JP2002534115A5 - - Google Patents

Description

[Claims]
(1)
(A) the sequences provided in SEQ ID Nos. 1-67, 131-481 and 833-888, and (b) the sequences listed in SEQ ID Nos. 1-67, 131-481 and 833-888. (C) reverse complements of the sequences listed under Nos. 1-67, 131-481 and 833-888, and (d) Nos. 1-67, 131-481 and Using the computer algorithm BLASTN, the reverse sequence of the sequence listed at position 833-888 and (e) the sequence provided at sequence ID Nos. 1-67, 131-481 and 833-888 using the computer algorithm BLASTN, % Of the sequence having the same nucleotide and (f) the sequence provided in SEQ ID NOs: 1-67, 131-481 and 833-888, the computer algorithm BL Using STN, the sequence having at least 60% identical nucleotides and (g) the sequence provided in SEQ ID NOs: 1-67, 131-481 and 833-888 using the computer algorithm BLASTN, (H) using a computer algorithm BLASTN, at least 90% identical to a sequence having nucleotides at least 75% identical to the sequence provided in SEQ ID NOs: 1-67, 131-481, and 833-888. (I) a 200-mer nucleotide sequence of the sequences listed in (a) to (d) above, and (j) 100 nucleotides of the sequence listed in (a) to (d) above. And (k) the 40-mer nucleotide of the sequences listed in (a) to (d) above. And de sequence comprises a sequence selected from the group consisting of a 20-mer nucleotide sequence of the sequence listed in (l) to (a) above no (d), isolated polynucleotide.
(2)
An isolated polypeptide encoded by the polynucleotide of claim 1.
(3)
(A) a sequence having at least 50% residue identity to the sequence of SEQ ID Nos. 68-130, 482-832 and 889-945, and (c) (a) A) a sequence having at least 70% identical residues to the sequence of (d) and (d) a sequence having at least 90% identical residues to the sequence of (a). An isolated polypeptide.
(4)
An isolated polynucleotide encoding the polypeptide of claim 3.
(5)
A DNA construct comprising the polynucleotide according to claim 1.
6.
A transgenic cell comprising the DNA construct according to claim 5.
7.
In the 5 ′ to 3 ′ direction, an open gene encoding (a) a gene promoter sequence and (b) at least a functional portion of a polypeptide encoded by the isolated polynucleotide of claim 1. A DNA construct comprising a reading frame and (c) a gene termination sequence.
Claim 8.
8. The DNA construct according to claim 7, wherein the open reading frame is in a sense direction.
9.
The DNA construct according to claim 7, wherein the open reading frame is in an antisense direction.
10.
The DNA construct according to claim 7, wherein the gene promoter sequence and the gene termination sequence are functional in a plant host.
11. The DNA construct according to claim 7, comprising a marker for identifying a transformed cell.
12. In the 5 'to 3' direction: (a) a gene promoter sequence; and (b) a non-coding region of the isolated polynucleotide of any one of claims 1 to 6 and 9. (C) a DNA construct comprising a gene termination sequence.
Claim 13
13. The DNA construct according to claim 12, wherein the non-coding region is in a sense direction.
14.
13. The DNA construct according to claim 12, wherein the non-coding region is in the antisense direction.
15.
13. The DNA construct according to claim 12, wherein the gene promoter sequence and the gene termination sequence are functional in a plant host.
16.
A transgenic plant cell comprising the DNA construct according to any one of claims 7 to 15.
17.
A plant comprising the transgenic plant cell according to claim 16, or a part thereof, a reed, or a progeny.
18.
18. The plant according to claim 17, wherein the plant is a woody plant.
(19)
19. The plant according to claim 18, wherein the plant is selected from the group consisting of eucalyptus, pine, acacia, poplar, sweet gum, teak and mahogany species.
20.
A method for modifying plant cell signaling, comprising stably incorporating the DNA construct according to any one of claims 7 to 15 into a plant genome.
21.
21. The method according to claim 20, wherein the plant is a woody plant.
22.
22. The method according to claim 21, wherein the plant is selected from the group consisting of eucalyptus, pine, acacia, poplar, sweet gum, teak and mahogany seeds.
23.
(A) transforming a plant cell with the DNA construct of any one of claims 7 to 15 to provide a transgenic cell; and (b) conditions leading to regeneration and growth of a mature plant. A method for producing a plant in which cell signaling is modified, comprising culturing the transgenic cell in step (a).
24.
24. The method according to claim 23, wherein the plant is a woody plant.
25.
25. The method of claim 24, wherein the plant is selected from the group consisting of eucalyptus, pine, acacia, poplar, sweet gum, teak and mahogany seeds.
26.
A method for modifying the activity of a plant polypeptide, comprising stably incorporating the DNA construct according to any one of claims 7 to 15 into a plant genome.
27.
The method according to claim 26, wherein the plant is a woody plant.
28.
28. The method of claim 27, wherein the plant is selected from the group consisting of eucalyptus, pine, acacia, poplar, sweet gum, teak and mahogany species.
29.
An isolated oligonucleotide probe or primer comprising at least 10 contiguous residues complementary to 10 contiguous residues of the nucleotide sequence recited in claim 1.
30.
A kit comprising a plurality of oligonucleotide probes or primers according to claim 29.
31.
A recording medium on which a plurality of polynucleotides are recorded, wherein at least one of the polynucleotides comprises the nucleotide sequence listed in claim 1.

DETAILED DESCRIPTION OF THE INVENTION
[0001]
Technical Field of the Invention
The present invention relates to modifying the response of plant cells to external signals such as environmental changes and cues during development. More specifically, the present invention provides isolated polynucleotides that encode polypeptides that are integral to the plant cell membrane and mediate cell signaling processes.
[0002]
Background of the invention
Plants grow through a given developmental program over their lifetime. This point is particularly remarkable in embryo development and flower development. There are a variety of signal molecules that are produced by certain cells of the plant and are ready for other cells to respond, especially in meristem areas. These signal molecules trigger a development program at a specific time that leads to the formation of a completely different, for example flower or cotyledon. In addition to the programmed pathways described above, plants are exposed to a variety of environmental stimuli, such as temperature changes, sunshine, hydration potential, mechanical damage wounds and pathogen attack. I have. Environmental factors, such as exposure to light, heat, cold, dryness, etc., regulate the expression of genes and the synthesis of proteins and other compounds essential for the proper response to environmental signals and the healthy development of plants through them. Activate. These responses are mediated by signal molecules as well as developmental pathways.
[0003]
To respond to these signal molecules, plant cells produce surface receptor proteins that serve as detectors, modulators and / or transducers of cellular signals. Intracellular transmission of signals is often transmitted via a phosphorylation cascade of molecules that culminates in the transcription of genes that trigger appropriate cellular responses for normal development or to counter challenges from the environment.
[0004]
One large class of receptor proteins is the single transmembrane family, which has several subclasses. These proteins are characterized by three domains: a recognition / binding domain for extracellular signals (or ligands), a domain that penetrates once through the cell membrane, and an intracellular signaling domain, usually a protein kinase. Single, but not all, transmembrane proteins in many plants belong to a subclass known as receptor-like kinases (RLKs). The intracellular kinase domains of plant RLK are all serine / threonine protein kinases, whereas the extracellular domain of RLK is of a different type. One type of RLK is characterized by the presence of an extracellular S domain first described in self-incompatibility-locus glycoproteins that inhibits self-pollination. The S domain is recognized by an array of 10 cysteine residues combined with other conserved residues. Another class of RLK has an extracellular domain identified by a leucine rich repeat (LRR) that is involved in protein-protein interactions. Binding of the ligand to the extracellular domain is followed by receptor dimerization, autophosphorylation, and activation of a series of intracellular proteins that serve to transmit signals to the nucleus. The structure of plant RLK is very similar to the receptors found in animal cell signaling pathways.
[0005]
One example of a plant PLK is the plant pathogen Xanthomonas oryzae pv. Xa21 gene that confers resistance to oryzae race 6. This gene has been cloned using genetic means to compare Xanthomonas susceptible and resistant rice lines (Song et al., Science 270: 1804-1806, 1995), and subsequently confers resistance to Xanthomonas in Arabidopsis as well. It was shown that. This 1025 amino acid protein exhibits a number of features similar to known protein domains including a signal peptide of the amino terminal 23 amino acid residues, indicating that this protein is localized at the plasma membrane. Amino acids 81 to 634 contain 23 incomplete repeats of the 24 amino acid LRR. Amino acids 651 to 676 encode a hydrophobic segment of 26 amino acids that may form a transmembrane domain. The C-terminal amino acids include those predicted to be intracellular serine threonine kinase domains that include 11 subdomains of 15 invariant amino acids typical of protein kinases. Subdomains VI and VIII display the specificity of serine threonine phosphorylation. Xa21 is highly similar to other RLKs, such as the Arabidopsis receptor-like kinase proteins RLK5 and TMK1, showing conservation of both LRR and protein kinase domains. It is unknown what protein Xa21 transmits its pathogen recognition signal to.
[0006]
Another family of membrane receptor molecules expressed on plant cells is histidine kinase (HK). HK has been known for some time in bacterial signaling systems, one of the two-component signaling systems. Bacterial HK serves as a detector molecule for extracellular signals such as changes in osmoticum, nutrients and toxicants. HK autophosphorylates to histidine residues in response to ligand binding. This phosphohistidine contributes its own phosphate group to the aspartate group of the second component of the two-component system known as the response regulator (RR). The phosphorylated RR binds to DNA in a sequence-specific manner and plays a role in directly activating a specific gene encoding a protein that mediates the response to the extracellular stimulus. In certain cases, HK has a complex structure. Specifically, these proteins contain an RR domain at the carboxyl terminus. The phosphohistidine of HK transfers its phosphate group to an aspartic acid residue in the active site of this RR domain. In these cases, since the RR domain is membrane-bound, the signal cannot be transmitted directly by the RR binding to DNA. Instead, the histidine phosphotransfer (HPt) protein plays a role in further signaling. The phosphoaspartic acid of the complex HK / RR protein contributes a phosphate group to the active site histidine of the HPt protein. The phosphohistidine of this HPt in turn donates a phosphate group to the true RR, which then activates gene expression in response to external signals.
[0007]
Like bacteria, plant cells have a two-element signaling system consisting of HK and RR with detector elements. Furthermore, complex HK proteins having an RR domain at the carboxyl terminus (hereinafter referred to as hybrid HK / RR proteins) are found in both bacteria and plants. HK proteins are distinguished by well-conserved amino acid motifs that occur in a specific order. The conserved regions from the amino terminus are identified as H, N, G1, F and G2 boxes. These motifs are usually found in the range of 200 to 250 amino acids of the protein. As in bacteria, upon receiving extracellular signals, HK autophosphorylates histidine residues contained in the H box. This phosphate group is then transferred to the RR. As an alternative, some HKs constitutively autophosphorylate their histidine residues and this activity is suppressed by binding of extracellular signals. It is believed that all HK phosphorylates RR as an obligate part of signal transduction. RR is characterized by the absolute conservation of aspartic acid and lysine residues phosphorylated by phosphohistidine of HK. Unlike bacteria, it has been shown that plant RRs do not bind DNA directly. Rather, the RRs of all plants examined to date appear to transmit signals at first glance to the protein kinase cascade, which eventually phosphorylates transcription factors to activate gene expression or not. Activate either. Like bacteria, plants also contain complex HKs containing an RR domain at the carboxyl terminus of the protein. As predicted based on this observation, the plant genome has also been found to also carry the HPt gene. However, it has not yet been directly shown that any plant HPt protein interacts with complex HK or soluble RR.
[0008]
The ethylene receptor (ETR1, Chang et al., Science 262: 539-544, 1993) is the most well known plant two-component signaling system. Ethylene is a well-known signaling molecule involved in regulating fertilization, senescence, dark / photomorphogenesis, and response to pathogens and mechanical damage, as well as regulating plant development. The ethylene receptor is a hybrid HK / RR protein. The signal is transmitted via the CTR1 protein, a Raf-like protein kinase. CTR1 is a negative regulator of downstream steps in the signaling pathway. Although the details of this pathway are not yet clear, HK is constitutively active in the absence of ethylene and always phosphorylates CTR1, which represses other genes in the ethylene response pathway. Binding of ethylene to ETR1 inhibits the HK function of the receptor, allowing activation of proteins downstream of the ethylene signaling cascade. This culminates in the activation of ethylene-responsive genes.
[0009]
Two RR genes, IBC6 and IBC7, which are induced in response to the plant growth regulator cytokinin, have recently been cloned and analyzed from Arabidopsis thaliana (Brandstatter and Kieber, Plant Cell 10: 1009-1019, 1998). IBC6 and IBC7 appear to be involved in the signaling of cytokinin signals in plants. This is particularly interesting in light of the fact that the gene encoding the hybrid HK / RR protein CKI1 (Kakimoto, Science 274: 982-985, 1996) produces a cytokinin-like effect when ectopically expressed in transgenic plants. . Thus, it appears that the two-element HK / RR system may be involved in cytokinin signaling. Cytokinins are known to regulate plant growth and development, including physiological events such as nutrient metabolism, leaf elongation and senescence, and lateral branching.
[0010]
Although polynucleotides encoding proteins involved in plant cell signaling have been isolated for certain plant species, genes encoding many such proteins have not yet been identified in a wide range of plant species. Thus, there is still a need in the art for materials that can be used effectively to alter cell signaling.
[0011]
Summary of the Invention
Briefly, the present invention provides polynucleotides isolated from Eucalyptus and pine and encoding a polypeptide involved in cell signaling, along with methods of using such oligonucleotides and polypeptides. Such polypeptides function as detector-modulators or receptor kinases. The isolated polynucleotides and polypeptides described above are effectively used to modify plant cell responses either in plant growth and development or under stress conditions caused by pathogens or environmental factors.
[0012]
In a first aspect, the present invention provides an isolated and purified polynucleotide available from Eucalyptus and pine, encoding an RLK, HK, RR, HPt or hybrid HK / RR protein. In one embodiment, the isolated polynucleotide comprises: (a) the sequence recited in SEQ ID NOs: 1-67, 131-481, and 833-888; (b) the sequences identified by SEQ ID NOs: 1-67. , 131-481 and 833-888, (c) the reverse complement of the sequences listed in SEQ ID Nos. 1-67, 131-481 and 833-888, (d) 40%, 60% as defined below with the reverse sequence of the sequences listed under SEQ ID Nos. 1-67, 131-481 and 833-888 and the sequences (e) (a) to (d) , A DNA sequence selected from the group consisting of sequences having sequences that are 75% or 90% identical.
[0013]
In a further aspect, there is provided an isolated polypeptide encoded by the DNA sequence of the present invention. In one embodiment, such a polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68-130, 482-832, and 889-945.
[0014]
In another aspect, the present invention provides a DNA construct comprising a polynucleotide of the present invention, alone or in combination with one or more of the polynucleotides disclosed herein or in combination with one or more known DNA sequences. Provided with a transgenic cell containing
[0015]
In a related aspect, the invention provides a DNA construct comprising, in the 5 'to 3' direction, a gene promoter sequence, an open reading frame encoding at least a functional portion of a polypeptide of the invention, and a gene termination sequence. I do. The open reading frames may be arranged in a sense direction or an antisense direction. There is also provided a gene construct comprising a non-coding region or a nucleotide sequence complementary to the non-coding region of a gene encoding an enzyme encoded by the polynucleotide, together with a gene promoter sequence and a gene terminator sequence. It is preferred that the gene promoter and termination sequence function in the host plant. The gene promoter and termination sequence are most preferably those of the original gene, but also those commonly used in the technical field of the present invention, such as other cauliflower mosaic virus (CaMV) promoters, such as the Kozak sequence or the omega enhancer. It can be effectively used in the present invention together with a simple enhancer and a terminator of the nopaline synthase gene of Agrobacterium tumefaciens bacterium. Tissue-specific promoters are used to target and express one or more desired tissues. The DNA construct may further include a marker for identification of the transformed cell.
[0016]
In a further aspect, a transgenic cell, preferably a plant cell, comprising the DNA construct of the present invention comprises an organism, preferably a plant, comprising such a transgenic cell, together with the fruit, seed or other product, derivative or progeny of such a plant. Provided. Such transgenic plants are also included in the present invention. As used herein, the term "zero remnant" means any part of a plant used for sexual or asexual reproduction or propagation, including cuttings.
[0017]
In yet another aspect, there is provided a method for modifying cell signaling in a target organism, such as a plant, comprising stably incorporating a DNA construct of the present invention into the genome of the plant. In a preferred embodiment, the target plant is a woody plant, preferably one selected from the group consisting of eucalyptus and pine species, most preferably one selected from the group consisting of Eucalyptus grandis and Pinus radiata. In a related aspect, a method for producing a target organism, such as a plant, having altered cell signaling, comprising transforming with a DNA construct of the present invention to provide a transgenic cell, wherein said transgene comprises Culturing the transgenic cells under conditions that lead to regeneration and growth of the mature plant.
[0018]
In yet a further aspect, the present invention provides a method for modifying the activity of a polypeptide in a target organism, such as a plant, comprising stably incorporating the DNA construct of the present invention into the genome of the plant. In a preferred embodiment, the target plant is a woody plant, preferably selected from the group consisting of Eucalyptus and pine species, and most preferably selected from the group consisting of Eucalyptus grandis and Pinus radiata.
[0019]
BRIEF DESCRIPTION OF THE DRAWINGS The above and further features of the invention and the manner in which it is obtained will become apparent and the invention is best understood by referring to the following detailed description. All cited references disclosed herein are incorporated by reference as if each were individually incorporated.
[0020]
Detailed description
The present invention provides isolated and purified polynucleotides encoding polypeptides involved in plant cell signaling. As noted above, cell signaling is known to play a critical role in plant growth and development and in the response of cells to external stimuli such as environmental factors and pathogens. Thus, transforming a plant with a polynucleotide encoding a polypeptide involved in cell signaling can be used to modify properties such as cell growth, differentiation, elongation and survival, disease resistance and nutrient metabolism. Is done.
[0021]
For example, the hybrid HK / RR protein ETR1 is known to be involved in ethylene signaling. Thus, altering the expression of ETR1 leads to alteration of ethylene-regulated physiological properties such as fruit ripening and leaf and flower senescence. Modification of the expression of this protein in transgenic plants is then used to extend the useful life of cut flowers by delaying senescence. In addition, alterations in ETR1 expression can be used to selectively promote reproductive senescence, resulting in the production of engineered sterile plants.
[0022]
The hybrid HK / RR protein CKI1 has been implicated in cytokinin signaling. Overexpression of this protein is known to produce cytokinin-like effects in mutant plants. Cytokinins have been shown to play important roles in lateral branching, expansion of expression, cell division, nutrient distribution and senescence delay among physiological phenomena. Thus, altered expression of CKI1 results in, for example, delayed senescence in a selected cell type or organ. This extends the shelf life of fruits and vegetables from harvest to consumption. As an alternative, altering CKI1 expression can be used to reduce branching frequency in forest tree species, resulting in intact, clear timber for hardwood furniture and plywood .
[0023]
The methods and materials of the present invention can be used to increase or decrease the amount of a particular plant cell polypeptide by incorporating additional copies of the gene encoding the polypeptide into the genome of a target organism, such as a plant. . Similarly, polypeptides can be increased or decreased by transforming a target organism with an antisense copy of such a gene.
[0024]
In one embodiment, the invention provides an isolated polynucleotide that encodes or partially encodes a plant polypeptide involved in cell signaling, wherein the polynucleotide is from Eucalyptus and pine. Specifically, the present invention is derived from Eucalyptus grandis (SEQ ID NO: 2, 8, 9, 11, 15, 18, 19, 21-25, 33, 34, 38, 131-301, 448-463, 848, 858-874 and 882-887) and derived from Pinus radiata (SEQ ID NOS: 1, 3-7, 10, 12-14, 16, 17, 20, 26-32, 35-37, 39-41, 302-). 447, 833-847, 875-881 and 888), and an isolated polynucleotide encoding RLK and derived from Eucalyptus grandis (SEQ ID NOS: 42, 48-52, 55-58, 67, 464-471, 474). 478 and 850-857) and from Pinus radiata (SEQ ID NOs: 43-47, 53; 54, 59-66, 472, 473, 479-481, and 849) of the two-component signaling system (HK, RR or hybrid HK / RR protein). . The complement of such an isolated polynucleotide, the reverse complement of such an isolated polynucleotide, and the reverse sequence of such an isolated polynucleotide are also provided, along with variants of such a sequence as defined below.
[0025]
In another embodiment, the invention provides an isolated polypeptide encoded by a polynucleotide of the invention. The amino acid sequences encoded by the DNA sequences of SEQ ID Nos. 1-59, 63, 64, 66, 67, 131-481, 833-848, 851 and 853-888 are sequence ID Nos. 68-130, respectively. 482-832 and 889-945. Thus, in certain embodiments, a polypeptide of the invention comprises an amino acid sequence selected from the group consisting of Nos. 68-130, 482-832, 889-945, and variants thereof.
[0026]
The polypeptides disclosed herein were derived from forest plant resources, mainly Eucalyptus grandis and Pinus radiata. Some polynucleotides of the present invention are "partial" sequences in that they do not refer to full-length genes encoding full-length polypeptides. The term "polynucleotide-encoded polypeptide" as used herein includes polypeptides encoded by nucleotide sequences, including partially isolated DNA sequences of the present invention. Such partial sequences can be extended by analyzing and sequencing various DNA libraries using primers and / or probes and well-known hybridization and / or PCR techniques. The subsequence can be extended until an open reading frame encoding the polypeptide, a full-length polynucleotide and / or a gene capable of expressing the polypeptide or another useful portion of the genome is identified. Such extended sequences, including full-length polynucleotides and genes, are those sequences in which the extended sequence comprises the identified sequence or a variant thereof or one contiguous portion (x-mer) of the sequences disclosed herein or a variant thereof. The sequences disclosed herein or variants thereof or one portion of the sequences disclosed herein or variants thereof are described as "corresponding to". Similarly, RNA sequences, reverse sequences, complementary sequences, antisense sequences and the like corresponding to the polynucleotides of the present invention have been identified as the sequences listed under SEQ ID Nos. 1-67, 131-481 and 833-888. Can be confirmed and obtained by routine procedures using the obtained cDNA sequence.
[0027]
As described above, the polynucleotides disclosed herein include an open reading frame (ORF) or a partial open reading frame encoding a polypeptide. In addition, open reading frames encoding the polypeptides may be identifiable in extended or full-length sequences corresponding to the sequences presented as SEQ ID NOs: 1-67, 131-481 and 833-888. . Open reading frames can be identified using techniques well known to those skilled in the art. These techniques include, for example, analysis of the location of known start and end codons, identification of the most likely reading frame based on codon frequency, and the like. Suitable tools and software for ORF analysis are available, for example, on the Internet at http: // www. ncbi. nlm. nih. gov / gorf / gorf. available at html. The open reading frame and portions of the open reading frame can be identified in the polynucleotides of the present invention. Once the partial open reading frame has been identified, the polynucleotide can be modified using techniques well known to those of skill in the art until a complete open reading frame polynucleotide can be identified. The area can be expanded. Thus, an open reading frame encoding a polypeptide can be identified using a polynucleotide of the present invention.
[0028]
Once an open reading frame has been identified in a polynucleotide of the invention, the open reading frame can be isolated and / or synthesized. An expressible DNA construct comprising the open reading frame and an appropriate promoter, initiator, terminator and the like well known to those skilled in the art can be constructed. Such a genetic construct is introduced into a host cell to express the polypeptide encoded in the open reading frame. Suitable host cells include various prokaryotic and eukaryotic cells, including plant cells, mammalian cells, bacterial cells, algae and the like.
[0029]
The polypeptide encoded by the polynucleotide of the present invention can be expressed and used in various assays to determine its biological activity. Such polypeptides can be used to generate antibodies, to isolate proteins or other compounds with which they interact, and to quantitatively determine the level of interacting proteins or other compounds.
[0030]
The term "polynucleotide" as used herein refers to a single-stranded or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases, and is defined as DNA and corresponding RNA molecules on both the sense and antisense strands and HnRNA. and cDNA, genomic DNA and recombinant DNA, as well as fully or partially synthesized polynucleotides. HnRNA contains introns and generally corresponds one-to-one with DNA molecules. mRNA corresponds to HnRNA and DNA molecules from which introns have been excised. A polynucleotide comprises the entire gene or a portion thereof. An operable antisense polynucleotide includes fragments of the corresponding polynucleotide, and the definition of "polynucleotide" includes all such operable antisense fragments. Antisense polynucleotides and techniques involving antisense polynucleotides are well known to those of skill in the art and are described, for example, in Robinson-Benion et al., Methods in Enzymol. 254 (23): 363-375, 1995 and Kawasaki et al., Artific. Organs 20 (8): 834-848, 1996.
[0031]
As used herein, the term "polypeptide" includes amino acid chains of any length, including full length, with amino acid residues covalently linked. The polypeptides of the present invention may be purified natural products or may be partially or wholly produced using recombinant or synthetic techniques.
[0032]
All of the polynucleotides and polypeptides described herein are isolated and purified under conditions commonly used by those skilled in the art.
[0033]
The definitions of the terms "complement", "reverse complement", and "reverse sequence" as used herein are best illustrated in the following examples. For the sequence 5'AGGACC3 ', the complement, reverse complement and reverse sequence are as follows.
Complement 3'TCCTGG5 '
Reverse complement 3'GGTCCT5 '
Reverse sequence 5'CCAGGA3 '
[0034]
As used herein, the term “variant” is at least about 40%, more preferably at least about 60%, even more preferably at least about 75%, and most preferably at least about 90% identical to a sequence of the present invention. Refers to residues (either nucleotides or amino acids) that exhibit sex. The percent identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, and determining the number of identical residues according to the invention or It is obtained by dividing by the total length in the query sequence and multiplying the result by 100.
[0035]
Publicly available computer algorithms can be used to align polynucleotide or polypeptide sequences, and the percentage of identical nucleotides in a particular region can be determined relative to another. Two representative algorithms for polynucleotide sequence alignment and similarity identification are the BLASTN and FASTA algorithms. The percentage of polypeptide sequence identity can be determined using the BLASTP algorithm. Both BLASTN and BLASTP software are available at / blast / executables / on NCBI's anonymous FTP server (ftp:https://ncbi.nlm.nih.gov). The BLASTN algorithm version 2.0.6 (September 16, 1998), set in default instructions and described in the instructions and distributed with the algorithm, is preferably used for the determination of variants according to the invention. . The use of the BLAST family of algorithms, including BLASTN and BLASTP, is described at the NCBI website URL (https://www.ncbi.nlm.nih.gov/BLAST/newblast.html) and Nucleic Res. By Altschul et al. 25: 3389-3402, 1997. The computer algorithm FASTA is available on the Internet at the ftp site ftp: // ftp. virginia. Available at edu / pub / fasta /. Version 3.1t11 of August 1998, set to the default parameter values described in the instructions and distributed with the algorithm, is preferably used for the determination of variants according to the invention. The use of the FASTA algorithm is described in Pearson WR and Lipman DJ Proc. Natl. Acad. Sci. ScL USA 85: 2444-2448, 1988, and Pearson WR Methods in Enzymol. 183: 63-98, 1990.
[0036]
The following running parameters are preferably used for alignment and similarity determination using BLASTN, which contribute to the E values and percent identity described below for polynucleotide sequences. The Unix (registered trademark) running command is “blastall p blastn deembldbe 10G0E0r1v30b30iqeriesqresults”, and parameters are as follows.
p Program name [Character string]
d database [character string]
e Expected value (E) [real number]
G The cost of opening the gap (zero invokes default action) [integer]
E Cost of expanding the gap (zero invokes default behavior) [integer]
r nucleotide match reward (blastn only) [integer]
v Number of one-line descriptions (V) [integer]
b Number of alignments to be displayed (B) [integer]
i Query File [File In]
o BLAST report output file [File Out] Optional
For BLASTP, a running parameter of "blastall blastp d swissprotdbe 10G0E0v30b30i queryseq results" is preferable.
p Program name [Character string]
d database [character string]
e Expected value (E) [real number]
G The cost of opening the gap (zero invokes default action) [integer]
E Cost of expanding the gap (zero invokes default behavior) [integer]
r nucleotide match reward (blastn only) [integer]
v Number of one-line descriptions (V) [integer]
b Number of alignments to be displayed (B) [integer]
i Query File [File In]
o BLAST report output file [File Out] Optional
[0037]
A "hit" to one or more database sequences by a query sequence, generated by BLASTN, BLASTP, FASTA or a similar algorithm, is identified by alignment of similar parts of the sequence. The hits are ordered by the degree of similarity and the length of the overlapping sequence. A hit to the database sequence generally means that it only overlaps part of the sequence length of the query sequence.
[0038]
The BLASTN and FASTA algorithms also calculate the "expected" (E) value of the alignment. The E-value indicates the number of hits that can be "expected" to be discovered over a certain number of adjacent sequences by chance when searching a certain size database. The E value is used as a threshold of significance to determine whether a hit to a database, such as the preferred EMBL database, represents true similarity. For example, an E value of 0.1 assigned to a certain polynucleotide hit can be expected to match by chance 0.1 times over aligned parts of sequences with similar scores in a database of the size of the EMBL database. Is interpreted as According to this criterion, a portion where the polynucleotide sequence is aligned and matched has a probability of being 90% identical. For sequences that have an E value of less than 0.01 across aligned matches, the probability of finding a match by chance in the EMBL database using the BLASTN or FASTA algorithm is less than 1%.
[0039]
According to one embodiment, a "variant" polynucleotide for each of the polynucleotides of the invention is a sequence having the same or less number of nucleic acids than each of the polynucleotides of the invention, and It is preferable to include an array having an E value of 0.01 or less in comparison. That is, the mutant polynucleotide has any probability that the probability of being the same as the polynucleotide of the present invention is 99% or more and the E value becomes 0.01 or less using the BLASTN or FASTA algorithm with the above parameter setting. Say.
[0040]
Variant polynucleotides will typically hybridize to the recited polynucleotide sequences under stringent conditions. As used herein, “stringent conditions” refers to pre-washing with a solution of 6 × SSC and 0.2% SDS and over night hybridization in 6 × SSC and 0.2% SDS at 65 ° C. And then washed twice in 1 × SSC and 0.1% SDS at 65 ° C. for 30 minutes each, and twice in 0.2 × SSC and 0.1% SDS at 65 ° C. for 30 minutes each. Refers to
[0041]
The present invention relates to polynucleotides that differ from the disclosed sequences, but also to polynucleotides that encode the same polypeptide as a result of the degeneracy of the genetic code with the polypeptide encoded by the polynucleotides disclosed herein. Allelic variants of the gene. Accordingly, as a result of conservative substitutions, include the polynucleotide sequences listed as SEQ ID Nos. 1-67, 131-481 and 833-888 or the complement, reverse sequence or a sequence different from the reverse complement of these sequences. Polynucleotides are contemplated and encompassed by the present invention. In addition, the sequences listed under SEQ ID NOs: 1-67, 131-481 and 833-888, or the complements of these sequences, as a result of deletions and / or insertions that total less than 10% of the previous sequence length Polynucleotides containing sequences that differ from the complement, reverse sequence or reverse complement are also contemplated by the present invention and are included in the present invention. Similarly, sequences that differ from the sequences listed in SEQ ID NOs: 68-130, 482-832 and 889-945 as a result of amino acid substitutions, insertions and / or deletions that total less than 10% of the total sequence length. Are contemplated by and encompassed by the present invention provided that the mutant polypeptide has activity in the lignin biosynthetic pathway.
[0042]
As used herein, the term “x-mer” refers to at least any of the polynucleotides identified as SEQ ID NOs: 1-67, 131-481, and 833-888, for a particular value of “x”. A polynucleotide comprising a specified number ("x") of contiguous residues. The value of x is from about 20 to about 600, depending on the particular sequence.
[0043]
The polynucleotide of the invention comprises at least the specified number of contiguous residues (x) of any of the polynucleotides identified as SEQ ID NOs: 1-67, 131-481, and 833-888, or variants thereof. -Mer). According to a preferred embodiment, the value of x is preferably 20 or more, more preferably 40 or more, still more preferably 60 or more, and most preferably 80 or more. Thus, the polynucleotides of the present invention were identified as polynucleotides identified as SEQ ID Nos. 1-67, 131-481 and 833-888 and as SEQ ID Nos. 1-67, 131-481 and 833-888. A 20-mer, 40-mer, 60-mer, 80-mer, 100-mer, 120-mer, 150-mer, 180-mer, 220-mer, 250-mer or 300-mer, 400-mer, 500-mer or Includes polynucleotides containing the 600 mer.
[0044]
The polynucleotides of the present invention may have been isolated by high-throughput sequencing of a cDNA library prepared from Eucalyptus grandis and Pinus radiata, as described in Examples 1 and 2 below. Alternatively, oligonucleotide probes based on the sequences provided in SEQ ID NOs: 1-67, 131-481 and 833-888 are synthesized and cDNA from Eucalyptus grandis and Pinus radiata using hybridization or PCR techniques. Alternatively, it is used to identify positive clones in either genomic DNA libraries. Probes may be shorter than the sequences provided herein, but are at least 10, preferably at least about 15, most preferably at least 20 nucleotides in length. Hybridization and PCR techniques suitable for use with such oligonucleotide probes are well known in the art of the present invention and are described in Sambrook et al., "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory. (Cold Spring Harbor, NY), including those taught in 1989. Positive clones are analyzed by restriction enzyme digestion, DNA sequencing and the like.
[0045]
Further, the DNA sequences of the present invention can be generated by synthetic means using techniques well known in the art of the present invention. Equipment for automated oligonucleotide synthesis is commercially available from suppliers such as Perkin Elmer / Applied Biosystems Division (Foster City, CA) and may be operated according to the manufacturer's instructions.
[0046]
In one embodiment, a DNA construct of the invention comprises an open reading frame encoding at least a functional portion of a polypeptide encoded by a polynucleotide of the invention or a variant thereof. As used herein, a “functional portion” of a polypeptide refers to a portion of the polypeptide that contains a binding site or a catalytic signaling site. Functional portions of a polypeptide can be determined by targeted mutational analysis using protocols well known in the art of the invention and by screening for the modified polypeptide product. Normally the functional portion is 10 to 20 amino acids long, but may be longer or shorter. The active site may be composed of distant portions present on one or more polypeptide chains and generally exhibits high binding affinity.
[0047]
An open reading frame is inserted in the DNA construct in a sense or antisense orientation such that transformation of the DNA construct into a target plant results in a change in the amount of polypeptide as compared to a wild-type plant. Transformation with a DNA construct that includes an open reading frame in the sense direction generally results in altered expression of the selected gene, and the gene construct that includes the open reading frame in the antisense direction. Transformation generally results in reduced expression of the selected gene. A population of plants transformed with a DNA construct that contains an open reading frame of the invention in either the sense or antisense orientation can be prepared using techniques well known to those of skill in the art to increase or decrease the expression of the gene in question. Are screened for, and plants having the desired phenotype are isolated.
[0048]
Alternatively, expression of genes involved in plant cell signaling can be inhibited by inserting a portion of the open reading frame of the invention in either sense or antisense orientation. Such portions need not be full length, but preferably comprise at least 25 residues, more preferably at least 50, of the polynucleotides of the present invention. However, full-length polynucleotides corresponding to longer or full open reading frames can also be used. The portion of the open reading frame need not be exactly the same as the endogenous sequence, provided that there is sufficient sequence similarity to achieve inhibition of the target gene. Thus, sequences from one species may be used to inhibit the expression of genes of a different species.
[0049]
In another embodiment, a DNA construct of the present invention comprises a DNA sequence that comprises or is complementary to a non-coding region of a gene encoding a polypeptide encoded by a polynucleotide of the present invention. Examples of non-coding regions that can be used effectively for such constructs include introns and 5 'non-coding leader sequences. Transformation of target plants with such DNA constructs can be performed, for example, in a manner similar to that discussed by Napoli et al., Plant Cell 2: 279-290, 1990 and de Carvalho Niebel et al., Plant Cell 7: 347-358, 1995. The process of cosuppression results in a reduction in the amount of isoprenoid compounds synthesized by the plant.
[0050]
Alternatively, regulation of polypeptide expression can be achieved by inserting an appropriate sequence or subsequence (eg, DNA or RNA) into the ribozyme construct (McIntyre CL and Manners JM, Transgenic Res. 5 (4): 257-262). , 1996). Ribozymes are synthetic RNA molecules that contain a hybridizing region complementary to two regions. Each region preferably comprises at least 5 or more contiguous nucleotides of an mRNA molecule encoded by one of the polynucleotides of the present invention. Ribozymes have very specific endonuclease activity and cleave the mRNA autocatalytically.
[0051]
The gene construct of the present invention further comprises a gene promoter sequence and a gene termination sequence operably linked to the DNA sequence to be transcribed, and controls gene expression. The gene promoter sequence is generally located at the 5 'end of the transcribed DNA sequence and is used to initiate transcription of the DNA sequence. The gene promoter sequence is generally found in the 5 'non-coding region of the gene, but may be located downstream of the open reading frame or in an intron (Luehrsen KR, Mol. Gen. Genet. 225: 81-). 93, 1991), for example, as in plant defense genes (Douglas et al., EMBO J. 10: 1767-1775, 1991).
[0052]
Various gene promoter sequences that can be effectively used in the DNA construct of the present invention are well known in the technical field of the present invention. The gene promoter sequence and the gene termination sequence may be endogenous in the target plant host, or may be exogenous when functional in the target host. For example, the promoter and termination sequences may be from other plant species, plant viruses, bacterial plasmids, and the like. Preferably, the gene promoter and termination sequence are derived from the sequences of the invention themselves.
[0053]
Factors affecting promoter selection include the desired tissue specificity of the construct and the timing of transcription and translation. For example, a constitutive promoter, such as the 35S cauliflower mosaic virus (CaMV35S) promoter, affects enzyme activity in all parts of the plant. Using a tissue specific promoter will produce the desired sense or antisense RNA only in the tissue of interest. In gene constructs using an inducible gene promoter sequence, the rate of RNA polymerase binding and transcription initiation can be varied by external stimuli such as light, heat, anaerobic stress, altered nutritional conditions, and the like. Temporally regulated promoters are used to influence variations in the rate of RNA polymerase binding and transcription initiation at specific times during the development of transformed cells. It is preferred to use the native promoter from the enzyme gene in question or a promoter from a gene which is expressed only in a particular tissue of the transformed organism, such as eucalyptus or pine. Other examples of gene promoters that can be usefully employed in the present invention are reviewed by mannopine synthase (mas), octopine synthase (ocs) and Chua et al., Science 244: 174181, 1989. Things included.
[0054]
The gene termination sequence located 3 'to the DNA sequence to be transcribed may be derived from the same gene as the gene promoter sequence or may be derived from a different gene. Many gene termination sequences well known to those skilled in the art, such as the 3 'end of the nopaline synthase gene of Agrobacterium tumefaciens, can be effectively used in the present invention. In one embodiment, the gene terminator sequence is from the native enzyme gene or the target species to be transformed.
[0055]
The DNA construct of the present invention may also include a selectable marker effective on plant cells so that a transformed cell containing the construct of the present invention can be detected. Such markers, well known in the art of the present invention, typically have resistance to one or more toxins. One example of such a marker is the NPTII gene, whose expression confers resistance to kanamycin or hygromycin, which is normally toxic to plant cells at moderate concentrations (Rogers et al., Weissbach A and Weissbach H, "Plant Molecules" Methods for Plant Molecular Biology, Academic Press Inc. (San Diego, CA), 1988. Transformed cells can be identified by their ability to grow in media containing the antibiotic in question. Alternatively, the presence of the desired construct in the transformed cells can be determined by other techniques well known in the art of the invention, such as Southern and Western blotting.
[0056]
Techniques for operatively linking the components of the DNA constructs of the present invention are well known in the art of the present invention and are described, for example, in Sambrook et al., "Molecular cloning: a laboratory manual" Cold. Spring Harbor Laboratory Press (Cold Spring Harbor, NY, 1989), which includes the use of synthetic linkers containing one or more restriction endonuclease sites. The DNA constructs of the present invention comprise a vector having one or more replication systems, such as E. coli, for example, which allows the resulting construct to be cloned and sequenced after each operation to determine the accuracy of the operation. They may be connected.
[0057]
The genetic construct of the present invention includes monocotyledonous angiosperms (eg, grass, corn, cereals, oats, wheat, barley) and dicotyledonous angiosperms (eg, Arabidopsis, tobacco, legumes, alfalfa, oak, eucalyptus, Maple) and gymnosperms (eg, Scots pine (Aronen, Finnish Forest Res. Papers, Vol. 595, 1996), white spruce (Ellis et al., Biotechnology 11: 84-89, 1993), and larch (Huang et al., Ind.) (vitro Cell 27: 201-207, 1991) .In a preferred embodiment, the DNA constructs of the present invention can be used for their stems to survive multiple years and to add woody tissue. Every year It is used for the transformation of woody plants, herein defined as trees or shrubs of increasing diameter, wherein the target plant is preferably selected from the group comprising eucalyptus and pine species, most preferably Eucalyptus grandis. Other species usefully transformed with the DNA constructs of the present invention include Pinus banksia, Pinus brutia, Pinus caribaea, Pinus clausa, Pinus controla, Pinus coulta, Pinus coulta, Pinus radiata. eldarica, Pinus ellioti, Pinus jeffreyi, Pinus lambertiana, Pinus monticol , Pinus nigra, Pinus palustrus, Pinus pinaster, Pinus ponderosa, Pinus resinosa, Pinus rigida, Pinus serotina, Pinus strobus, Pinus sylvestris, Pinus taeda, and pine such as Pinus virginiana, Abies amabilis, Abies balsamea, Abies concolor, Abies grandis , Abies lasiocarpa, Abies magnifica, Abies procera, Chamacecyparis lawsoniona, Chamacecyparis nototkatensis, Champaeciperis typhois des, Huniperus virginiana, Larix decidua, Larix laricina, Larix leptolepis, Larix occidentalis, Larix siberica, Libocedrus decurrens, Picea abies, Picea engelmanni, Picea glauca, Picea mariana, Picea pungens, Picea rubens, Picea sitchensis, Pseudotsuga menziesii, Sequoia gigantea, Sequoia sempervirens, Taxodium distichum, Tsuga canadensis, Tsuga heterophylla, Tsuga ertensiana, Thuja occidentalis, and other gymnosperms such as Thuja plicata, Eucalyptus alba, Eucalyptus bancroftii, Eucalyptus botyroides, Eucalyptus bridgesiana, Eucalyptus calophylla, Eucalyptus camaldulensis, Eucalyptus citriodora, Eucalyptus cladocalyx, Eucalyptus coccifera, Eucalyptus curtisii, Eucalyptus dalrympleana, Eucalyptus degupta, Eucalyptus delagensis, Euca yptus diversicolor, Eucalyptus dunnii, Eucalyptus ficifolia, Eucalyptus globulus, Eucalyptus gomphocephala, Eucalyptus gunnii, Eucalyptus henryi, Eucalyptus laevopinea, Eucalyptus macarthurii, Eucalyptus macrorhyncha, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus megacarpa, Eucalyptus melliodora, Eucalyptus nicholii, Eucalyptus nitens, Eucalyptus nov -anglica, Eucalyptus obliqua, Eucalyptus obtusiflora, Eucalyptus oreades, Eucalyptus pauciflora, Eucalyptus polybractea, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus robusta, Eucalyptus rudis, Eucalyptus saligna, Eucalyptus sideroxylon, Eucalyptus stuartiana, Eucalyptus tereticornis, Eucalyptus torelliana, Eucalyptus urnigera, Eucalyptus urophylla , ucalyptus viminalis, Eucalyptus viridis, Eucalyptus wandoo, and eucalyptus such as Eucalyptus Youmanni, including any one of the hybrid of the seed, but not limited to.
[0058]
Techniques for stably incorporating a DNA construct into the genome of a target plant are well known in the art of the present invention, and include Agrobacterium tumefaciens-mediated transduction, electroporation, protoplast fusion, and reproductive organ injection. Methods, immature embryo injection, high velocity projectile introduction, and the like. The choice of technique will depend on the target plant to be transformed. For example, dicotyledonous plants and certain monocotyledonous and gymnosperm plants can be transformed by Agrobacterium Ti plasmid technology, for example, as described by Bevan (Nucleic Acids Res. 12: 8711-8721, 1984). . Targets for the introduction of the gene constructs of the present invention include tissues such as leaf tissue, sprayed cells, protoplasts, seeds, embryos, meristem regions, cotyledons, hypocotyls and the like. One method of transforming eucalyptus and pine is pollen (see, for example, Aronen, Finnish Forest Res. Papers, Vol. 595: 53, 1996) or biolistic using readily reproducible embryonic tissue. Is the way.
[0059]
Once the cells have been transformed, cells having the DNA construct of the present invention integrated into the genome can be selected with a marker such as the kanamycin resistance marker described above. The transgenic cells are cultured in a suitable medium for regenerating the whole plant using techniques well known in the art of the present invention. In the case of protoplasts, the cell wall is regenerated under appropriate osmotic conditions. In the case of seeds or embryos, a suitable germination or callus induction medium is used. For explants, an appropriate regeneration medium is used. Plant regeneration is well established for many species. For a review of forest tree regeneration, see Dunstan et al., "Somatic Embryogenesis in Woody Plants," (Thorpe TA Editing, In Vitro Embryogenesis of Plants in Plants). See Plant Science and Biotechnology in Agriculture 20 (12): 471-540, 1995). A specific protocol for spruce regeneration is described in Roberts'"Somatic Embryogenesis of Spruce" (Regenbaugh K, edited by Shensee: Improving Artificial Seed Harvest (Spruce). Synthetic: applications of synthetic seed to crop improvement), published by CRC Press, Chapter 23, pages 427-449, 1993). The resulting transformed plants may be propagated sexually or asexually to obtain transgenic plants of the next generation or later using techniques well known in the art.
[0060]
As described above, RNA production in target cells can be controlled by selecting a promoter sequence. A target plant is transformed with two or more constructs of the invention, modifies two or more polypeptide activities, affects polypeptide activity in two or more tissues, or expresses two or more polypeptides upon expression. May affect activity. Similarly, a gene construct may be constructed to include two or more open reading frames encoding a polypeptide of the invention, or to include two or more non-coding regions of a gene encoding such a polypeptide. Good. The polynucleotides of the present invention may also be used in combination with other known sequences that encode a polypeptide involved in plant cell signaling.
[0061]
The isolated polynucleotides of the invention are involved in cell signaling from other plant species using techniques well known in the art of the invention as routinely used in DNA hybridization and PCR techniques. It can also be used as a probe to isolate a DNA sequence encoding a polypeptide.
[0062]
The polynucleotides, polypeptides and antibodies against such polypeptides of the invention can be used to screen for molecules that interact with such polynucleotides and / or polypeptides and thereby alter cell signaling. Techniques for performing such assays are well known in the art. Similarly, the polynucleotides and polypeptides of the present invention may be used in studies designed to elucidate cell signaling pathways.
[0063]
The following examples are given by way of illustration and not by way of limitation.
[0064]
Example 1
Isolation and characterization of a cDNA clone from Eucalyptus grandis
A Eucalyptus grandis cDNA expression library was constructed and screened as follows.
[0065]
mRNA was extracted from specific plant tissues, such as stem xylem, using the protocol of Chang et al. (Plant Molecular Biology Reporter 11: 113-116, 1993) with minor modifications. Specifically, the sample was dissolved in CPC-RNAXB (100 mM Tris-Cl, pH 8.0, 25 mM EDTA, 2.0 M NaCl, 2% CTAB, 2% PVP, and 0.05% spermidine trihydrochloride), and chloroform was dissolved. : Isoamyl alcohol was extracted with a mixture of 24: 1. The mRNA was precipitated with ethanol and the total RNA preparation was purified using a poly (A) quick mRNA isolation kit (Stratagene, La Jolla, CA). The cDNA expression library was constructed by synthesizing reverse transcriptase from the purified mRNA using a ZAP Express cDNA synthesis kit (Stratagene) according to the manufacturer's protocol, and then inserting the cDNA clone into lambda ZAP. The obtained cDNA was packaged with Gigapack II packaging extract (Stratagene) using 1 μl of sample DNA in 5 μl of ligation mix. Mass excision of the library was performed using XL1-Blue MRF 'cells and XLOLR cells (Stratagene) with ExAssist helper phage (Stratagene). The excised phagemid was diluted in NZY broth (Gibco BRL, Gaithersburg, MD) and plated on LB-kanamycin agar plates containing X-gal and isopropylthio-beta-galactoside (IPTG).
[0066]
Of the colonies plated and picked for DNA minipreps, 99% contained inserts suitable for sequencing. Positive clones were cultured in NZY broth containing kanamycin, and cDNA was purified by alkaline lysis and polyethylene glycol (PEG) precipitation. 1% agarose gel was used to screen for chromosomal contamination in the sequencing template. Dye primer sequences were prepared using a Turbo Catalyst 800 machine (Perkin Elmer / Applied Biosystems Division, Foster City, CA) according to the manufacturer's protocol.
[0067]
The DNA sequence of the positive clones was obtained using a Perkin Elmer / Applied Biosystems Division Prism 377 sequencer. cDNA clones were first sequenced from the 5 'end and, optionally, also from the 3' end. For some clones, internal sequences were obtained by designing primers that hybridize to the termini of known sequences and using them as sequencing primers to extend the amount of sequence information. As an alternative, the internal sequence may be obtained by generating a "nested" deletion clone of the gene of interest using published methods (Henikoff, Gene 28: 351-359, 1984). Was done.
[0068]
Sequenced cDNA sequences were compared to known sequences in the EMBL database (Release 58, March 1999) using the computer algorithms FASTA and / or BLASTN. Multiple alignments of redundant sequences were used to assemble reliable consensus sequences. Based on similarities to known sequences from other plant species, the isolated DNA was identified as RLK (SEQ ID NOs: 2, 8, 9, 11, 15, 18, 19, 21-25, 33, 34, 38, 131-301, 448-463, 848, 858-874 and 882-887) or at least one component of a two-component signaling system (HK, RR or hybrid HK / RR protein, SEQ ID NO: 42; 48-52, 55-58, 67, 464-471, 474-478, and 850-857). Sequence ID Nos. 2, 8, 9, 11, 15, 18, 19, 21-25, 33, 34 and 38 have less than 10% of residues identical to the known sequence (by the determination method described above). Could not be found. The sequence identification numbers 848, 854, 855, 856, 859, 860, 862, 863, 864, 865, 866, 867, 868, 869, 871, 872, 873, 874, 882, 883 and 885 are the sequences, respectively. ID Nos. 232, 467, 468, 48, 282, 288, 488, 453, 289, 268, 297, 278, 290, 449, 299, 301, 270, 269, 276, 454, and 300 The sequence identification numbers 848, 854-856, 859, 860, 862-865, 868, 869 and 871-874 are full-length sequences.
[0069]
Example 2
Isolation and characterization of cDNA clone from Pinus radiata
A Pinus radiata cDNA expression library was constructed from specific tissues such as xylem as described in Example 1 and screened. The DNA sequence of the positive clone was obtained using the Perkin Elmer / Applied Biosystems Division Prism 377 sequencer using forward and reverse primers, and the determined sequence was compared to known sequences in the database as described above.
[0070]
The isolated DNA sequence based on similarity to known sequences from other plant species was RLK (SEQ ID NOs: 1, 3-7, 10, 12-14, 16, 17, 20, 26-32, 35-37, 39-41, 302-447, 833-847, 875-881 and 888) or at least one component of a two-component signaling system (HK, RR or hybrid HK / RR protein, SEQ ID NO: 43-47, 53, 54, 59-66, 472, 473, 479-481, and 849). The sequences of SEQ ID NOs: 3-7, 10, 12-14, 16, 17, 20, 26, 28-32, 35-37 and 39-41 are identical to the known sequences (as determined above). The residue was found to be less than 10%. The sequence with SEQ ID NO: 480 was found to contain a predicted unspliced intron, and translation was spliced into two open reading frames. The amino acid sequences encoded in these two open reading frames are provided in SEQ ID NOs: 830 and 831. SEQ ID Nos. 411, 413, 317, 421, 415, 434 and 416 represent extended sequences of SEQ ID Nos. 26, 17, 28, 39, 16, 30 and 41, respectively. The sequence identification numbers 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 875, 876, 877, 878, 879, 880 and 888 are the sequence identification numbers 411, 413, 317, 29, 421, 415, 434, 416, 35, 37, 36, 40, 438, 426, 445, 418, 435, 411, and 427. , 844 and 878-881 are full length sequences.
[0071]
Example 3
Use of the ethylene receptor gene to modify plant growth
Transformation of tobacco plants with the Pinus radiata ethylene receptor gene homolog is performed as follows. DNA constructs including the sense and antisense constructs containing the DNA sequence containing the coding region of the ethylene receptor homolog (SEQ ID NO: 43) derived from Pinus radiata were constructed and published methods (An, Binary Vectors (Binary Vectors)). .), Edited by Gelvin SB and Schilperort RA, plant molecular biology manual (Plant Molecular Biology Manual), published by Kluer Academic Publishers, Dordrecht, 1988), and directly transformed into Agrobacterium agobacterium by Escherichia coli using the Agrobacterium bacterium. . Sense DNA constructs were made by cloning the cDNA insert of the PBK-CMV plasmid into the pART7 plasmid, and then cloning the NotS digested 35S-insert-OCS3'UTR fragment from the pART7 vector into the pART27 plant expression vector. (Gleave, Plant Mol. Biol. 20: 1203-1207, 1992). The presence and integrity of the transgenic construct is confirmed by restriction enzyme digestion and DNA sequencing.
[0072]
Sections of tobacco (Nicotiana tabacum cv. Samsun) leaves are transformed with the sense and antisense ethylene receptor constructs described above using a method based on the method of Horsch et al., Science 227: 1229-1231, 1985. Transformed plants containing the appropriate constructs are identified using Southern blot experiments. Expression of the Pinus ethylene receptor homolog in the transformed plant is confirmed by isolating total RNA from each of the independent transformed plant strains created using the sense and antisense constructs. The RNA sample is analyzed in a Northern blot experiment to determine the level of foreign gene expression in each transformant. For each of the transformed plant strains produced with the sense and antisense constructs, the expression level of the ethylene receptor polypeptide encoded by the Pinus ethylene receptor gene and the endogenous tobacco ethylene receptor gene was determined to be wild. The expression level of the plant in the type control experiment is compared.
[0073]
Although the present invention has been described in some detail for purposes of illustration for purposes of clarity, changes and modifications may be made without departing from the scope of the invention, which is intended to be limited solely by the appended claims. sell.