala?Met?Trp?Lys?Asp?Val?Leu?Lys?Lys?Ile?Gly?Thr?Val?Ala?Leu 1 5 10 15cat?gca?ggg?aag?gcc?gcg?ctt?gga?gca?gta?gcc?gac?acc?atc?tcg?cag 96His?Ala?Gly?Lys?Ala?Ala?Leu?Gly?Ala?Val?Ala?Asp?Thr?Ile?Ser?Gln

20 25 30taa 99<210>28<211>32<212>PRT<213>Phyllomedusa?bicolor<400>28Met?Ala?Met?Trp?Lys?Asp?Val?Leu?Lys?Lys?Ile?Gly?Thr?Val?Ala?Leu 1 5 10 15His?Ala?Gly?Lys?Ala?Ala?Leu?Gly?Ala?Val?Ala?Asp?Thr?Ile?Ser?Gln

20 25 30＜210〉29＜211〉57＜212〉DNA＜213〉＜220〉＜223〉：PCR＜400〉29atggccatgt ggaaagacgt tctgaaaaag atcggtactg tcgccctcca tgcaggg 57＜210〉30＜211〉63＜212〉DNA＜213〉＜220〉＜223〉：PCR＜400〉30ttactgcgag atggtgtcgg ctactgctcc aagcgcggcc ttccctgcat ggagggcgac 60agt 63＜210〉31＜211〉31＜212〉DNA＜213〉＜220〉＜223〉：PCR＜400〉31tctagaggta ccatggccat gtggaaagac g 31＜210〉32＜211〉38＜212〉DNA＜213〉＜220〉＜223〉：PCR＜400〉32caagcttctg cagagctctt actgcgagat ggtgtcgg 38＜210〉33＜211〉60＜212〉DNA＜213〉Rana temporaria＜220〉＜221〉CDS＜222〉 ( 1 ) .. ( 57 )＜400〉33atg gcc tct aga cat atg ttt ctg ccc cta atc ggg agg gtt ctc tcg 48Met Ala Ser Arg His Met Phe Leu Pro Leu Ile Gly Arg Val Leu Ser 1 5 10 15gga atc ctg taa 60Gly Ile Leu＜210〉34＜211〉19＜212〉PRT＜213〉Rana temporaria＜400〉34Met Ala Ser Arg His Met Phe Leu Pro Leu Ile Gly Arg Val Leu Ser 1 5 10 15Gly Ile Leu＜210〉35＜211〉45＜212〉DNA＜213〉＜220〉＜223〉：PCR＜400〉35atgtttctgc ccctaatcgg gagggttctc tcgggaatcc tgtaa 45＜210〉36＜211〉45＜212〉DNA＜213〉＜220〉＜223〉：PCR＜400〉36ttacaggatt cccgagagaa ccctcccgat taggggcaga aacat 45＜210〉37＜211〉30＜212〉DNA＜213〉＜220〉＜223〉：PCR＜400〉37ggtacctcta gacatatgtt tctgccccta 30＜210〉38＜211〉29＜212〉DNA＜213〉＜220〉＜223〉：PCR＜400〉38ctgcagagct cttacaggat tcccgagag 29＜210〉39＜211〉4＜212〉PRT＜213〉Phyllomedusa bicolor＜400〉39Ala Met Trp Lys 1＜210〉40＜211〉4＜212〉PRT＜213〉＜220〉＜223〉：＜400〉40Ala Ser Arg His 1＜210〉41＜211〉4＜212〉PRT＜213〉＜220〉＜223〉：＜400〉41Ala Leu Trp Lys 1

Claims

1. transgenic plant of expressing cationic peptide, described cationic peptide is selected from:

(a) temporin; With

(b)dermaseptin。

2. transgenic plant that comprise recombinant nucleic acid molecules, wherein said nucleic acid molecule encoding is selected from following peptide:

(a) temporin; With

(b)dermaseptin。

3. according to the transgenic plant of claim 2, wherein said peptide comprises a kind of aminoacid sequence, and this sequence is selected from the aminoacid sequence of being listed by SEQ ID:3-14 and 17-26.

4. according to the transgenic plant of claim 3, wherein said peptide comprises that further the N-terminal peptide of 2-25 amino acid length extends.

5. according to the transgenic plant of claim 4, wherein said N-terminal peptide extension is selected from AMWK, ASRH and ALWK.

6. transgenic plant that comprise recombinant nucleic acid molecules, wherein said nucleic acid molecule encoding fusogenic peptide, this peptide have the following formula that is selected from:

(a) P-D; With

(b)P-T，

Wherein D is the dermaseptin peptide, and T is the temporin peptide, and P is a negatively charged ion proparea peptide.

7. transgenic plant that comprise recombinant nucleic acid molecules, wherein said nucleic acid molecule encoding fusogenic peptide, this peptide have the following formula that is selected from:

(a) P-S-D; With

(b)P-S-T，

Wherein D is the dermaseptin peptide, and T is the temporin peptide, and P is a negatively charged ion proparea peptide, and S is a spacer peptide.

8. transgenic plant that comprise nucleic acid molecule, a kind of peptide that is selected from following aminoacid sequence that comprises of described nucleic acid molecule encoding:

(a) SEQ ID:3-14 and fragment thereof;

(b) replace and the different aminoacid sequence of (a) specified aminoacid sequence by one or more conserved amino acids; With

(c) have and (a) aminoacid sequence of at least 40% identity of specified aminoacid sequence,

Wherein said peptide has the biologic activity of dermaseptin.

9. according to the transgenic plant of claim 8, wherein said peptide further comprises the negatively charged ion proparea peptide that is operably connected with the N-end of described peptide.

10. transgenic plant that comprise nucleic acid molecule, a kind of peptide that is selected from following aminoacid sequence that comprises of described nucleic acid molecule encoding:

(a) SEQ ID:17-26 and its fragment;

(c) have and (a) aminoacid sequence of at least 50% identity of specified aminoacid sequence,

Wherein said peptide has the biologic activity of temporin.

11. according to the transgenic plant of claim 8, wherein said peptide further comprises the negatively charged ion proparea peptide that is operably connected with the N-end of described peptide.

12. transgenic plant that comprise recombinant nucleic acid molecules, a kind of peptide that comprises aminoacid sequence of described nucleic acid molecule encoding, described sequence is selected from SEQ ID:28 and 34.

13. a method of producing the biologic activity cationic peptide comprises: the transgenic plant according to claim 1 are provided; With separating out at least one biologic activity cationic peptide from this plant.