CA2301257A1 - The ire gene regulating the root-hair growth in arabidopsis - Google Patents
The ire gene regulating the root-hair growth in arabidopsis Download PDFInfo
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Abstract
Disclosed herein is a gene coding for a protein having a plant tip growth regulating activity, and a promoter for the gene. Various plants with different morphologies, such as those having elongated root hairs, can be created. Further, any gene may be expressed specifically at a site in which plant tip growth is performed.
Description
SPECIFICATION
The IRE gene regulating the root-hair growth in Arabidopsis BACKGROUND OF THE INVENTION
Field of the Invention The present invention relates to a gene coding for a protein that is involved in regulation of tip growth in plants, and also to the promoter of the gene.
Prior Art Unlike other cells, it is known that root hairs of plants elongate in a particular manner called tip growth. Pollen tubes of higher plants also perform tip growth. Tip growth is also observed in those eukaryotic cells that hold cell walls in ferns, mosses, algae, fungi and so forth. A number of mutants that show abnormalities in the number or shape of root hairs have been isolated from Arabidopsis. hy5, phyB, rhd2 and tipl are known as mutants with abnormal root-hair lengths (Genes &
Development, Vol. 11, p. 2983-2995 (1997); Plant Cell, Vol. 5, p.
147-157 (1993); Plant Cell, Vol. 2, p. 235-243 (1990); Plant Physiology, Vol. 103, p. 979-985 (1993)). These mutants except tipl show various phenotypes in addition to root-hair elongation, suggesting that roles of these genes are not restricted just to root hairs or to tip growth. It is therefore unclear how these genes are involved in root-hair elongation. Since the tipl mutant shows abnormalities in elongation both of root hairs and pollen tubes, tipl is considered as a tip growth-impaired mutant.
The molecular function of this gene product has not yet been revealed. As described above, those genes that specifically function in regulation of the root-hair elongation or tip growth have remained to be seen.
SUMMARY OF THE INVENTION
The present invention aims to isolate a novel gene regulating tip growth such as root-hair elongation and to use the gene for providing plants that show altered tip growth rates.
The present inventors have made great efforts to solve the above mentioned problems and isolated a short root-hair mutant of Arabidopsis. They have cloned the gene responsible for the phenotype. Furthermore, the cloned gene has been introduced into plants and the effect of the gene has been confirmed. Thus, the present invention has been completed.
The first aspect of the present invention relates to a gene coding for a protein (a) or (b) mentioned below:
(a) a protein represented by the amino acid sequence as set forth in SEQ ID NO.: 2; or (b) a protein represented by an amino acid sequence having one or more amino acids deleted from, substituted in, modified in or added to the amino acid sequence as set forth in SEQ ID NO.: 2, and having a tip growth regulating activity.
The second aspect of the present invention relates to a DNA (a) or (b) as mentioned below:
(a) a DNA represented by the nucleotide sequence as set forth in SEQ ID NO.: 3; or (b) a DNA represented by a nucleotide sequence having one or more nucleotides deleted from, substituted in or added to the nucleotide sequence as set forth in SEQ ID NO.: 3, and having a function as a promoter.
This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No.82402/1999, which is a priority document of the present application.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows root hairs of Arabidopsis: wild type (A), the ire mutant (B), and an ire mutant in which the wild-type IRE
gene is introduced (C).
Figure 2 shows elongation properties of root hairs of wild type (A) and the ire mutant (B).
Figure 3 shows a physical map of the genome around the IRE
locus.
Figure 4 shows the exon regions of the IRE gene.
Figure 5 shows expression patterns of the GUS gene under the control of the IRE promoter.
Figure 6 shows the nucleotide sequence of the IRE promoter.
DESCRIPTION OF THE INVENTION
The present invention is hereinafter described in detail.
(1) First Invention (Structural Gene) The first invention relates to a gene coding for a protein (a) or (b) as mentioned below and encompasses the protein encoded by the gene, mutant genes, organisms carrying these genes:
(a) a protein represented by the amino acid sequence as set forth in SEQ ID NO.: 2; or (b) a protein represented by an amino acid sequence having one or more amino acids deleted from, substituted for, modified in or added to the amino acid sequence as set forth in SEQ ID NO.:
2, and having a tip growth regulating activity.
The term "one or more" used herein means the number of amino acids which can be deleted or otherwise altered according to the technology conventionally used at the time of filing the present application such as the site-directed mutagnenesis.
Chemical modification of amino acid residues in a protein is commonly carried out in the art: see, for example, Hirs, C.H.W. and Timasheff, S.N., eds, (1977) Methods in Enzymology, Vol. 47, p. 407-498, Academic Press, New York.
The gene coding for the amino acid sequence as set forth in SET ID No.: 2 modifies the elongation of root hairs. This is shown by the fact that the mutant impaired this gene displays shorter root hairs. This effect is restricted in root hairs because the mutant shows the normal elongation of root hair-bearing cells. Thus we have named this gene IRE (Incomplete Root-hair Elongation). No gene products that have a tip growth specific function have not been found.
The IRE gene can be cloned according to the method of Japanese Patent Application Laying Open No. 10-4978 as described below.
First, genomic DNA of Arabidopsis is fragmented with an appropriate restriction enzyme and the DNA fragments are ligated into an appropriate vector for a genomic DNA library. This library is amplified by transforming into a host microorganism.
The isolation of DNA may be carried out in a conventional method using cesium chloride and ethidum bromide. The restriction enzyme is not particularly limited and may include, for example, Sau3AI. The vector is also not particularly limited and may include, for example, a DASH II. The host microorganism may be determined depending upon the vector used. For example, 1 Dash II requires Escherichia coli XL-1 Blue MRA (P2).
Then, the above mentioned genomic DNA library is screened for the genomic region of the IRE locus. A DNA fragment that is adjacent to the T-DNA insertion in the ire mutant is used for the probe. Since the ire gene is disrupted by the insertion in the genome of the ire mutant, the adjacent region includes a part of the IRE gene.
Positive clones selected by the above mentioned probe are purified and the insert genomic DNA fragments are separated. A
genomic fragment is used for a probe to screen a cDNA library.
The cDNA library may be prepared according to a conventional procedure. That is, the total RNA is isolated from plant tissues and polyA+ RNA is purified using oligo(dT) resins. cDNA
is synthesized from the template polyA+ using a reverse transcriptase. The synthesized cDNA is ligated into an appropriate vector for a cDNA library. The library is amplified by transforming into a host microorganism. Positive clones in the cDNA library contain the IRE gene.
Capabilities of the hybridization between one DNA sequence and others under stringent conditions may be determined, for example, by the following method. First one DNA fragment is blotted and immobilized onto a nylon membrane. Then, this membrane is prehybridized by the solution containing 6X SSC, O.O1M EDTA, 5X Denhardt's solution, 0.5% SDS, 100 ~g/ml denatured salmon DNA at 65~C for one hour or more. A hybridized solution is prepared by adding labeled DNA fragments or labeled RNA transcripts of a DNA template in the prehybidization solution. The prehybridized nylon membrane is hybridized in the hybridization solution at 65 ~C for 3 to 16 hours. Thereafter, it is washed in a solution containing 2X SSC and 0.1% SDS at 65 'C for 30 minutes. Then, it is further washed in 2X SSC, 0.1%
SDS solution at room temperature for 15 minutes. Further, it washed in 0.1X SSC, 0.1% SDS solution at 65 ~C for 30 minutes.
Then, the signal on the membrane is detected by a suitable method depending the labeling reagent.
In order to express genes of the first invention (the IRE
gene and modified genes) in a host, the genes may be inserted into an appropriate expression vector and the recombinant vector may be introduced into the host. Hosts are not particularly limited. Plants are preferable but bacteria, yeast and animals may be used. Species of plants used as the host are not particularly limited but those species of soybean, rape, and cotton may be preferable. Any expression vector may be used as far as it contains both a promoter for the expression and a marker gene that enable the handling the host. For example, plant tissues preferably require vectors that carry the cauliflower mosaic virus 35S promoter because of the wide application of species. pBI121 vector (Clontech) is an example.
Methods to introduce a vector into the host are not particularly limited and may be done depending upon the host organism. An Agrobacterium-mediated method is preferable for introducing into plant tissues although those methods using electroporation, particle gun may also be applicable.
Since the gene of the first invention has a function to modify tip growth such as elongation or root hairs, the following utilities may be considered.
I) Applications using the tip-growth modifying activity in root hairs.
Introducing the gene of the first invention into a plant and expressing it therein can let the plant have root hairs with different lengths which can provide drought resistance of the plant. Bacteria in Rhizobium are parasites of leguminous plants and they enable the plants nitrogen fixation. Those bacteria are shown to infect only elongating root hairs. Thus plants that have altered root-hair growth by using the gene can be expected to improve the infectious efficiency of the bacteria.
II) Applications using the tip-growth modifying activity in other than root hairs.
In higher plants, pollen tubes emerge from pollen grains (male gametophytes) and elongate to female gametophytes while the fertilization. It is expected that the manipulation of the gene expression in pollen tubes may alter the fertilization timing and efficiency. Namely, plants (male gametophytes) with either promoting or retarding fertilization ability may be created. Cotton fibers, which are the raw material of the common fiber, cotton yarn, elongate with a kind of tip growth in cotton fruits. Thus it is also expected to improve the quality of cotton fiber.
(2) Second Invention (Promoter) The second invention relates to a DNA (a) or (b) as mentioned below and encompasses promoters, expression vectors, organisms carrying these DNA sequences:
(a) a DNA represented by the base sequence as set forth in SEQ
ID NO.: 3; or (b) a DNA represented by a base sequence having one or more bases deleted from, substituted for or added to the base sequence as set forth in SEQ ID NO.: 3, and having a function as a promoter.
The term "one or more" used herein means the number of nucleotide sequences which can be deleted or otherwise altered according to the technology conventionally used at the time of filing the present application such as the site-directed mutagnenesis.
Since the DNA of the second invention exists in the upstream region of the gene of the first invention, it may be cloned according to the method of Japanese Patent Application Laying Open No. 10-4978 as in the case of the gene of the first invention.
The DNA of second invention function as a promoter in such tip-growing tissues as root hairs and pollen grains (pollen tubes). Recombinant genes that have any gene under this DNA
sequence can be expressed specifically in root hairs and/or pollen grains. Thus useful biological functions depending upon root hairs and/or pollen grains can be introduces.
EXAMPLES
The present inventions are illustrated in more detailed by way of examples which in no way limit the scope of the present invention.
Example 1: Production of transgenic plants for the isolation of IRE
Transgenic plants for the isolation of IRE were created according to the method of Example 1 of Japanese Patent Application Laying Open No. 10-4978.
Seeds of Arabidopsis (variety: Wassilewskija [WS]) were sterilized and sown on an agar medium. These seeds were incubated at 4 'C in darkness for two to four days. This cold treatment prompted breakage of seed dormancy and made the germination uniform and enhanced the flowering timing.
Thereafter, seeds were germinated and grown under continuous light at 22 ~C. The seed sterilization was carried out by using a sterilization solution containing 10 ~ of Hitar (Kao) and 0.02 Triton-X 100. Seeds were mixed and vortexed in the sterilization solution and allowed to stand at room temperature for three to five minutes, and washed five times with sterilized water. The agar medium contained 1/2 x Arabidopsis nutrient salt solution and 1.5 ~ agar (Nakarai, special grade). lx Arabidopsis nutrient salts solution was prepared by following:
985 ml distilled water or deionized water, 5 ml of 1M KN03, 2 ml of 1M MgS04, 2 ml of 1M Ca(N03)2, 2.5 ml of 20mM Fe-EDTA, 1 ml of trace element solution, 2.5 ml of K-P04 buffer (pH 5.5)]
(Hideaki Shiroishi et al., (1991), Gendai-Kagaku vo120, Plant Biotechnology II, p. 38). The agar medium was autoclaved and poured into petri dishes. This concentration of agar prevented Arabidopsis roots from penetrating in the agar medium. It made he observation of the root morphology easier. The light source was built with two commonly used 40 W fluorescent lamps and one Homolux fluorescent lamp (National). Plants were grown under this light source at an about 30cm distance. The intensity of the light was about 3000 lux.
Forty individuals of Arabidopsis at three weeks after the seed sowing were inoculated with Agrobacterium. The inoculation was carried out according to the modified in planta method (Plant Journal, Vol. 5, p. 551-558 (1994)). In this method, floral stems of plants were wounded in order to promote the infection. Agrobacterium tumefaciens strain C58C1rif (Nucleic Acids Research, Vol. 13, p. 6981-6998 (1985)) was obtained from Velten, J. et al. and used. This strain carries pGV3850 HPT as the intermediate Ti plasmid and between the right- and left-borders, there is a hygromycin phosphotransferase gene driven by the cauliflower mosaic virus 35S promoter on this vector that acts as the selection marker for plants.
After the inoculation of Agrobacterium, those plants were transplanted on soil of the 1:1 mixture of vermiculite and perlite. After 1.5 to 2 months of transplantation, seeds (T1 seeds) were harvested. Those seeds were sterilized in a similar manner to the above mentioned, and sown on a hygromycin containing medium (1X Ganborg B5 mixed salts for culture medium, 1~ sucrose, 0.8~ agar, 10 mg/1 hygromycin B). Individuals showing hygromycin resistance were selected and transplanted on soil. The soil was mentioned above. After 1.5 to 2 months from the transplantation, self-pollinated seeds (T2 seeds) were harvested.
Example 2: Screening for root-morphology mutants The screening was carried out according to Example 2 of Japanese Patent Application Laying Open No. 10-4978. The T2 seeds obtained in Example 1 were sterilized, sown on an agar medium and grown. The sterilization and the agar medium were as above mentioned in Example 1. To facilitate morphological observation of roots, the agar medium was placed in a transparent plastic Petri dish (Eiken Kizai Kabushiki Kaisha, No.
2 square Petri dish, 14 cm x 10 cm).
The morphology of roots was observed through transmitted light by means of a stereoscopic microscope OLYMPUS SZH-IDDL.
Morphologies of roots in about 300 lines of transgenic Arabidopsis were examined and ire was isolated as an abnormal root-hair length mutant. The length of root hairs was smaller in the ire mutant (Fig. 1B) than in wild type (Fig. 1A).
However, the density of root hairs and the distance between root hairs were identical, indicating that this mutation specifically reduces the length of root hairs. A detailed analysis revealed that the elongation of root hairs ceased earlier in the mutant (Fig. 2B) than in wild type (Fig. 2A). This cessation in the mutant resulted in 60 ~ of root-hair length of wild type.
Example 3: Genetic studies of the ire mutant According to the method in Japanese Patent Application Laying Open No. 10-4978, the ire mutant and wild type were cross-pollinated. The phenotype (length of root hairs) of wild type, ire and their F1 progeny was examined (Table 1).
Length of root hairs t i ne (11m + SE) Wild type 396 ~ 17 Ire 232 ~ 25 F1 progeny and wild type 452 ~ 32 As shown in Table 1, the Fl progeny showed root hairs of a normal length like wild type, indicating that the ire mutation is recessive. Among 711 individuals of selfed progeny of the F1 plants (F2 progeny), 165 individuals (23~) showed the short root-hair phenotype, suggesting that this phenotype is caused by a single recessive locus.
Example 4: Preparatory studies for the cloning of the IRE gene The ire mutant was isolated as a T-DNA insertion line carrying the selection marker. In order to examine if the inserted T-DNA disrupted the IRE gene or not, selfed F3 seeds of 213 mutants in the F2 progeny were subjected to the linkage analysis between the phenotype and the selection marker gene (hygromycin resistance gene). All F3 progeny of every mutant showed the hygromycin resistance on the selection medium (1x Gamborg B5 mixed salts for medium, 1~ sucrose, 0.8~ agar, 10 mg/1 hygromycin B). Namely, each mutant in the F2 progeny carried the T-DNA homozygously. Thus it was suggested that the inserted T-DNA is closely linked with the IRE locus and probably disrupts the gene. Therefore, genomic regions flanking the T-DNA insertion should include the IRE gene.
Example 5: Cloning of the IRE gene A genomic DNA fragment adjacent to the inserted T-DNA was amplified and cloned by the TAIL-PCR method using the genome DNA of the ire mutant as a template (Plant Journal, Vol.8, p.457-463 (1995)). The sequences used in the PCR are below: a first specific primer 5'-CACATCATCTCATTGATGCTTGGT-3' (24mer); a second specific primer 5'-CATAGATGCACTCGAAATCAGCC-3' (23mer); a third specific primer 5'-GTGTTATTAAGTTGTCTAAGCGTC-3' (24mer); and arbitrary primers 5'-(A/T)GTG(A/T/G/C)AG(A/T)A(A/T/G/C)CA(A/T/G/C)AGA-3' (l6mer). A 0.7 kb PCR fragment was amplified using this set of primers. The template genomic DNA was extracted according to Example 4 of Japanese Patent Application Laying Open No. 10-4978.
An Arabidopsis genomic library was screened using the amplified 0.7 kb PCR fragment as a probe. Those genomic clones were isolated that covered an about 10 kb region in which a sequence hybridized by the PCR fragment was contained. This genomic region was divided into several fragments and subcloned into pBluescript II KS+
(Stratagene) to determine the nucleotide sequence. The genomic region underlined in Fig. 3 was used as a probe to screen a cDNA
library. A cDNA clone was isolated in this screening. The screening of the genomic and cDNA libraries as well as the method of determination of nucleotide sequences was as described in Example 7 of Japanese Patent Application Laying Open No. 10-4978.
The isolated cDNA was only about 1 kb in length. In order to isolate a full length cDNA, a PCR method was tried. cDNA that had been synthesized for the cDNA library was used as a template and several primer sets designed based on the genome sequence were used for the PCR. Two PCR fragments were amplified when using the following sets of primers and the PCR fragments and the isolated cDNA were joined into a full length cDNA. One of the PCR fragments, which is composed of 2.2 kb at the 5' region of the full length cDNA, was amplified using following primers: f2384 f2384:
CAACCGCTTCTCTGTAATC and r4950: AGCCTTCCTATCCTGAATG. The other fragment, which is composed of 1.2 kb at the 3' region of the full length cDNA was amplified using following primers: f4897:
TCATGATTGAGCAGTTGGA and r6898: CCGAGCAAGTGTGTCC.
The nucleotide sequence of the full length cDNA was determined. The full length except for polyA sequence consists of 3842 by (SEQ ID NO.: 1) and the possible largest ORF (Open Reading Frame) consists of 1168 amino acid residues (SEQ ID NO.:
2). A stop codon locates in front of the start codon in frame of this ORF. The correspondence between the cDNA and the genome of IRE gene is shown in Fig. 4. The IRE gene consists of 17 exons and the T-DNA was inserted into the promoter region in the ire mutant. The wild-type genomic region as shown in Fig. 4 was introduced into the ire mutant. A vector was reconstructed from pBI121 (Clontech) by removing the b-Glucuronidase (hereinafter referred to GUS) gene and replaced with the genomic region. pBI
121 carries a gene for kanamycin resistance and transformed plants show the resistance against kanamycin. The construction of vector and gene transfer into plants were carried out according to the method of Example 8 of Japanese Patent Application Laying Open No. 10-4978; however, the media for selecting bacteria and plants were supplemented with 50 mg/1 kanamycin sulfate (Meiji Seika). As shown in Fig. 1C, the transformed plant showed longer root hairs than the untransformed plant (the ire mutant), indicating that this gene has an ability to increase the length of root hairs.
Example 6: Tissue-specific activity of the promoter region of the IRE gene The DNA fragment as set forth in Fig. 6 (SEQ ID NO.: 3) was obtained by digesting with restriction enzymes XhoI and BamHI. This fragment was ligated into SalI-BamHI sites of pBI101-2 vector (Clontech). This recombinant vector was used for the transformation into wild-type plants (using the method of Example 5 above). Restriction enzymes used were purchased from Takara Shuzo. The GUS gene was to be expressed under the control of the IRE gene promoter. The sequence as shown in Fig.
6 corresponds to the region spanning between XhoI and BamHI
sites in Fig. 4. The sequence of white letters in Fig. 6 corresponds to the first exon of the IRE gene where the start codon ATG is enclosed with the rectangle. The sequences with dotted underlines show exons of another gene adjacent to the IRE
gene. To observe the activity of the expressed GUS, the transformants were treated with an X-Gluc solution (5.7 mM X-Gluc (5-bromo-4-chloro-3-indolyl-~-glucuronide), 1.5 mM
K3Fe (CN) 6, 1 . 5 mM K4Fe (CN) 6, 0. 9~ Triton X-100) . Transformant plants were soaked in the X-Gluc solution, subjected to a vacuum treatment, and then incubated at 37 ~C overnight to develop the color. Reagents used were purchased from Wako Pure Chemical.
The GUS activities were substantially shown in only the root and pollen grains. As shown by arrows in Fig. 5B, the GUS activity was found in pollen grains in the anther. Pollen grains are germinated on a stigma and elongate pollen tubes. The GUS
activity was also found in elongating pollen tubes (Fig. 5C, arrow). The foregoing indicates that the promoter of IRE gene as set forth in SEQ ID NO.: 3 has activities in such tip-growing cells as root hairs and pollen tubes.
The present inventions) provides) a structural gene coding a protein that is involved in regulation of tip growth in plants, and its promoter. Various plants with different morphologies such as those showing prolonged root hairs, can be created by utilizing the structural gene of the present invention. Further, any gene may be expressed specifically in tissues that are tip-growing by utilizing the promoter of the present invention.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: BIOMOLECULAR ENGINEERING RESEARCH INSTITUTE
(ii) TITLE OF INVENTION: THE IRE GENE REGULATING THE ROOT-JAIR GROWTH IN
ARABIDOPSIS
(iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text) (vi) CURRENT APPLICATION DATA:
2 O (A) APPLICATION NUMBER: CA 2,301,257 (B) FILING DATE: 24-MAR-2000 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: JP 82402/1999 (B) FILING DATE: 25-MAR-1999 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SMART & BIGGAR
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 72813-119 3O (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-232-2486 (B) TELEFAX: (613)-232-8440 (2) INFORMATION ID N0:1:
FOR
SEQ
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 842base rs 3 pai (B) TYPE: leicacid nuc (C) STRANDEDNESS: double (D) TOPOLOGY:linear (ii)MOLECULE TYPE:cDNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM:Arabidopsisthaliana lO (ix)FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION:124..3627 (xi)SEQUENCE DESCRIPTI ON: ID
SEQ N0:1:
TTATG ATAAAATCAC AACAAACAAA
GTGTAAATAT ATAATTCTCT
GAAAA
GAA CCT
Met Ser Thr Thr ProSer GluAsnAsp ArgAspPro Gln Glu Pro O ACT TCC
Pro Thr Thr Ile Ser ProThr ThrAsnAla LysLeuLeu Lys Thr Ser CCT CAT
Lys Ile Pro Ala Ile PheArg SerAspLys GluGlyGlu Asp Pro His GAC ACT
Glu Gln Ala Lys Thr GluVal ThrGluLeu AlaGlyGlu Gly Asp Thr TCG ATT
Pro Met Ser His Asp ProGlu LeuAlaPro SerSerLeu Gly Ser Ile ACG TCT
Leu Asn His Ile Arg LysSer ProAlaPro SerProLeu Arg Thr Ser CCT TCT
Phe Ser Ser Ala Thr LeuIle ProGlyGln AspAspLys Asp Pro Ser CCG GGT
Val Ala Lys Glu Lys ArgVal ValValAsp AlaArgAla Asp Pro Gly Ala Arg Ala Arg Trp Pro Ile Pro Pro His Gln Pro Asp Gln Gly Lys Lys Val Gln Trp Ser Gln Ser Lys Ser Gln Arg Val Pro Ala Asn Ser Asn Pro Gly Val Glu Ser Thr His Val Gly Leu Ala Lys Glu Thr Gln Ser Pro Arg Phe Gln Ala Ile Leu Arg Val Thr Ser Gly Arg Lys Lys Lys Ala His Asp Ile Lys Ser Phe Ser His Glu Leu Asn Ser Lys Gly Val Arg Pro Phe Pro Val Trp Arg Ser Arg Ala Val Gly His Met Glu Glu Ile Met Ala Ala Ile Arg Thr Lys Phe Asp Lys Gln Lys Glu Asp 3~ Val Asp Ala Asp Leu Gly Val Phe Ala Gly Tyr Leu Val Thr Thr Leu Glu Ser Thr Pro Glu Ser Asn Lys Glu Leu Arg Val Gly Leu Glu Asp Leu Leu Val Glu Ala Arg Gln Cys Ala Thr Met Pro Ala Ser Glu Phe Trp Leu Lys Cys Glu Gly Ile Val Gln Lys Leu Asp Asp Lys Arg Gln Glu Leu Pro Met Gly Gly Leu Lys Gln Ala His Asn Arg Leu Leu Phe Ile Leu Thr Arg Cys Asn Arg Leu Val Gln Phe Arg Lys Glu Ser Gly Tyr Val Glu Glu His Ile Leu Gly Met His Gln Leu Ser Asp Leu Gly Val Tyr Pro Glu Gln Met Val Glu Ile Ser Arg Gln Gln Asp Leu Leu Arg Glu Lys Glu Ile Gln Lys Ile Asn Glu Lys Gln Asn Leu Ala Gly Lys Gln Asp Asp Gln Asn Ser Asn Ser Gly Ala Asp Gly Val Glu Val Asn Thr Ala Arg Ser Thr Asp Ser Thr Ser Ser Asn Phe Arg Met Ser Ser Trp Lys Lys Leu Pro Ser Ala Ala Glu Lys Asn Arg Ser Leu Asn Asn Thr Pro Lys Ala Lys Gly Glu Ser Lys Ile Gln Pro Lys Val Tyr Gly Asp Glu Asn Ala Glu Asn Leu His Ser Pro Ser Gly Gln Pro Ala Ser Ala Asp Arg Ser Ala Leu Trp Gly Phe Trp Ala Asp His Gln Cys Val Thr Tyr Asp Asn Ser Met Ile Cys Arg Ile Cys Glu Val Glu Ile Pro Val Val His Val Glu Glu His Ser Arg Ile Cys Thr Ile Ala Asp Arg Cys Asp Leu Lys Gly Ile Asn Val Asn Leu Arg Leu Glu Arg Val Ala Glu Ser Leu Glu Lys Ile Leu Glu Ser Trp Thr Pro Lys Ser Ser Val Thr Pro Arg Ala Val Ala Asp Ser Ala Arg Leu Ser Asn Ser Ser Arg Gln Glu Asp Leu Asp Glu Ile Ser Gln Arg Cys Ser Asp Asp Met Leu Asp Cys Val Pro Arg Ser Gln Asn Thr Phe Ser Leu Asp Glu Leu Asn Ile Leu Asn Glu Met Ser Met Thr Asn Gly Thr Lys Asp Ser Ser Ala Gly Ser Leu Thr Pro Pro Ser Pro Ala Thr Pro Arg Asn Ser Gln Val Asp Leu Leu Leu Ser Gly Arg Lys Thr Ile Ser Glu Leu Glu Asn Tyr Gln Gln Ile Asn Lys Leu Leu Asp Ile Ala Arg Ser Val Ala Asn Val Asn Val Cys Gly Tyr Ser Ser Leu Asp Phe Met Ile Glu Gln Leu Asp Glu Leu Lys Tyr Val Ile Gln Asp Arg Lys Ala Asp Ala Leu Val Val Glu Thr Phe Gly Arg Arg Ile Glu Lys Leu Leu Gln Glu Lys Tyr Ile Glu Leu Cys Gly Leu Ile Asp Asp Glu Lys Val Asp Ser Ser Asn Ala Met Pro Asp Glu Glu Ser Ser Ala Asp Glu Asp Thr Val Arg Ser Leu Arg Ala Ser Pro Leu Asn Pro Arg Ala Lys Asp Arg Thr Ser Ile Glu Asp Phe Glu Ile Ile Lys Pro Ile Ser Arg Gly Ala Phe Gly Arg Val Phe Leu Ala Lys Lys Arg Ala Thr Gly Asp Leu Phe Ala Ile Lys Val Leu Lys Lys Ala Asp Met Ile Arg Lys Asn Ala Val Glu Ser Ile Leu Ala Glu Arg Asn Ile Leu Ile Ser Val Arg Asn Pro Phe Val Val Arg Phe Phe Tyr Ser Phe Thr Cys Arg Glu Asn Leu Tyr Leu Val Met Glu Tyr Leu Asn Gly Gly Asp Leu Phe Ser Leu Leu Arg Asn Leu Gly Cys Leu Asp Glu Asp Met Ala Arg Ile Tyr Ile Ala Glu Val Val Leu Ala Leu Glu Tyr Leu His Ser Val Asn Ile Ile His Arg Asp Leu Lys Pro Asp Asn Leu Leu Ile Asn Gln Asp Gly His Ile Lys Leu Thr Asp Phe Gly Leu Ser Lys Val Gly Leu Ile Asn Ser Thr Asp Asp Leu Ser Gly Glu Ser Ser Leu Gly Asn Ser Gly Phe Phe Ala Glu Asp Gly Ser Lys Ala Gln His Ser Gln Gly Lys Asp Ser Arg Lys Lys His Ala Val Val Gly Thr Pro Asp Tyr Leu Ala Pro Glu Ile Leu Leu Gly Met Gly His Gly Lys Thr Ala Asp Trp Trp Ser Val Gly Val Ile Leu Phe Glu Val Leu Val Gly Ile Pro Pro Phe Asn Ala Glu Thr Pro Gln Gln Ile Phe Glu Asn Ile Ile Asn Arg Asp Ile Pro Trp Pro Asn Val Pro Glu Glu Ile Ser Tyr Glu Ala His Asp Leu Ile Asn Lys Leu Leu Thr Glu Asn Pro Val Gln Arg Leu Gly Ala Thr Gly Ala Gly Glu Val Lys Gln His His Phe Phe Lys Asp Ile Asn Trp Asp Thr Leu Ala Arg Gln Lys Ala Met Phe Val Pro Ser Ala Glu Pro Gln Asp Thr Ser Tyr Phe Met Ser Arg Tyr Ile Trp Asn Pro Glu Asp Glu Asn Val His Gly Gly Ser Asp Phe Asp Asp Leu Thr Asp Thr Cys Ser Ser Ser Ser Phe Asn Thr Gln Glu Glu Asp Gly Asp Glu Cys Gly Ser Leu Ala Glu Phe Gly Asn Gly Pro Asn Leu Ala Val Lys Tyr Ser Phe Ser Asn Phe Ser Phe Lys Asn Leu Ser Gln Leu Ala Ser Ile Asn Tyr Asp Leu Val Leu Lys Asn Ala Lys Glu Ser Val Glu Ala Ser Asn Gln Ser Ala Pro Arg Pro Glu Thr lO GAAATTACAA GATTTTTTCA AATAAATCTA AAAAAATCTT GAATT 3842 (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1168 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide 2 O (vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ser Thr Thr Glu Pro Ser Pro Glu Asn Asp Arg Asp Pro Gln Pro Thr Thr Ile Ser Thr Pro Thr Ser Thr Asn Ala Lys Leu Leu Lys Lys Ile Pro Ala Ile Pro Phe Arg His Ser Asp Lys Glu Gly Glu Asp Glu Gln Ala Lys Thr Asp Glu Val Thr Thr Glu Leu Ala Gly Glu Gly Pro Met Ser His Asp Ser Pro Glu Ile Leu Ala Pro Ser Ser Leu Gly Leu Asn His Ile Arg Thr Lys Ser Ser Pro Ala Pro Ser Pro Leu Arg Phe Ser Ser Ala Thr Pro Leu Ile Ser Pro Gly Gln Asp Asp Lys Asp Val Ala Lys Glu Lys Pro Arg Val Gly Val Val Asp Ala Arg Ala Asp Ala Arg Ala Arg Trp Pro Ile Pro Pro His Gln Pro Asp Gln Gly Lys Lys Val Gln Trp Ser Gln Ser Lys Ser Gln Arg Val Pro Ala Asn Ser Asn Pro Gly Val Glu Ser Thr His Val Gly Leu Ala Lys Glu Thr Gln Ser Pro Arg Phe Gln Ala Ile Leu Arg Val Thr Ser Gly Arg Lys Lys Lys Ala His Asp Ile Lys Ser Phe Ser His Glu Leu Asn Ser Lys Gly Val Arg Pro Phe Pro Val Trp Arg Ser Arg Ala Val Gly His Met Glu Glu Ile Met Ala Ala Ile Arg Thr Lys Phe Asp Lys Gln Lys Glu Asp Val Asp Ala Asp Leu Gly Val Phe Ala Gly Tyr Leu Val Thr Thr Leu Glu Ser Thr Pro Glu Ser Asn Lys Glu Leu Arg Val Gly Leu Glu Asp Leu Leu Val Glu Ala Arg Gln Cys Ala Thr Met Pro Ala Ser Glu Phe Trp Leu Lys Cys Glu Gly Ile Val Gln Lys Leu Asp Asp Lys Arg Gln Glu Leu Pro Met Gly Gly Leu Lys Gln Ala His Asn Arg Leu Leu Phe Ile Leu Thr Arg Cys Asn Arg Leu Val Gln Phe Arg Lys Glu Ser Gly Tyr Val Glu Glu His Ile Leu Gly Met His Gln Leu Ser Asp Leu Gly Val Tyr Pro Glu Gln Met Val Glu Ile Ser Arg Gln Gln Asp Leu Leu Arg Glu Lys Glu Ile Gln Lys Ile Asn Glu Lys Gln Asn Leu Ala Gly Lys Gln Asp Asp Gln Asn Ser Asn Ser Gly Ala Asp Gly Val Glu Val Asn Thr Ala Arg Ser Thr Asp Ser Thr Ser Ser Asn Phe Arg Met Ser Ser Trp Lys Lys Leu Pro Ser Ala Ala Glu Lys Asn Arg Ser Leu Asn Asn Thr Pro Lys Ala Lys Gly Glu Ser Lys Ile Gln Pro Lys Val Tyr Gly Asp Glu Asn Ala Glu Asn Leu His Ser Pro Ser Gly Gln Pro Ala Ser Ala Asp Arg Ser Ala Leu Trp Gly Phe Trp Ala Asp His Gln Cys Val Thr Tyr Asp Asn Ser Met Ile Cys Arg Ile Cys Glu Val Glu Ile Pro Val Val His Val Glu Glu His Ser Arg Ile Cys Thr Ile Ala Asp Arg Cys Asp Leu Lys Gly Ile Asn Val Asn Leu Arg Leu Glu Arg Val Ala Glu Ser Leu Glu Lys Ile Leu Glu Ser Trp Thr Pro Lys Ser Ser Val Thr Pro Arg Ala Val Ala Asp Ser Ala Arg Leu Ser Asn Ser Ser Arg Gln Glu Asp Leu Asp Glu Ile Ser Gln Arg Cys Ser Asp Asp Met Leu Asp Cys Val Pro Arg Ser Gln Asn Thr Phe Ser Leu Asp Glu Leu Asn Ile Leu Asn Glu Met Ser Met Thr Asn Gly Thr Lys Asp Ser Ser Ala Gly Ser Leu Thr Pro Pro Ser Pro Ala Thr Pro Arg Asn Ser Gln Val Asp Leu Leu Leu Ser Gly Arg Lys Thr Ile Ser Glu Leu Glu Asn Tyr Gln Gln Ile Asn Lys Leu Leu Asp Ile Ala Arg Ser Val Ala Asn Val Asn Val Cys Gly Tyr Ser Ser Leu Asp Phe Met Ile Glu Gln Leu Asp Glu Leu Lys Tyr Val Ile Gln Asp Arg Lys Ala Asp Ala Leu Val Val 4 0 Glu Thr Phe Gly Arg Arg Ile Glu Lys Leu Leu Gln Glu Lys Tyr Ile Glu Leu Cys Gly Leu Ile Asp Asp Glu Lys Val Asp Ser Ser Asn Ala Met Pro Asp Glu Glu Ser Ser Ala Asp Glu Asp Thr Val Arg Ser Leu Arg Ala Ser Pro Leu Asn Pro Arg Ala Lys Asp Arg Thr Ser Ile Glu Asp Phe Glu Ile Ile Lys Pro Ile Ser Arg Gly Ala Phe Gly Arg Val Phe Leu Ala Lys Lys Arg Ala Thr Gly Asp Leu Phe Ala Ile Lys Val Leu Lys Lys Ala Asp Met Ile Arg Lys Asn Ala Val Glu Ser Ile Leu Ala Glu Arg Asn Ile Leu Ile Ser Val Arg Asn Pro Phe Val Val Arg Phe Phe Tyr Ser Phe Thr Cys Arg Glu Asn Leu Tyr Leu Val Met Glu Tyr Leu Asn Gly Gly Asp Leu Phe Ser Leu Leu Arg Asn Leu Gly Cys Leu Asp Glu Asp Met Ala Arg Ile Tyr Ile Ala Glu Val Val Leu Ala Leu Glu Tyr Leu His Ser Val Asn Ile Ile His Arg Asp Leu Lys Pro Asp Asn Leu Leu Ile Asn Gln Asp Gly His Ile Lys Leu Thr Asp Phe Gly Leu Ser Lys Val Gly Leu Ile Asn Ser Thr Asp Asp Leu Ser Gly Glu Ser Ser Leu Gly Asn Ser Gly Phe Phe Ala Glu Asp Gly Ser Lys Ala Gln His Ser Gln Gly Lys Asp Ser Arg Lys Lys His Ala Val Val Gly Thr Pro Asp Tyr Leu Ala Pro Glu Ile Leu Leu Gly Met Gly His Gly Lys Thr Ala Asp Trp Trp Ser Val Gly Val Ile Leu Phe Glu Val Leu Val Gly Ile Pro Pro Phe Asn Ala Glu Thr Pro Gln Gln Ile Phe Glu Asn Ile Ile Asn Arg Asp Ile Pro Trp Pro Asn Val Pro Glu Glu Ile Ser Tyr Glu Ala His Asp Leu Ile Asn Lys Leu Leu Thr Glu Asn 4 0 Pro Val Gln Arg Leu Gly Ala Thr Gly Ala Gly Glu Val Lys Gln His His Phe Phe Lys Asp Ile Asn Trp Asp Thr Leu Ala Arg Gln Lys Ala Met Phe Val Pro Ser Ala Glu Pro Gln Asp Thr Ser Tyr Phe Met Ser Arg Tyr Ile Trp Asn Pro Glu Asp Glu Asn Val His Gly Gly Ser Asp Phe Asp Asp Leu Thr Asp Thr Cys Ser Ser Ser Ser Phe Asn Thr Gln Glu Glu Asp Gly Asp Glu Cys Gly Ser Leu Ala Glu Phe Gly Asn Gly Pro Asn Leu Ala Val Lys Tyr Ser Phe Ser Asn Phe Ser Phe Lys Asn Leu Ser Gln Leu Ala Ser Ile Asn Tyr Asp Leu Val Leu Lys Asn Ala Lys Glu Ser Val Glu Ala Ser Asn Gln Ser Ala Pro Arg Pro Glu Thr (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1270 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
(2) INFORMATION FOR SEQID
N0:4:
(i) SEQUENCE ARACTERISTIC S:
CH
(A) : pairs base (B) nucleic acid TYPE:
(C) single STRANDEDNESS:
(D) linear TOPOLOGY:
(ii)MOLECULE cDNA
TYPE:
(vi)ORIGINAL (A) Arabidopsis thaliana SOURCE: ORGANISM:
(xi)SEQUENCE EQ ID
DESCRIPTION: N0:4:
S
ACG
Met Ser Thr Glu ProSerPro GluAsnAsp ArgAspPro GlnPro Thr TCT
2 Thr Thr Ile Thr ProThrSer ThrAsnAla LysLeuLeu LysLys 0 Ser ATT
Ile Pro Ala Pro PheArgHis SerAspLys GluGlyGlu AspGlu Ile ACG
Gln Ala Lys Asp GluValThr ThrGluLeu AlaGlyGlu GlyPro Thr GAT
Met Ser His Ser ProGluIle LeuAlaPro SerSerLeu GlyLeu Asp AGA
Asn His Ile Thr LysSerSer ProAlaPro SerProLeu ArgPhe Arg ACG
4 Ser Ser Ala Pro LeuIleSer ProGlyGln AspAspLys AspVal 0 Thr AAA
Ala Lys Glu Pro ArgValGly ValValAsp AlaArgAla AspAla Lys TGG
Arg Ala Arg Pro IleProPro HisGlnPro AspGlnGly LysLys Trp Val Gln Trp Ser Gln Ser Lys Ser Gln Arg Val Pro Ala Asn Ser Asn Pro Gly Val Glu Ser Thr His Val Gly Leu Ala Lys Glu Thr Gln Ser Pro Arg Phe Gln Ala Ile Leu Arg Val Thr Ser Gly Arg Lys Lys Lys Ala His Asp Ile Lys Ser Phe Ser His Glu Leu Asn Ser Lys Gly Val Arg Pro Phe Pro Val Trp Arg Ser Arg Ala Val Gly His Met Glu Glu Ile Met Ala Ala Ile Arg Thr Lys Phe Asp Lys Gln Lys Glu Asp Val Asp Ala Asp Leu Gly Val Phe Ala Gly Tyr Leu Val Thr Thr Leu Glu Ser Thr Pro Glu Ser Asn Lys Glu Leu Arg Val Gly Leu Glu Asp Leu Leu Val Glu Ala Arg Gln Cys Ala Thr Met Pro Ala Ser Glu Phe Trp Leu Lys Cys Glu Gly Ile Val Gln Lys Leu Asp Asp Lys Arg Gln Glu Leu Pro Met Gly Gly Leu Lys Gln Ala His Asn Arg Leu Leu Phe Ile Leu Thr Arg Cys Asn Arg Leu Val Gln Phe Arg Lys Glu Ser Gly Tyr Val Glu Glu His Ile Leu Gly Met His Gln Leu Ser Asp Leu Gly Val Tyr Pro Glu Gln Met Val Glu Ile Ser Arg Gln Gln Asp Leu Leu Arg Glu Lys Glu Ile Gln Lys Ile Asn Glu Lys Gln Asn Leu Ala Gly Ile Gln Asp Asp Gln Asn Ser Asn Ser Gly Ala Asp Gly Val Glu Val Asn Thr Ala Arg Ser Thr Asp Ser Thr Ser Ser Asn Phe Arg Met Ser Ser Trp Lys Lys Leu Pro Ser Ala Ala Glu Lys Asn Arg Ser Leu Asn Asn Thr Pro Lys Ala Lys Gly Glu Ser Lys Ile Gln Pro Lys Val Tyr Gly Asp Glu Asn Ala Glu Asn Leu His Ser Pro Ser Gly Gln Pro Ala Ser Ala Asp Arg Ser Ala Leu Trp Gly Phe Trp Ala Asp His Gln Cys Val Thr Tyr Asp Asn Ser Met Ile Cys Arg Ile Cys Glu Val Glu Ile Pro Val Val His Val Glu Glu His Ser Arg Ile Cys Thr Ile Ala Asp Arg Cys Asp Leu Lys Gly Ile Asn Val Asn Leu Arg Leu Glu Arg Val Ala Glu Ser Leu Glu Lys Ile Leu Glu Ser Trp Thr Pro Lys Ser Ser Val Thr Pro Arg Ala Val Ala Asp Ser Ala Arg Leu Ser Asn Ser Ser Arg Gln Glu Asp Leu Asp Glu Ile Ser Gln Arg Cys Ser Asp Asp Met Leu Asp Cys Val Pro Arg Ser Gln Asn Thr Phe Ser Leu Asp Glu Leu Asn Ile Leu Asn Glu Met Ser Met Thr Asn Gly Thr Lys Asp Ser Ser Ala Gly Ser Leu Thr Pro Pro Ser Pro Ala Thr Pro Arg Asn Ser Gln Val Asp Leu Leu Leu Ser Gly Arg Lys Thr Ile Ser Glu Leu Glu Asn Tyr Gln Gln Ile Asn Lys Leu Leu Asp Ile Ala Arg Ser Val Ala Asn Val Asn Val Cys Gly Tyr Ser Ser Leu Asp Phe Met Ile Glu Gln Leu Asp Glu Leu Lys Tyr Val Ile Gln Asp Arg Lys Ala Asp Ala Leu Val Val Glu Thr Phe Gly Arg Arg Ile Glu Lys Leu Leu Gln Glu Lys Tyr Ile Glu Leu Cys Gly Leu Ile Asp Asp Glu Lys Val Asp Ser Ser Asn Ala Met Pro Asp Glu Glu Ser Ser Ala Asp Glu Asp Thr Val Arg Ser Leu Arg Ala Ser Pro Leu Asn Pro Arg Ala Lys Asp Arg Thr Ser Ile Glu Asp Phe Glu Ile Ile Lys Pro Ile Ser Arg Gly Ala Phe Gly Arg Val Phe Leu Ala Lys Lys Arg Ala Thr Gly Asp Leu Phe Ala Ile Lys Val Leu Lys Lys Ala Asp Met Ile Arg Lys Asn Ala Val Glu Ser Ile Leu Ala Glu Arg Asn Ile Leu Ile Ser Val Arg Asn Pro Phe Val Val Arg Phe Phe Tyr Ser Phe Thr Cys Arg Glu Asn Leu Tyr Leu Val Met Glu Tyr Leu Asn Gly Gly Asp Leu Phe Ser Leu Leu Arg Asn Leu Gly Cys Leu Asp Glu Asp Met Ala Arg Ile Tyr Ile Ala Glu Val Val Leu Ala Leu Glu Tyr Leu His Ser Val Asn Ile Ile His Arg Asp Leu Lys Pro Asp Asn Leu Leu Ile Asn Gln Asp Gly His Ile Lys Leu Thr Asp Phe Gly Leu Ser Lys Val Gly Leu Ile Asn Ser Thr Asp Asp Leu Ser Gly Glu Ser Ser Leu Gly Asn Ser Gly Phe Phe Ala Glu Asp Gly Ser Lys Ala Gln His Ser Gln Gly Lys Asp Ser Arg Lys Lys His Ala Val Val Gly Thr Pro Asp Tyr Leu Ala Pro Glu Ile Leu Leu Gly Met Gly His Gly Lys Thr Ala Asp Trp Trp Ser Val Gly Val Ile Leu Phe Glu Val Leu Val Gly Ile Pro Pro Phe Asn Ala Glu Thr Pro Gln Gln Ile Phe Glu Asn Ile Ile Asn Arg Asp Ile Pro Trp Pro Asn Val Pro Glu Glu Ile Ser Tyr Glu Ala His Asp Leu Ile Asn Lys Leu Leu Thr Glu Asn Pro Val Gln Arg Leu Gly Ala Thr Gly Ala Gly Glu Val Lys Gln His His Phe Phe Lys Asp Ile Asn Trp Asp Thr Leu Ala Arg Gln Lys Ala Met Phe Val Pro Ser Ala Glu Pro Gln Asp Thr Ser Tyr Phe Met Ser Arg Tyr Ile Trp Asn Pro Glu Asp Glu Asn Val His Gly Gly Ser Asp Phe Asp Asp Leu Thr Asp Thr Cys Ser Ser Ser Ser Phe Asn Thr Gln Glu Glu Asp Gly Asp Glu Cys Gly Ser Leu Ala Glu Phe Gly Asn Gly Pro Asn Leu Ala Val Lys Tyr Ser Phe Ser Asn Phe Ser Phe Lys Asn Leu Ser Gln Leu Ala Ser Ile Asn Tyr Asp Leu Val Leu Lys Asn Ala Lys Glu Ser Val Glu Ala Ser Asn Gln Ser Ala Pro Arg Pro Glu Thr
The IRE gene regulating the root-hair growth in Arabidopsis BACKGROUND OF THE INVENTION
Field of the Invention The present invention relates to a gene coding for a protein that is involved in regulation of tip growth in plants, and also to the promoter of the gene.
Prior Art Unlike other cells, it is known that root hairs of plants elongate in a particular manner called tip growth. Pollen tubes of higher plants also perform tip growth. Tip growth is also observed in those eukaryotic cells that hold cell walls in ferns, mosses, algae, fungi and so forth. A number of mutants that show abnormalities in the number or shape of root hairs have been isolated from Arabidopsis. hy5, phyB, rhd2 and tipl are known as mutants with abnormal root-hair lengths (Genes &
Development, Vol. 11, p. 2983-2995 (1997); Plant Cell, Vol. 5, p.
147-157 (1993); Plant Cell, Vol. 2, p. 235-243 (1990); Plant Physiology, Vol. 103, p. 979-985 (1993)). These mutants except tipl show various phenotypes in addition to root-hair elongation, suggesting that roles of these genes are not restricted just to root hairs or to tip growth. It is therefore unclear how these genes are involved in root-hair elongation. Since the tipl mutant shows abnormalities in elongation both of root hairs and pollen tubes, tipl is considered as a tip growth-impaired mutant.
The molecular function of this gene product has not yet been revealed. As described above, those genes that specifically function in regulation of the root-hair elongation or tip growth have remained to be seen.
SUMMARY OF THE INVENTION
The present invention aims to isolate a novel gene regulating tip growth such as root-hair elongation and to use the gene for providing plants that show altered tip growth rates.
The present inventors have made great efforts to solve the above mentioned problems and isolated a short root-hair mutant of Arabidopsis. They have cloned the gene responsible for the phenotype. Furthermore, the cloned gene has been introduced into plants and the effect of the gene has been confirmed. Thus, the present invention has been completed.
The first aspect of the present invention relates to a gene coding for a protein (a) or (b) mentioned below:
(a) a protein represented by the amino acid sequence as set forth in SEQ ID NO.: 2; or (b) a protein represented by an amino acid sequence having one or more amino acids deleted from, substituted in, modified in or added to the amino acid sequence as set forth in SEQ ID NO.: 2, and having a tip growth regulating activity.
The second aspect of the present invention relates to a DNA (a) or (b) as mentioned below:
(a) a DNA represented by the nucleotide sequence as set forth in SEQ ID NO.: 3; or (b) a DNA represented by a nucleotide sequence having one or more nucleotides deleted from, substituted in or added to the nucleotide sequence as set forth in SEQ ID NO.: 3, and having a function as a promoter.
This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No.82402/1999, which is a priority document of the present application.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows root hairs of Arabidopsis: wild type (A), the ire mutant (B), and an ire mutant in which the wild-type IRE
gene is introduced (C).
Figure 2 shows elongation properties of root hairs of wild type (A) and the ire mutant (B).
Figure 3 shows a physical map of the genome around the IRE
locus.
Figure 4 shows the exon regions of the IRE gene.
Figure 5 shows expression patterns of the GUS gene under the control of the IRE promoter.
Figure 6 shows the nucleotide sequence of the IRE promoter.
DESCRIPTION OF THE INVENTION
The present invention is hereinafter described in detail.
(1) First Invention (Structural Gene) The first invention relates to a gene coding for a protein (a) or (b) as mentioned below and encompasses the protein encoded by the gene, mutant genes, organisms carrying these genes:
(a) a protein represented by the amino acid sequence as set forth in SEQ ID NO.: 2; or (b) a protein represented by an amino acid sequence having one or more amino acids deleted from, substituted for, modified in or added to the amino acid sequence as set forth in SEQ ID NO.:
2, and having a tip growth regulating activity.
The term "one or more" used herein means the number of amino acids which can be deleted or otherwise altered according to the technology conventionally used at the time of filing the present application such as the site-directed mutagnenesis.
Chemical modification of amino acid residues in a protein is commonly carried out in the art: see, for example, Hirs, C.H.W. and Timasheff, S.N., eds, (1977) Methods in Enzymology, Vol. 47, p. 407-498, Academic Press, New York.
The gene coding for the amino acid sequence as set forth in SET ID No.: 2 modifies the elongation of root hairs. This is shown by the fact that the mutant impaired this gene displays shorter root hairs. This effect is restricted in root hairs because the mutant shows the normal elongation of root hair-bearing cells. Thus we have named this gene IRE (Incomplete Root-hair Elongation). No gene products that have a tip growth specific function have not been found.
The IRE gene can be cloned according to the method of Japanese Patent Application Laying Open No. 10-4978 as described below.
First, genomic DNA of Arabidopsis is fragmented with an appropriate restriction enzyme and the DNA fragments are ligated into an appropriate vector for a genomic DNA library. This library is amplified by transforming into a host microorganism.
The isolation of DNA may be carried out in a conventional method using cesium chloride and ethidum bromide. The restriction enzyme is not particularly limited and may include, for example, Sau3AI. The vector is also not particularly limited and may include, for example, a DASH II. The host microorganism may be determined depending upon the vector used. For example, 1 Dash II requires Escherichia coli XL-1 Blue MRA (P2).
Then, the above mentioned genomic DNA library is screened for the genomic region of the IRE locus. A DNA fragment that is adjacent to the T-DNA insertion in the ire mutant is used for the probe. Since the ire gene is disrupted by the insertion in the genome of the ire mutant, the adjacent region includes a part of the IRE gene.
Positive clones selected by the above mentioned probe are purified and the insert genomic DNA fragments are separated. A
genomic fragment is used for a probe to screen a cDNA library.
The cDNA library may be prepared according to a conventional procedure. That is, the total RNA is isolated from plant tissues and polyA+ RNA is purified using oligo(dT) resins. cDNA
is synthesized from the template polyA+ using a reverse transcriptase. The synthesized cDNA is ligated into an appropriate vector for a cDNA library. The library is amplified by transforming into a host microorganism. Positive clones in the cDNA library contain the IRE gene.
Capabilities of the hybridization between one DNA sequence and others under stringent conditions may be determined, for example, by the following method. First one DNA fragment is blotted and immobilized onto a nylon membrane. Then, this membrane is prehybridized by the solution containing 6X SSC, O.O1M EDTA, 5X Denhardt's solution, 0.5% SDS, 100 ~g/ml denatured salmon DNA at 65~C for one hour or more. A hybridized solution is prepared by adding labeled DNA fragments or labeled RNA transcripts of a DNA template in the prehybidization solution. The prehybridized nylon membrane is hybridized in the hybridization solution at 65 ~C for 3 to 16 hours. Thereafter, it is washed in a solution containing 2X SSC and 0.1% SDS at 65 'C for 30 minutes. Then, it is further washed in 2X SSC, 0.1%
SDS solution at room temperature for 15 minutes. Further, it washed in 0.1X SSC, 0.1% SDS solution at 65 ~C for 30 minutes.
Then, the signal on the membrane is detected by a suitable method depending the labeling reagent.
In order to express genes of the first invention (the IRE
gene and modified genes) in a host, the genes may be inserted into an appropriate expression vector and the recombinant vector may be introduced into the host. Hosts are not particularly limited. Plants are preferable but bacteria, yeast and animals may be used. Species of plants used as the host are not particularly limited but those species of soybean, rape, and cotton may be preferable. Any expression vector may be used as far as it contains both a promoter for the expression and a marker gene that enable the handling the host. For example, plant tissues preferably require vectors that carry the cauliflower mosaic virus 35S promoter because of the wide application of species. pBI121 vector (Clontech) is an example.
Methods to introduce a vector into the host are not particularly limited and may be done depending upon the host organism. An Agrobacterium-mediated method is preferable for introducing into plant tissues although those methods using electroporation, particle gun may also be applicable.
Since the gene of the first invention has a function to modify tip growth such as elongation or root hairs, the following utilities may be considered.
I) Applications using the tip-growth modifying activity in root hairs.
Introducing the gene of the first invention into a plant and expressing it therein can let the plant have root hairs with different lengths which can provide drought resistance of the plant. Bacteria in Rhizobium are parasites of leguminous plants and they enable the plants nitrogen fixation. Those bacteria are shown to infect only elongating root hairs. Thus plants that have altered root-hair growth by using the gene can be expected to improve the infectious efficiency of the bacteria.
II) Applications using the tip-growth modifying activity in other than root hairs.
In higher plants, pollen tubes emerge from pollen grains (male gametophytes) and elongate to female gametophytes while the fertilization. It is expected that the manipulation of the gene expression in pollen tubes may alter the fertilization timing and efficiency. Namely, plants (male gametophytes) with either promoting or retarding fertilization ability may be created. Cotton fibers, which are the raw material of the common fiber, cotton yarn, elongate with a kind of tip growth in cotton fruits. Thus it is also expected to improve the quality of cotton fiber.
(2) Second Invention (Promoter) The second invention relates to a DNA (a) or (b) as mentioned below and encompasses promoters, expression vectors, organisms carrying these DNA sequences:
(a) a DNA represented by the base sequence as set forth in SEQ
ID NO.: 3; or (b) a DNA represented by a base sequence having one or more bases deleted from, substituted for or added to the base sequence as set forth in SEQ ID NO.: 3, and having a function as a promoter.
The term "one or more" used herein means the number of nucleotide sequences which can be deleted or otherwise altered according to the technology conventionally used at the time of filing the present application such as the site-directed mutagnenesis.
Since the DNA of the second invention exists in the upstream region of the gene of the first invention, it may be cloned according to the method of Japanese Patent Application Laying Open No. 10-4978 as in the case of the gene of the first invention.
The DNA of second invention function as a promoter in such tip-growing tissues as root hairs and pollen grains (pollen tubes). Recombinant genes that have any gene under this DNA
sequence can be expressed specifically in root hairs and/or pollen grains. Thus useful biological functions depending upon root hairs and/or pollen grains can be introduces.
EXAMPLES
The present inventions are illustrated in more detailed by way of examples which in no way limit the scope of the present invention.
Example 1: Production of transgenic plants for the isolation of IRE
Transgenic plants for the isolation of IRE were created according to the method of Example 1 of Japanese Patent Application Laying Open No. 10-4978.
Seeds of Arabidopsis (variety: Wassilewskija [WS]) were sterilized and sown on an agar medium. These seeds were incubated at 4 'C in darkness for two to four days. This cold treatment prompted breakage of seed dormancy and made the germination uniform and enhanced the flowering timing.
Thereafter, seeds were germinated and grown under continuous light at 22 ~C. The seed sterilization was carried out by using a sterilization solution containing 10 ~ of Hitar (Kao) and 0.02 Triton-X 100. Seeds were mixed and vortexed in the sterilization solution and allowed to stand at room temperature for three to five minutes, and washed five times with sterilized water. The agar medium contained 1/2 x Arabidopsis nutrient salt solution and 1.5 ~ agar (Nakarai, special grade). lx Arabidopsis nutrient salts solution was prepared by following:
985 ml distilled water or deionized water, 5 ml of 1M KN03, 2 ml of 1M MgS04, 2 ml of 1M Ca(N03)2, 2.5 ml of 20mM Fe-EDTA, 1 ml of trace element solution, 2.5 ml of K-P04 buffer (pH 5.5)]
(Hideaki Shiroishi et al., (1991), Gendai-Kagaku vo120, Plant Biotechnology II, p. 38). The agar medium was autoclaved and poured into petri dishes. This concentration of agar prevented Arabidopsis roots from penetrating in the agar medium. It made he observation of the root morphology easier. The light source was built with two commonly used 40 W fluorescent lamps and one Homolux fluorescent lamp (National). Plants were grown under this light source at an about 30cm distance. The intensity of the light was about 3000 lux.
Forty individuals of Arabidopsis at three weeks after the seed sowing were inoculated with Agrobacterium. The inoculation was carried out according to the modified in planta method (Plant Journal, Vol. 5, p. 551-558 (1994)). In this method, floral stems of plants were wounded in order to promote the infection. Agrobacterium tumefaciens strain C58C1rif (Nucleic Acids Research, Vol. 13, p. 6981-6998 (1985)) was obtained from Velten, J. et al. and used. This strain carries pGV3850 HPT as the intermediate Ti plasmid and between the right- and left-borders, there is a hygromycin phosphotransferase gene driven by the cauliflower mosaic virus 35S promoter on this vector that acts as the selection marker for plants.
After the inoculation of Agrobacterium, those plants were transplanted on soil of the 1:1 mixture of vermiculite and perlite. After 1.5 to 2 months of transplantation, seeds (T1 seeds) were harvested. Those seeds were sterilized in a similar manner to the above mentioned, and sown on a hygromycin containing medium (1X Ganborg B5 mixed salts for culture medium, 1~ sucrose, 0.8~ agar, 10 mg/1 hygromycin B). Individuals showing hygromycin resistance were selected and transplanted on soil. The soil was mentioned above. After 1.5 to 2 months from the transplantation, self-pollinated seeds (T2 seeds) were harvested.
Example 2: Screening for root-morphology mutants The screening was carried out according to Example 2 of Japanese Patent Application Laying Open No. 10-4978. The T2 seeds obtained in Example 1 were sterilized, sown on an agar medium and grown. The sterilization and the agar medium were as above mentioned in Example 1. To facilitate morphological observation of roots, the agar medium was placed in a transparent plastic Petri dish (Eiken Kizai Kabushiki Kaisha, No.
2 square Petri dish, 14 cm x 10 cm).
The morphology of roots was observed through transmitted light by means of a stereoscopic microscope OLYMPUS SZH-IDDL.
Morphologies of roots in about 300 lines of transgenic Arabidopsis were examined and ire was isolated as an abnormal root-hair length mutant. The length of root hairs was smaller in the ire mutant (Fig. 1B) than in wild type (Fig. 1A).
However, the density of root hairs and the distance between root hairs were identical, indicating that this mutation specifically reduces the length of root hairs. A detailed analysis revealed that the elongation of root hairs ceased earlier in the mutant (Fig. 2B) than in wild type (Fig. 2A). This cessation in the mutant resulted in 60 ~ of root-hair length of wild type.
Example 3: Genetic studies of the ire mutant According to the method in Japanese Patent Application Laying Open No. 10-4978, the ire mutant and wild type were cross-pollinated. The phenotype (length of root hairs) of wild type, ire and their F1 progeny was examined (Table 1).
Length of root hairs t i ne (11m + SE) Wild type 396 ~ 17 Ire 232 ~ 25 F1 progeny and wild type 452 ~ 32 As shown in Table 1, the Fl progeny showed root hairs of a normal length like wild type, indicating that the ire mutation is recessive. Among 711 individuals of selfed progeny of the F1 plants (F2 progeny), 165 individuals (23~) showed the short root-hair phenotype, suggesting that this phenotype is caused by a single recessive locus.
Example 4: Preparatory studies for the cloning of the IRE gene The ire mutant was isolated as a T-DNA insertion line carrying the selection marker. In order to examine if the inserted T-DNA disrupted the IRE gene or not, selfed F3 seeds of 213 mutants in the F2 progeny were subjected to the linkage analysis between the phenotype and the selection marker gene (hygromycin resistance gene). All F3 progeny of every mutant showed the hygromycin resistance on the selection medium (1x Gamborg B5 mixed salts for medium, 1~ sucrose, 0.8~ agar, 10 mg/1 hygromycin B). Namely, each mutant in the F2 progeny carried the T-DNA homozygously. Thus it was suggested that the inserted T-DNA is closely linked with the IRE locus and probably disrupts the gene. Therefore, genomic regions flanking the T-DNA insertion should include the IRE gene.
Example 5: Cloning of the IRE gene A genomic DNA fragment adjacent to the inserted T-DNA was amplified and cloned by the TAIL-PCR method using the genome DNA of the ire mutant as a template (Plant Journal, Vol.8, p.457-463 (1995)). The sequences used in the PCR are below: a first specific primer 5'-CACATCATCTCATTGATGCTTGGT-3' (24mer); a second specific primer 5'-CATAGATGCACTCGAAATCAGCC-3' (23mer); a third specific primer 5'-GTGTTATTAAGTTGTCTAAGCGTC-3' (24mer); and arbitrary primers 5'-(A/T)GTG(A/T/G/C)AG(A/T)A(A/T/G/C)CA(A/T/G/C)AGA-3' (l6mer). A 0.7 kb PCR fragment was amplified using this set of primers. The template genomic DNA was extracted according to Example 4 of Japanese Patent Application Laying Open No. 10-4978.
An Arabidopsis genomic library was screened using the amplified 0.7 kb PCR fragment as a probe. Those genomic clones were isolated that covered an about 10 kb region in which a sequence hybridized by the PCR fragment was contained. This genomic region was divided into several fragments and subcloned into pBluescript II KS+
(Stratagene) to determine the nucleotide sequence. The genomic region underlined in Fig. 3 was used as a probe to screen a cDNA
library. A cDNA clone was isolated in this screening. The screening of the genomic and cDNA libraries as well as the method of determination of nucleotide sequences was as described in Example 7 of Japanese Patent Application Laying Open No. 10-4978.
The isolated cDNA was only about 1 kb in length. In order to isolate a full length cDNA, a PCR method was tried. cDNA that had been synthesized for the cDNA library was used as a template and several primer sets designed based on the genome sequence were used for the PCR. Two PCR fragments were amplified when using the following sets of primers and the PCR fragments and the isolated cDNA were joined into a full length cDNA. One of the PCR fragments, which is composed of 2.2 kb at the 5' region of the full length cDNA, was amplified using following primers: f2384 f2384:
CAACCGCTTCTCTGTAATC and r4950: AGCCTTCCTATCCTGAATG. The other fragment, which is composed of 1.2 kb at the 3' region of the full length cDNA was amplified using following primers: f4897:
TCATGATTGAGCAGTTGGA and r6898: CCGAGCAAGTGTGTCC.
The nucleotide sequence of the full length cDNA was determined. The full length except for polyA sequence consists of 3842 by (SEQ ID NO.: 1) and the possible largest ORF (Open Reading Frame) consists of 1168 amino acid residues (SEQ ID NO.:
2). A stop codon locates in front of the start codon in frame of this ORF. The correspondence between the cDNA and the genome of IRE gene is shown in Fig. 4. The IRE gene consists of 17 exons and the T-DNA was inserted into the promoter region in the ire mutant. The wild-type genomic region as shown in Fig. 4 was introduced into the ire mutant. A vector was reconstructed from pBI121 (Clontech) by removing the b-Glucuronidase (hereinafter referred to GUS) gene and replaced with the genomic region. pBI
121 carries a gene for kanamycin resistance and transformed plants show the resistance against kanamycin. The construction of vector and gene transfer into plants were carried out according to the method of Example 8 of Japanese Patent Application Laying Open No. 10-4978; however, the media for selecting bacteria and plants were supplemented with 50 mg/1 kanamycin sulfate (Meiji Seika). As shown in Fig. 1C, the transformed plant showed longer root hairs than the untransformed plant (the ire mutant), indicating that this gene has an ability to increase the length of root hairs.
Example 6: Tissue-specific activity of the promoter region of the IRE gene The DNA fragment as set forth in Fig. 6 (SEQ ID NO.: 3) was obtained by digesting with restriction enzymes XhoI and BamHI. This fragment was ligated into SalI-BamHI sites of pBI101-2 vector (Clontech). This recombinant vector was used for the transformation into wild-type plants (using the method of Example 5 above). Restriction enzymes used were purchased from Takara Shuzo. The GUS gene was to be expressed under the control of the IRE gene promoter. The sequence as shown in Fig.
6 corresponds to the region spanning between XhoI and BamHI
sites in Fig. 4. The sequence of white letters in Fig. 6 corresponds to the first exon of the IRE gene where the start codon ATG is enclosed with the rectangle. The sequences with dotted underlines show exons of another gene adjacent to the IRE
gene. To observe the activity of the expressed GUS, the transformants were treated with an X-Gluc solution (5.7 mM X-Gluc (5-bromo-4-chloro-3-indolyl-~-glucuronide), 1.5 mM
K3Fe (CN) 6, 1 . 5 mM K4Fe (CN) 6, 0. 9~ Triton X-100) . Transformant plants were soaked in the X-Gluc solution, subjected to a vacuum treatment, and then incubated at 37 ~C overnight to develop the color. Reagents used were purchased from Wako Pure Chemical.
The GUS activities were substantially shown in only the root and pollen grains. As shown by arrows in Fig. 5B, the GUS activity was found in pollen grains in the anther. Pollen grains are germinated on a stigma and elongate pollen tubes. The GUS
activity was also found in elongating pollen tubes (Fig. 5C, arrow). The foregoing indicates that the promoter of IRE gene as set forth in SEQ ID NO.: 3 has activities in such tip-growing cells as root hairs and pollen tubes.
The present inventions) provides) a structural gene coding a protein that is involved in regulation of tip growth in plants, and its promoter. Various plants with different morphologies such as those showing prolonged root hairs, can be created by utilizing the structural gene of the present invention. Further, any gene may be expressed specifically in tissues that are tip-growing by utilizing the promoter of the present invention.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: BIOMOLECULAR ENGINEERING RESEARCH INSTITUTE
(ii) TITLE OF INVENTION: THE IRE GENE REGULATING THE ROOT-JAIR GROWTH IN
ARABIDOPSIS
(iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text) (vi) CURRENT APPLICATION DATA:
2 O (A) APPLICATION NUMBER: CA 2,301,257 (B) FILING DATE: 24-MAR-2000 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: JP 82402/1999 (B) FILING DATE: 25-MAR-1999 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SMART & BIGGAR
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 72813-119 3O (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-232-2486 (B) TELEFAX: (613)-232-8440 (2) INFORMATION ID N0:1:
FOR
SEQ
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 842base rs 3 pai (B) TYPE: leicacid nuc (C) STRANDEDNESS: double (D) TOPOLOGY:linear (ii)MOLECULE TYPE:cDNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM:Arabidopsisthaliana lO (ix)FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION:124..3627 (xi)SEQUENCE DESCRIPTI ON: ID
SEQ N0:1:
TTATG ATAAAATCAC AACAAACAAA
GTGTAAATAT ATAATTCTCT
GAAAA
GAA CCT
Met Ser Thr Thr ProSer GluAsnAsp ArgAspPro Gln Glu Pro O ACT TCC
Pro Thr Thr Ile Ser ProThr ThrAsnAla LysLeuLeu Lys Thr Ser CCT CAT
Lys Ile Pro Ala Ile PheArg SerAspLys GluGlyGlu Asp Pro His GAC ACT
Glu Gln Ala Lys Thr GluVal ThrGluLeu AlaGlyGlu Gly Asp Thr TCG ATT
Pro Met Ser His Asp ProGlu LeuAlaPro SerSerLeu Gly Ser Ile ACG TCT
Leu Asn His Ile Arg LysSer ProAlaPro SerProLeu Arg Thr Ser CCT TCT
Phe Ser Ser Ala Thr LeuIle ProGlyGln AspAspLys Asp Pro Ser CCG GGT
Val Ala Lys Glu Lys ArgVal ValValAsp AlaArgAla Asp Pro Gly Ala Arg Ala Arg Trp Pro Ile Pro Pro His Gln Pro Asp Gln Gly Lys Lys Val Gln Trp Ser Gln Ser Lys Ser Gln Arg Val Pro Ala Asn Ser Asn Pro Gly Val Glu Ser Thr His Val Gly Leu Ala Lys Glu Thr Gln Ser Pro Arg Phe Gln Ala Ile Leu Arg Val Thr Ser Gly Arg Lys Lys Lys Ala His Asp Ile Lys Ser Phe Ser His Glu Leu Asn Ser Lys Gly Val Arg Pro Phe Pro Val Trp Arg Ser Arg Ala Val Gly His Met Glu Glu Ile Met Ala Ala Ile Arg Thr Lys Phe Asp Lys Gln Lys Glu Asp 3~ Val Asp Ala Asp Leu Gly Val Phe Ala Gly Tyr Leu Val Thr Thr Leu Glu Ser Thr Pro Glu Ser Asn Lys Glu Leu Arg Val Gly Leu Glu Asp Leu Leu Val Glu Ala Arg Gln Cys Ala Thr Met Pro Ala Ser Glu Phe Trp Leu Lys Cys Glu Gly Ile Val Gln Lys Leu Asp Asp Lys Arg Gln Glu Leu Pro Met Gly Gly Leu Lys Gln Ala His Asn Arg Leu Leu Phe Ile Leu Thr Arg Cys Asn Arg Leu Val Gln Phe Arg Lys Glu Ser Gly Tyr Val Glu Glu His Ile Leu Gly Met His Gln Leu Ser Asp Leu Gly Val Tyr Pro Glu Gln Met Val Glu Ile Ser Arg Gln Gln Asp Leu Leu Arg Glu Lys Glu Ile Gln Lys Ile Asn Glu Lys Gln Asn Leu Ala Gly Lys Gln Asp Asp Gln Asn Ser Asn Ser Gly Ala Asp Gly Val Glu Val Asn Thr Ala Arg Ser Thr Asp Ser Thr Ser Ser Asn Phe Arg Met Ser Ser Trp Lys Lys Leu Pro Ser Ala Ala Glu Lys Asn Arg Ser Leu Asn Asn Thr Pro Lys Ala Lys Gly Glu Ser Lys Ile Gln Pro Lys Val Tyr Gly Asp Glu Asn Ala Glu Asn Leu His Ser Pro Ser Gly Gln Pro Ala Ser Ala Asp Arg Ser Ala Leu Trp Gly Phe Trp Ala Asp His Gln Cys Val Thr Tyr Asp Asn Ser Met Ile Cys Arg Ile Cys Glu Val Glu Ile Pro Val Val His Val Glu Glu His Ser Arg Ile Cys Thr Ile Ala Asp Arg Cys Asp Leu Lys Gly Ile Asn Val Asn Leu Arg Leu Glu Arg Val Ala Glu Ser Leu Glu Lys Ile Leu Glu Ser Trp Thr Pro Lys Ser Ser Val Thr Pro Arg Ala Val Ala Asp Ser Ala Arg Leu Ser Asn Ser Ser Arg Gln Glu Asp Leu Asp Glu Ile Ser Gln Arg Cys Ser Asp Asp Met Leu Asp Cys Val Pro Arg Ser Gln Asn Thr Phe Ser Leu Asp Glu Leu Asn Ile Leu Asn Glu Met Ser Met Thr Asn Gly Thr Lys Asp Ser Ser Ala Gly Ser Leu Thr Pro Pro Ser Pro Ala Thr Pro Arg Asn Ser Gln Val Asp Leu Leu Leu Ser Gly Arg Lys Thr Ile Ser Glu Leu Glu Asn Tyr Gln Gln Ile Asn Lys Leu Leu Asp Ile Ala Arg Ser Val Ala Asn Val Asn Val Cys Gly Tyr Ser Ser Leu Asp Phe Met Ile Glu Gln Leu Asp Glu Leu Lys Tyr Val Ile Gln Asp Arg Lys Ala Asp Ala Leu Val Val Glu Thr Phe Gly Arg Arg Ile Glu Lys Leu Leu Gln Glu Lys Tyr Ile Glu Leu Cys Gly Leu Ile Asp Asp Glu Lys Val Asp Ser Ser Asn Ala Met Pro Asp Glu Glu Ser Ser Ala Asp Glu Asp Thr Val Arg Ser Leu Arg Ala Ser Pro Leu Asn Pro Arg Ala Lys Asp Arg Thr Ser Ile Glu Asp Phe Glu Ile Ile Lys Pro Ile Ser Arg Gly Ala Phe Gly Arg Val Phe Leu Ala Lys Lys Arg Ala Thr Gly Asp Leu Phe Ala Ile Lys Val Leu Lys Lys Ala Asp Met Ile Arg Lys Asn Ala Val Glu Ser Ile Leu Ala Glu Arg Asn Ile Leu Ile Ser Val Arg Asn Pro Phe Val Val Arg Phe Phe Tyr Ser Phe Thr Cys Arg Glu Asn Leu Tyr Leu Val Met Glu Tyr Leu Asn Gly Gly Asp Leu Phe Ser Leu Leu Arg Asn Leu Gly Cys Leu Asp Glu Asp Met Ala Arg Ile Tyr Ile Ala Glu Val Val Leu Ala Leu Glu Tyr Leu His Ser Val Asn Ile Ile His Arg Asp Leu Lys Pro Asp Asn Leu Leu Ile Asn Gln Asp Gly His Ile Lys Leu Thr Asp Phe Gly Leu Ser Lys Val Gly Leu Ile Asn Ser Thr Asp Asp Leu Ser Gly Glu Ser Ser Leu Gly Asn Ser Gly Phe Phe Ala Glu Asp Gly Ser Lys Ala Gln His Ser Gln Gly Lys Asp Ser Arg Lys Lys His Ala Val Val Gly Thr Pro Asp Tyr Leu Ala Pro Glu Ile Leu Leu Gly Met Gly His Gly Lys Thr Ala Asp Trp Trp Ser Val Gly Val Ile Leu Phe Glu Val Leu Val Gly Ile Pro Pro Phe Asn Ala Glu Thr Pro Gln Gln Ile Phe Glu Asn Ile Ile Asn Arg Asp Ile Pro Trp Pro Asn Val Pro Glu Glu Ile Ser Tyr Glu Ala His Asp Leu Ile Asn Lys Leu Leu Thr Glu Asn Pro Val Gln Arg Leu Gly Ala Thr Gly Ala Gly Glu Val Lys Gln His His Phe Phe Lys Asp Ile Asn Trp Asp Thr Leu Ala Arg Gln Lys Ala Met Phe Val Pro Ser Ala Glu Pro Gln Asp Thr Ser Tyr Phe Met Ser Arg Tyr Ile Trp Asn Pro Glu Asp Glu Asn Val His Gly Gly Ser Asp Phe Asp Asp Leu Thr Asp Thr Cys Ser Ser Ser Ser Phe Asn Thr Gln Glu Glu Asp Gly Asp Glu Cys Gly Ser Leu Ala Glu Phe Gly Asn Gly Pro Asn Leu Ala Val Lys Tyr Ser Phe Ser Asn Phe Ser Phe Lys Asn Leu Ser Gln Leu Ala Ser Ile Asn Tyr Asp Leu Val Leu Lys Asn Ala Lys Glu Ser Val Glu Ala Ser Asn Gln Ser Ala Pro Arg Pro Glu Thr lO GAAATTACAA GATTTTTTCA AATAAATCTA AAAAAATCTT GAATT 3842 (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1168 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide 2 O (vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ser Thr Thr Glu Pro Ser Pro Glu Asn Asp Arg Asp Pro Gln Pro Thr Thr Ile Ser Thr Pro Thr Ser Thr Asn Ala Lys Leu Leu Lys Lys Ile Pro Ala Ile Pro Phe Arg His Ser Asp Lys Glu Gly Glu Asp Glu Gln Ala Lys Thr Asp Glu Val Thr Thr Glu Leu Ala Gly Glu Gly Pro Met Ser His Asp Ser Pro Glu Ile Leu Ala Pro Ser Ser Leu Gly Leu Asn His Ile Arg Thr Lys Ser Ser Pro Ala Pro Ser Pro Leu Arg Phe Ser Ser Ala Thr Pro Leu Ile Ser Pro Gly Gln Asp Asp Lys Asp Val Ala Lys Glu Lys Pro Arg Val Gly Val Val Asp Ala Arg Ala Asp Ala Arg Ala Arg Trp Pro Ile Pro Pro His Gln Pro Asp Gln Gly Lys Lys Val Gln Trp Ser Gln Ser Lys Ser Gln Arg Val Pro Ala Asn Ser Asn Pro Gly Val Glu Ser Thr His Val Gly Leu Ala Lys Glu Thr Gln Ser Pro Arg Phe Gln Ala Ile Leu Arg Val Thr Ser Gly Arg Lys Lys Lys Ala His Asp Ile Lys Ser Phe Ser His Glu Leu Asn Ser Lys Gly Val Arg Pro Phe Pro Val Trp Arg Ser Arg Ala Val Gly His Met Glu Glu Ile Met Ala Ala Ile Arg Thr Lys Phe Asp Lys Gln Lys Glu Asp Val Asp Ala Asp Leu Gly Val Phe Ala Gly Tyr Leu Val Thr Thr Leu Glu Ser Thr Pro Glu Ser Asn Lys Glu Leu Arg Val Gly Leu Glu Asp Leu Leu Val Glu Ala Arg Gln Cys Ala Thr Met Pro Ala Ser Glu Phe Trp Leu Lys Cys Glu Gly Ile Val Gln Lys Leu Asp Asp Lys Arg Gln Glu Leu Pro Met Gly Gly Leu Lys Gln Ala His Asn Arg Leu Leu Phe Ile Leu Thr Arg Cys Asn Arg Leu Val Gln Phe Arg Lys Glu Ser Gly Tyr Val Glu Glu His Ile Leu Gly Met His Gln Leu Ser Asp Leu Gly Val Tyr Pro Glu Gln Met Val Glu Ile Ser Arg Gln Gln Asp Leu Leu Arg Glu Lys Glu Ile Gln Lys Ile Asn Glu Lys Gln Asn Leu Ala Gly Lys Gln Asp Asp Gln Asn Ser Asn Ser Gly Ala Asp Gly Val Glu Val Asn Thr Ala Arg Ser Thr Asp Ser Thr Ser Ser Asn Phe Arg Met Ser Ser Trp Lys Lys Leu Pro Ser Ala Ala Glu Lys Asn Arg Ser Leu Asn Asn Thr Pro Lys Ala Lys Gly Glu Ser Lys Ile Gln Pro Lys Val Tyr Gly Asp Glu Asn Ala Glu Asn Leu His Ser Pro Ser Gly Gln Pro Ala Ser Ala Asp Arg Ser Ala Leu Trp Gly Phe Trp Ala Asp His Gln Cys Val Thr Tyr Asp Asn Ser Met Ile Cys Arg Ile Cys Glu Val Glu Ile Pro Val Val His Val Glu Glu His Ser Arg Ile Cys Thr Ile Ala Asp Arg Cys Asp Leu Lys Gly Ile Asn Val Asn Leu Arg Leu Glu Arg Val Ala Glu Ser Leu Glu Lys Ile Leu Glu Ser Trp Thr Pro Lys Ser Ser Val Thr Pro Arg Ala Val Ala Asp Ser Ala Arg Leu Ser Asn Ser Ser Arg Gln Glu Asp Leu Asp Glu Ile Ser Gln Arg Cys Ser Asp Asp Met Leu Asp Cys Val Pro Arg Ser Gln Asn Thr Phe Ser Leu Asp Glu Leu Asn Ile Leu Asn Glu Met Ser Met Thr Asn Gly Thr Lys Asp Ser Ser Ala Gly Ser Leu Thr Pro Pro Ser Pro Ala Thr Pro Arg Asn Ser Gln Val Asp Leu Leu Leu Ser Gly Arg Lys Thr Ile Ser Glu Leu Glu Asn Tyr Gln Gln Ile Asn Lys Leu Leu Asp Ile Ala Arg Ser Val Ala Asn Val Asn Val Cys Gly Tyr Ser Ser Leu Asp Phe Met Ile Glu Gln Leu Asp Glu Leu Lys Tyr Val Ile Gln Asp Arg Lys Ala Asp Ala Leu Val Val 4 0 Glu Thr Phe Gly Arg Arg Ile Glu Lys Leu Leu Gln Glu Lys Tyr Ile Glu Leu Cys Gly Leu Ile Asp Asp Glu Lys Val Asp Ser Ser Asn Ala Met Pro Asp Glu Glu Ser Ser Ala Asp Glu Asp Thr Val Arg Ser Leu Arg Ala Ser Pro Leu Asn Pro Arg Ala Lys Asp Arg Thr Ser Ile Glu Asp Phe Glu Ile Ile Lys Pro Ile Ser Arg Gly Ala Phe Gly Arg Val Phe Leu Ala Lys Lys Arg Ala Thr Gly Asp Leu Phe Ala Ile Lys Val Leu Lys Lys Ala Asp Met Ile Arg Lys Asn Ala Val Glu Ser Ile Leu Ala Glu Arg Asn Ile Leu Ile Ser Val Arg Asn Pro Phe Val Val Arg Phe Phe Tyr Ser Phe Thr Cys Arg Glu Asn Leu Tyr Leu Val Met Glu Tyr Leu Asn Gly Gly Asp Leu Phe Ser Leu Leu Arg Asn Leu Gly Cys Leu Asp Glu Asp Met Ala Arg Ile Tyr Ile Ala Glu Val Val Leu Ala Leu Glu Tyr Leu His Ser Val Asn Ile Ile His Arg Asp Leu Lys Pro Asp Asn Leu Leu Ile Asn Gln Asp Gly His Ile Lys Leu Thr Asp Phe Gly Leu Ser Lys Val Gly Leu Ile Asn Ser Thr Asp Asp Leu Ser Gly Glu Ser Ser Leu Gly Asn Ser Gly Phe Phe Ala Glu Asp Gly Ser Lys Ala Gln His Ser Gln Gly Lys Asp Ser Arg Lys Lys His Ala Val Val Gly Thr Pro Asp Tyr Leu Ala Pro Glu Ile Leu Leu Gly Met Gly His Gly Lys Thr Ala Asp Trp Trp Ser Val Gly Val Ile Leu Phe Glu Val Leu Val Gly Ile Pro Pro Phe Asn Ala Glu Thr Pro Gln Gln Ile Phe Glu Asn Ile Ile Asn Arg Asp Ile Pro Trp Pro Asn Val Pro Glu Glu Ile Ser Tyr Glu Ala His Asp Leu Ile Asn Lys Leu Leu Thr Glu Asn 4 0 Pro Val Gln Arg Leu Gly Ala Thr Gly Ala Gly Glu Val Lys Gln His His Phe Phe Lys Asp Ile Asn Trp Asp Thr Leu Ala Arg Gln Lys Ala Met Phe Val Pro Ser Ala Glu Pro Gln Asp Thr Ser Tyr Phe Met Ser Arg Tyr Ile Trp Asn Pro Glu Asp Glu Asn Val His Gly Gly Ser Asp Phe Asp Asp Leu Thr Asp Thr Cys Ser Ser Ser Ser Phe Asn Thr Gln Glu Glu Asp Gly Asp Glu Cys Gly Ser Leu Ala Glu Phe Gly Asn Gly Pro Asn Leu Ala Val Lys Tyr Ser Phe Ser Asn Phe Ser Phe Lys Asn Leu Ser Gln Leu Ala Ser Ile Asn Tyr Asp Leu Val Leu Lys Asn Ala Lys Glu Ser Val Glu Ala Ser Asn Gln Ser Ala Pro Arg Pro Glu Thr (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1270 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
(2) INFORMATION FOR SEQID
N0:4:
(i) SEQUENCE ARACTERISTIC S:
CH
(A) : pairs base (B) nucleic acid TYPE:
(C) single STRANDEDNESS:
(D) linear TOPOLOGY:
(ii)MOLECULE cDNA
TYPE:
(vi)ORIGINAL (A) Arabidopsis thaliana SOURCE: ORGANISM:
(xi)SEQUENCE EQ ID
DESCRIPTION: N0:4:
S
ACG
Met Ser Thr Glu ProSerPro GluAsnAsp ArgAspPro GlnPro Thr TCT
2 Thr Thr Ile Thr ProThrSer ThrAsnAla LysLeuLeu LysLys 0 Ser ATT
Ile Pro Ala Pro PheArgHis SerAspLys GluGlyGlu AspGlu Ile ACG
Gln Ala Lys Asp GluValThr ThrGluLeu AlaGlyGlu GlyPro Thr GAT
Met Ser His Ser ProGluIle LeuAlaPro SerSerLeu GlyLeu Asp AGA
Asn His Ile Thr LysSerSer ProAlaPro SerProLeu ArgPhe Arg ACG
4 Ser Ser Ala Pro LeuIleSer ProGlyGln AspAspLys AspVal 0 Thr AAA
Ala Lys Glu Pro ArgValGly ValValAsp AlaArgAla AspAla Lys TGG
Arg Ala Arg Pro IleProPro HisGlnPro AspGlnGly LysLys Trp Val Gln Trp Ser Gln Ser Lys Ser Gln Arg Val Pro Ala Asn Ser Asn Pro Gly Val Glu Ser Thr His Val Gly Leu Ala Lys Glu Thr Gln Ser Pro Arg Phe Gln Ala Ile Leu Arg Val Thr Ser Gly Arg Lys Lys Lys Ala His Asp Ile Lys Ser Phe Ser His Glu Leu Asn Ser Lys Gly Val Arg Pro Phe Pro Val Trp Arg Ser Arg Ala Val Gly His Met Glu Glu Ile Met Ala Ala Ile Arg Thr Lys Phe Asp Lys Gln Lys Glu Asp Val Asp Ala Asp Leu Gly Val Phe Ala Gly Tyr Leu Val Thr Thr Leu Glu Ser Thr Pro Glu Ser Asn Lys Glu Leu Arg Val Gly Leu Glu Asp Leu Leu Val Glu Ala Arg Gln Cys Ala Thr Met Pro Ala Ser Glu Phe Trp Leu Lys Cys Glu Gly Ile Val Gln Lys Leu Asp Asp Lys Arg Gln Glu Leu Pro Met Gly Gly Leu Lys Gln Ala His Asn Arg Leu Leu Phe Ile Leu Thr Arg Cys Asn Arg Leu Val Gln Phe Arg Lys Glu Ser Gly Tyr Val Glu Glu His Ile Leu Gly Met His Gln Leu Ser Asp Leu Gly Val Tyr Pro Glu Gln Met Val Glu Ile Ser Arg Gln Gln Asp Leu Leu Arg Glu Lys Glu Ile Gln Lys Ile Asn Glu Lys Gln Asn Leu Ala Gly Ile Gln Asp Asp Gln Asn Ser Asn Ser Gly Ala Asp Gly Val Glu Val Asn Thr Ala Arg Ser Thr Asp Ser Thr Ser Ser Asn Phe Arg Met Ser Ser Trp Lys Lys Leu Pro Ser Ala Ala Glu Lys Asn Arg Ser Leu Asn Asn Thr Pro Lys Ala Lys Gly Glu Ser Lys Ile Gln Pro Lys Val Tyr Gly Asp Glu Asn Ala Glu Asn Leu His Ser Pro Ser Gly Gln Pro Ala Ser Ala Asp Arg Ser Ala Leu Trp Gly Phe Trp Ala Asp His Gln Cys Val Thr Tyr Asp Asn Ser Met Ile Cys Arg Ile Cys Glu Val Glu Ile Pro Val Val His Val Glu Glu His Ser Arg Ile Cys Thr Ile Ala Asp Arg Cys Asp Leu Lys Gly Ile Asn Val Asn Leu Arg Leu Glu Arg Val Ala Glu Ser Leu Glu Lys Ile Leu Glu Ser Trp Thr Pro Lys Ser Ser Val Thr Pro Arg Ala Val Ala Asp Ser Ala Arg Leu Ser Asn Ser Ser Arg Gln Glu Asp Leu Asp Glu Ile Ser Gln Arg Cys Ser Asp Asp Met Leu Asp Cys Val Pro Arg Ser Gln Asn Thr Phe Ser Leu Asp Glu Leu Asn Ile Leu Asn Glu Met Ser Met Thr Asn Gly Thr Lys Asp Ser Ser Ala Gly Ser Leu Thr Pro Pro Ser Pro Ala Thr Pro Arg Asn Ser Gln Val Asp Leu Leu Leu Ser Gly Arg Lys Thr Ile Ser Glu Leu Glu Asn Tyr Gln Gln Ile Asn Lys Leu Leu Asp Ile Ala Arg Ser Val Ala Asn Val Asn Val Cys Gly Tyr Ser Ser Leu Asp Phe Met Ile Glu Gln Leu Asp Glu Leu Lys Tyr Val Ile Gln Asp Arg Lys Ala Asp Ala Leu Val Val Glu Thr Phe Gly Arg Arg Ile Glu Lys Leu Leu Gln Glu Lys Tyr Ile Glu Leu Cys Gly Leu Ile Asp Asp Glu Lys Val Asp Ser Ser Asn Ala Met Pro Asp Glu Glu Ser Ser Ala Asp Glu Asp Thr Val Arg Ser Leu Arg Ala Ser Pro Leu Asn Pro Arg Ala Lys Asp Arg Thr Ser Ile Glu Asp Phe Glu Ile Ile Lys Pro Ile Ser Arg Gly Ala Phe Gly Arg Val Phe Leu Ala Lys Lys Arg Ala Thr Gly Asp Leu Phe Ala Ile Lys Val Leu Lys Lys Ala Asp Met Ile Arg Lys Asn Ala Val Glu Ser Ile Leu Ala Glu Arg Asn Ile Leu Ile Ser Val Arg Asn Pro Phe Val Val Arg Phe Phe Tyr Ser Phe Thr Cys Arg Glu Asn Leu Tyr Leu Val Met Glu Tyr Leu Asn Gly Gly Asp Leu Phe Ser Leu Leu Arg Asn Leu Gly Cys Leu Asp Glu Asp Met Ala Arg Ile Tyr Ile Ala Glu Val Val Leu Ala Leu Glu Tyr Leu His Ser Val Asn Ile Ile His Arg Asp Leu Lys Pro Asp Asn Leu Leu Ile Asn Gln Asp Gly His Ile Lys Leu Thr Asp Phe Gly Leu Ser Lys Val Gly Leu Ile Asn Ser Thr Asp Asp Leu Ser Gly Glu Ser Ser Leu Gly Asn Ser Gly Phe Phe Ala Glu Asp Gly Ser Lys Ala Gln His Ser Gln Gly Lys Asp Ser Arg Lys Lys His Ala Val Val Gly Thr Pro Asp Tyr Leu Ala Pro Glu Ile Leu Leu Gly Met Gly His Gly Lys Thr Ala Asp Trp Trp Ser Val Gly Val Ile Leu Phe Glu Val Leu Val Gly Ile Pro Pro Phe Asn Ala Glu Thr Pro Gln Gln Ile Phe Glu Asn Ile Ile Asn Arg Asp Ile Pro Trp Pro Asn Val Pro Glu Glu Ile Ser Tyr Glu Ala His Asp Leu Ile Asn Lys Leu Leu Thr Glu Asn Pro Val Gln Arg Leu Gly Ala Thr Gly Ala Gly Glu Val Lys Gln His His Phe Phe Lys Asp Ile Asn Trp Asp Thr Leu Ala Arg Gln Lys Ala Met Phe Val Pro Ser Ala Glu Pro Gln Asp Thr Ser Tyr Phe Met Ser Arg Tyr Ile Trp Asn Pro Glu Asp Glu Asn Val His Gly Gly Ser Asp Phe Asp Asp Leu Thr Asp Thr Cys Ser Ser Ser Ser Phe Asn Thr Gln Glu Glu Asp Gly Asp Glu Cys Gly Ser Leu Ala Glu Phe Gly Asn Gly Pro Asn Leu Ala Val Lys Tyr Ser Phe Ser Asn Phe Ser Phe Lys Asn Leu Ser Gln Leu Ala Ser Ile Asn Tyr Asp Leu Val Leu Lys Asn Ala Lys Glu Ser Val Glu Ala Ser Asn Gln Ser Ala Pro Arg Pro Glu Thr
Claims (38)
1. A protein (a) or (b):
(a) a protein represented by the amino acid sequence as set forth in SEQ ID NO.: 2; or (b) a protein represented by an amino acid sequence having one or more amino acids deleted from, substituted for, modified in or added to the amino acid sequence as set forth in SEQ ID NO.:
(a) a protein represented by the amino acid sequence as set forth in SEQ ID NO.: 2; or (b) a protein represented by an amino acid sequence having one or more amino acids deleted from, substituted for, modified in or added to the amino acid sequence as set forth in SEQ ID NO.:
2, and having a tip growth regulating activity.
2. A gene coding for a protein of claim 1.
2. A gene coding for a protein of claim 1.
3. The gene of claim 2, represented by the nucleotide sequence as set forth in SEQ ID NO.: 4.
4. A gene which is capable of hybridizing to a DNA
complementary with the gene of claim 3 under stringent conditions and codes for a protein having a tip growth regulating activity.
complementary with the gene of claim 3 under stringent conditions and codes for a protein having a tip growth regulating activity.
5. A host into which any of the genes of claims 2 to 4 has been introduced.
6. A plant into which any of the genes of claims 2 to 4 has been introduced.
7. A method for regulating tip growth of a plant comprising introducing any of the genes of claims 2 to 4 into the plant.
8. The plant of claim 6 wherein the plant is Arabidopsis.
9. The method for regulating tip growth of a plant according to claim 7, wherein the plant is Arabidopsis.
10. A DNA (a) or (b):
(a) a DNA represented by the nucleotide sequence as set forth in SEQ ID NO.: 3; or (b) a DNA represented by a nucleotide sequence having one or more nucleotides deleted from, substituted for or added to the nucleotide sequence as set forth in SEQ ID NO.: 3, and having a function as a promoter.
(a) a DNA represented by the nucleotide sequence as set forth in SEQ ID NO.: 3; or (b) a DNA represented by a nucleotide sequence having one or more nucleotides deleted from, substituted for or added to the nucleotide sequence as set forth in SEQ ID NO.: 3, and having a function as a promoter.
11. A DNA which is capable of hybridizing to a DNA
complementary with the DNA of claim 10 under stringent conditions and has a function as a promoter.
complementary with the DNA of claim 10 under stringent conditions and has a function as a promoter.
12. The DNA of claim 10 or 11, wherein the expression of the function as a promoter is tissue specific.
13. The DNA of any of claims 10 to 12, wherein a host in which the function as a promoter is expressed is a plant.
14. The DNA of claim 13, wherein the expression of the function as a promoter is tip growth portion specific.
15. The DNA of claim 14, wherein the tip growth portion is root-hair and/or pollen gains of a plant.
16. A gene expression vector comprising the DNA of any of claims 10 to 15.
17. A gene expression vector comprising a gene ligated downstream from the DNA of any of claims 10 to 15.
18. A method for expressing a gene comprising ligating the gene downstream from the DNA of any of claims 10 to 15.
19. The gene expression vector of claim 17, wherein the expression of the gene is tissue specific.
20. The method for expressing a gene of claim 18, wherein the expression of the gene is tissue specific.
21. The gene expression vector of claim 17 or 19, wherein a host in which the gene is expressed is a plant.
22. The method for expressing a gene of claim 18 or 20, wherein a host in which the gene is expressed is a plant.
23. The gene expression vector of claim 21, wherein the expression of the gene is tip growth portion specific.
24. The method for expressing a gene of claim 22, wherein the expression of the gene is tip growth portion specific.
25. The gene expression vector of claim 23, wherein the tip growth portion is root-hair and/or pollen gains of a plant.
26. The method for expressing a gene of claim 24, wherein the tip growth portion is root-hair and/or pollen gains of a plant.
27. The gene expression vector of claim 17, 19, 21, 23 or 25, wherein the gene ligated is a gene derived from a plant.
28. The method for expressing a gene of claim 18, 20, 22, 24 or 26, wherein the gene ligated is a gene derived from a plant.
29. The gene expression vector of claim 17, 19, 21, 23 or 25, wherein the gene ligated is a gene derived from an organism other than plants.
30. The method for expressing a gene of claim 18, 20, 22, 24 or 26, wherein the gene ligated is a gene derived from an organism other than plants.
31. A host into which a gene expression vector of claim 16 or a gene expression vector of claim 17, 19, 21, 23, 25, 27 or 29 has been introduced.
32. A plant into which a gene expression vector of claim 16 or a gene expression vector of claim 17, 19, 21, 23, 25, 27 or 29 has been introduced.
33. The plant of claim 32 wherein the plant is Arabidopsis.
34. A method for regulating tip growth of a plant comprising eliminating the activity and/or expression of a protein by deleting, substituting or adding one or more nucleotides in the nucleotide sequence comprising the DNA of any of claims 2 to 4, 10 and 11.
35. A plant in which the tip growth is regulated by the method of claim 34.
36. The method for regulating tip growth of a plant according to claim 34 wherein the plant is Arabidopsis.
37. The plant in which the tip growth is regulated according to claim 35, wherein the plant is Arabidopsis.
38. A method for screening for a gene regulating the tip growth of a plant, comprising using the plant of any of claims 6, 8, 32, 33, 35 and 37.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11082402A JP2000270873A (en) | 1999-03-25 | 1999-03-25 | Ire gene which controls the elongation of the root hair of arabidopsis thaliana |
| JP82402/1999 | 1999-03-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2301257A1 true CA2301257A1 (en) | 2000-09-25 |
Family
ID=13773613
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002301257A Abandoned CA2301257A1 (en) | 1999-03-25 | 2000-03-24 | The ire gene regulating the root-hair growth in arabidopsis |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP2000270873A (en) |
| CA (1) | CA2301257A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20010099064A (en) * | 2001-08-22 | 2001-11-09 | 정명식 | AWI31 gene which regulates vascular strand formation in Arabidopsis thaliana |
| EP1498027B1 (en) | 2002-04-08 | 2012-05-09 | Toyo Boseki Kabushiki Kaisha | Plant with improved organogenesis and method of constructing the same |
| EP1930434B1 (en) | 2005-09-26 | 2012-02-22 | Nippon Soda Co., Ltd. | Protoporphyrinogen oxidase having activity of imparting resistance against acifluorfen and gene thereof |
| KR101202615B1 (en) | 2010-07-06 | 2012-11-19 | 서울대학교산학협력단 | Method of controlling plant root hair growth by expression of auxin transporters and the plants thereof |
-
1999
- 1999-03-25 JP JP11082402A patent/JP2000270873A/en active Pending
-
2000
- 2000-03-24 CA CA002301257A patent/CA2301257A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| JP2000270873A (en) | 2000-10-03 |
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