阅读说明：本技术 拟南芥tir2基因在提高植物盐胁迫抗性中的应用 (Application of arabidopsis TIR2 gene in improving salt stress resistance of plants ) 是由 黄金光 蔡慧娴 郑成超 于 2020-12-15 设计创作，主要内容包括：本发明提供了拟南芥TIR2基因在提高植物盐胁迫抗性中的应用。所述TIR2基因的互作蛋白包括IAA4和IAA8,本发明通过基因工程技术获得了拟南芥TIR2基因的过量表达载体,并由此获得了拟南芥的过量表达株系,经过筛选后最终获得拟南芥TIR2基因过量表达纯合株系；所述拟南芥TIR2基因过量表达纯合株系表现为耐盐,具有很好的植物盐胁迫抗性,不仅能够增加种子萌发率和子叶展开率,缩短种子萌发和子叶展开时间,而且可以提高成植物幼苗根系的长度。本发明通过实验确认了拟南芥TIR2基因在提高植物盐胁迫抗性方面具有潜在的应用价值,并为利用拟南芥TIR2基因培育耐盐、高产的农作物品种奠定良好的理论和应用基础。(The present invention provides Arabidopsis thaliana TIR2 Application of the gene in improving salt stress resistance of plants. The above-mentioned TIR2 The gene interacting protein includes IAA4 and IAA8, and the present invention obtains Arabidopsis thaliana through gene engineering technology TIR2 The overexpression vector of the gene and the overexpression strain of the arabidopsis are obtained from the overexpression vector, and the arabidopsis is finally obtained after screening TIR2 Gene over-expression homozygous lines; the Arabidopsis thaliana TIR2 The gene over-expression homozygous strain is salt-tolerant, has good plant salt stress resistance, can increase the seed germination rate and the cotyledon expansion rate, shortens the seed germination and cotyledon expansion time, and can improve the length of the root system of the adult plant seedling. The invention confirms the arabidopsis through experiments TIR2 The gene has potential application value in the aspect of improving the salt stress resistance of plantsAnd is obtained by using Arabidopsis thaliana TIR2 The gene lays a good theoretical and application foundation for cultivating salt-tolerant and high-yield crop varieties.)

1. Arabidopsis thalianaTIR2Application of the gene in improving salt stress resistance of plants.

2. Arabidopsis thaliana of claim 1TIR2The application of the gene in improving the salt stress resistance of the plant is characterized in that the application method comprises the following steps: will contain the Arabidopsis thalianaTIR2The gene over-expression vector is transformed into a plant by an inflorescence infection method to obtain the plant with salt stress resistance.

3. Arabidopsis thaliana of claim 1TIR2The application of the gene in improving the salt stress resistance of plants is characterized by comprising the arabidopsis thalianaTIR2The seed germination rate of the plant over-expression homozygous strain of the gene under salt stress is obviously higher than that of a wild type strain and a mutant strain.

4. Arabidopsis thaliana of claim 1TIR2The application of the gene in improving the salt stress resistance of plants is characterized by comprising the arabidopsis thalianaTIR2The expansion rate of cotyledon of the plant over-expression homozygous strain of the gene under salt stress is obviously higher than that of a wild strain and a mutant strain.

5. Arabidopsis thaliana of claim 1TIR2The application of the gene in improving the salt stress resistance of plants is characterized by comprising the arabidopsis thalianaTIR2The length of the seedling root system of the gene plant over-expression homozygous strain is obviously higher than that of a wild type strain and a mutant strain under the salt stress.

6. Arabidopsis thaliana of claim 2TIR2Gene for improving salt stress of plantsUse in resistance, characterized in that said overexpression vector comprises pBI121-TIR2, which is digested doubly by a recombinant plasmid, said Arabidopsis thaliana being subjected toTIR2The gene is obtained after being connected to a 35S promoter in a vector plasmid.

7. Arabidopsis thaliana of claim 6TIR2Use of a gene for increasing salt stress resistance in a plant, wherein said recombinant plasmid comprises pMD18-T-TIR2 produced by amplifying said Arabidopsis thalianaTIR2The gene product is obtained by cloning to a cloning vector after recovery and purification.

8. Arabidopsis thaliana of claim 7TIR2Application of gene in improving salt stress resistance of plant, and is characterized in that arabidopsis thaliana isTIR2The gene amplification primers include:

TIR2-F: ATGGTGAAACTGGAGAACTCGAGG；

TIR2-R: AAGGTCAATGCTTTTAATGAGCTTCATGTTG。

9. arabidopsis thaliana of claim 1TIR2Application of gene in improving salt stress resistance of plant, and is characterized in that arabidopsis thaliana isTIR2Interacting proteins of genes include IAA4 and IAA 8.

10. Arabidopsis thaliana of claim 1TIR2The application of the gene in improving the salt stress resistance of the plant is characterized in that the plant is arabidopsis thaliana.

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to arabidopsis thalianaTIR2Application of the gene in improving salt stress resistance of plants.

Background

Salt stress is one of the abiotic stress factors affecting crop yield. The large-area saline-alkali soil and salinized soil lead most crop varieties to be influenced by salt damages of different degrees in the growth process and are difficult to exert yield potential. The salt resistance of plants is a complex quantitative character determined by multiple genes, the salt tolerance modes and salt tolerance mechanisms of different plants are different, and the salt tolerance reactions of tissues or cells of the plants are also different. With the continuous reduction of the cultivated land area and the continuous increase of the population, the salt tolerance mechanism of the crops is researched, the salt tolerance variety is cultivated, and the method has important practical significance for developing and effectively utilizing the saline-alkali soil.

Phytohormones refer to a trace amount of organic substances which are synthesized in the plant body and can be transported from the place of production to other parts, resulting in a significant effect on growth and development. Auxin is the first discovered plant hormone and has important regulation and control functions on the processes of plant cell division, cell growth and differentiation, lateral root formation, tropism, flowering, embryonic development and the like. Auxin is synthesized at the parts where the plant grows actively, comprises young parts such as stem tips, terminal buds, young leaves, developing seeds, meristems of main root tips, developing lateral roots and the like, and is transported to other parts from the synthesized parts to play a role. An endogenous auxin commonly found in plants is indole-3-acetic acid (IAA). The biosynthesis of IAA in plants is largely divided into two categories, tryptophan-dependent and tryptophan-independent. Among them, the tryptophan-dependent pathway is a major synthetic pathway, including indole-3-pyruvate (IPA) pathway, indole-3-acetamide (IAM) pathway, and indole-3-acetaldoxime (IAOx) pathway. The TIR2 protein is localized to the site of auxin production and it has been found that the TIR2 protein functions as a tryptophan aminotransferase which is believed to convert tryptophan to indole-3-pyruvate (IPA), the precursor of auxin. However, at present, no report about the function and action of the coding gene of the key enzyme of the auxin synthetic pathway in plant salt stress is found.

Disclosure of Invention

In view of the above-described state of the art, it is an object of the present invention to provide Arabidopsis thalianaTIR2GeneThe invention relates to the application of improving the salt stress resistance of plantsTIR2Gene acquisitionTIR2A gene overexpression vector is obtainedTIR2The gene over-expression homozygous strain has good salt stress resistance.

In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:

the present invention provides Arabidopsis thalianaTIR2Application of the gene in improving salt stress resistance of plants.

Further, the application method comprises the following steps: will contain the Arabidopsis thalianaTIR2The gene over-expression vector is transformed into a plant by an inflorescence infection method to obtain the plant with salt stress resistance.

Further, it comprises the Arabidopsis thalianaTIR2The seed germination rate of the plant over-expression homozygous strain of the gene under salt stress is obviously higher than that of a wild type strain and a mutant strain.

Further, it comprises the Arabidopsis thalianaTIR2The seed germination time of the plant over-expression homozygous strain of the gene under salt stress is obviously shorter than that of a wild type strain and a mutant strain.

Further, it comprises the Arabidopsis thalianaTIR2The expansion rate of cotyledon of the plant over-expression homozygous strain of the gene under salt stress is obviously higher than that of a wild strain and a mutant strain.

Further, it comprises the Arabidopsis thalianaTIR2The length of the seedling root system of the gene plant over-expression homozygous strain is obviously higher than that of a wild type strain and a mutant strain under the salt stress.

Further, the overexpression vector comprises pBI121-TIR2, and the Arabidopsis thaliana is subjected to double enzyme digestion by a recombinant plasmidTIR2The gene is obtained after being connected to a 35S promoter in a vector plasmid.

Further, the recombinant plasmid comprises pMD18-T-TIR2, which is obtained by amplifying the Arabidopsis thalianaTIR2The gene product is obtained by cloning to a cloning vector after recovery and purification.

Further, the Arabidopsis thalianaTIR2The gene amplification primers include:

TIR2-F: ATGGTGAAACTGGAGAACTCGAGG；

TIR2-R: AAGGTCAATGCTTTTAATGAGCTTCATGTTG。

further, the Arabidopsis thalianaTIR2Interacting proteins of genes include IAA4 and IAA 8.

Further, the plant is arabidopsis thaliana.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the invention adopts gene engineering technology toTIR2The gene is over-expressed, then an over-expression vector is obtained, the vector is transformed into a strain, and the strain is obtained by screeningTIR2The gene over-expression homozygous strain further obviously improves the salt stress resistance of the plant, so that the plant is more salt-tolerant.TIR2The gene over-expression not only improves the germination quantity and speed of plant seeds under salt stress, shortens the time of cotyledon expansion, improves the expansion rate of the cotyledon, but also increases the growth length of the root system of the plant seedling, and is more beneficial to the growth and development of the plant in a high-salt environment. The invention confirms the arabidopsis for the first time through experimentsTIR2The gene can improve salt stress of plants, and its interacting proteins include IAA4 and IAA8, and is provided byTIR2The application of the gene in salt stress can be considered as having potential application value for improving the salt stress of plants, and the invention also utilizes the geneTIR2The gene lays a good theoretical and application foundation for cultivating salt-tolerant and high-yield crop varieties.

Drawings

FIG. 1:TIR2electrophoresis chart for detecting expression quantity of gene over-expression homozygous strain.

FIG. 2: wild type strain WT, mutant straintir2-3AndTIR2seed germination and cotyledon unfolding results of an over-expression homozygous strain OE 141; A. seed germination and cotyledon expansion phenotype; b: seed germination rate; c: the cotyledon expansion rate.

FIG. 3: wild type strain WT, mutant straintir2-3AndTIR2the seedling root system growth result of the over-expression homozygous strain OE 141; A. seedling root growth phenotype; b: root length statistics.

FIG. 4: yeast two-hybrid experimental verificationTIR2Interaction of genes IAA4 and IAA 8.

Detailed Description

The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. Experimental procedures in the following examples, where specific conditions are not noted, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer; reagents or materials not specified in detail are all commercially available products.

Example 1: arabidopsis thalianaTIR2Obtaining of Gene transgenic line OE141

First, Arabidopsis thalianaTIR2Obtaining of Gene mutants

Obtained in Arabidopsis thaliana databaseTIR2The CDS sequence of the gene is 1173 nt in length and codes protein containing 391 amino acids, the nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid series is shown as SEQ ID NO. 2. Arabidopsis thalianaTIR2Gene T-DNA insertion mutant (tir2-3) Obtaining from Arabidopsis resource center, screening, expressing, analyzing and finally identifying homozygote.

Second, Arabidopsis thalianaTIR2Obtaining of Gene transgenic line OE141

1. Arabidopsis thalianaTIR2Obtaining of Gene-overexpressing lines

(1) Construction of constitutive expression promoter 35S drivenTIR2The overexpression vector of (3) (35S:TIR2): according to Arabidopsis thalianaTIR2CDS sequence of the gene, gene-specific primers were designed, and the sequences were as follows (5 '-3'):

TIR2-F: ATGGTGAAACTGGAGAACTCGAGG（SEQ ID NO:3）；

TIR2-R: AAGGTCAATGCTTTTAATGAGCTTCATGTTG（SEQ ID NO:4）；

amplification by PCR techniqueTIR2The gene, and the amplified product is recovered and purified and then cloned on a pMD18-T Simple cloning vector to obtain pMD18-T-TIR2Recombinant plasmids;

(2) pMD18-T-TIR2After the recombinant plasmid is confirmed by plasmid PCR sequencing, restriction endonuclease is usedBamHI andSaci vs pMD18-T-TIR2Carrying out double enzyme digestion on the recombinant plasmid and the pBI121 plasmidTIR2Gene ligation into pBI121 behind the 35S promoter to obtain the overexpression vector pBI121-TIR2；

(3) Transforming the excessive expression vector pBI121-TIR2 into agrobacterium GV3101 competence, and transforming arabidopsis thaliana by inflorescence infection method to obtainTIR2Gene overexpression lines.

2、TIR2Screening of transgenic Positive homozygous lines

(1) After infection of the individual plantsTIR2Seeds of gene overexpression lines as T₀Generation;

(2) will T₀Culturing the generation seeds on a culture medium containing kanamycin (the final concentration is 43 mg/L), selecting green seedlings in a vermiculite culture medium, and collecting seeds T from a single plant₁Generation;

(3) will T₁Culturing the generation seeds on a culture medium containing kanamycin, and selecting the progeny green seedlings: the yellow seedling separation ratio is 3: 1 in a vermiculite culture medium, the seeds received by a single plant are T₂Generation;

(4) will T₂Culturing the seeds in culture medium containing kanamycin, selecting the strains with green seedlings as the descendants, and culturing in vermiculite culture medium to obtain seedsTIR2The seeds of homozygous transgenic lines with genes overexpressed were designated OE4, OE28, OE62, OE65 and OE141, respectively.

3. RT-PCR detection

To pairTIR2And (3) carrying out RT-PCR experiments on seeds of the gene transgenic positive homozygous lines, wherein primers used in the RT-PCR experiments are (5 '-3'):

35S-F: ATAGAGGACCTAACAGAACTCGCCG（SEQ ID NO:5）；

TIR2-R: AAGGTCAATGCTTTTAATGAGCTTCATGTTG（SEQ ID NO:4）；

the results show (figure 1) that,TIR2the gene exhibited a higher level of expression in the over-expressed homozygote strain OE141, which was therefore selected for subsequent studies.

Example 2 Arabidopsis thaliana under salt stressTIR2Effect of genes on seed Germination and cotyledon expansion

1. Preparation of the culture Medium

(1) Configuration of 1/2MS (-) medium for plant tissue culture: weighing 2.37 g/L of medicine MS and 15g/L of cane sugar, dissolving in ultrapure water, fixing the volume to 1000 ml, and adjusting the pH to 5.80 by using 1M KOH solution and dilute hydrochloric acid;

preparation of a high-salt culture medium: on the basis of the 1/2MS (-) culture medium formula, 58.44g/L sodium chloride is added to prepare a high-salt culture medium containing 150 mM NaCl.

(2) Subpackaging: each 200 ml of the liquid medium was dispensed into a 250 ml Erlenmeyer flask, and 1.6 g of agarose was added thereto, followed by autoclaving at 121 ℃ for 20 minutes.

(3) After sterilization, when the culture medium is cooled to 50-60 ℃, pouring the culture medium into glass plates, and subpackaging 10 glass plates per 200 ml. After the medium has solidified, the concentration is marked and sealed in a sterile bag for use.

2. Seed germination and cotyledon expansion detection

Wild type strain WT, T-DNA mutant strain (tir2-3) And selecting an over-expression homozygous strain (OE 141) as a treatment material, selecting arabidopsis thaliana seeds with the same period, consistent size and full seeds, and disinfecting for 5 hours by using chlorine.

Dividing the culture medium into 2 groups, respectively adding 1/2MS (-) culture medium and high-salt culture medium as a control group CK and a salt stress group, equally dividing a culture dish into three regions, sowing a strain seed in each region, sowing 28 seeds in each region, marking the culture dish, sowing 4 seeds in each treatment in parallel, sealing the culture dish, layering in dark at 4 ℃ for three days, then culturing in a light incubator at 22 ℃, and counting the germination rate every 12 hours. Germination conditions are shown in fig. 2A, and the germination conditions of the 3 seeds in the Control (CK) group are better than those in the salt stress group at the same time, which indicates that the salt stress can significantly reduce the germination of the arabidopsis seeds; and in the group of salt stress,TIR2the seed germination of the gene over-expression homozygous lines is obviously superior to that of mutant lines and wild-type lines. Germination rate results are shown in fig. 2B, where all seeds have germinated in 2d under normal (CK) conditions; and under the condition of salt stress,TIR2pure gene overexpressionThe resultant strain basically and completely germinates at 3.5 days, the germination rate is 99%, the wild type strain needs 5 days to basically and completely germinate, the germination rate is 98.6%, while the mutant strain slowly germinates, and does not completely germinate by the 7 th day. Counting the expansion number of cotyledons of each strain after one week of germination and counting the expansion rate of the cotyledons, wherein the results are shown in FIG. 2C, and compared with a control group, the expansion rate of the cotyledons of a salt stress group is reduced, which indicates that the expansion of the cotyledons of the arabidopsis thaliana can be obviously reduced due to the salt stress; and in the group of salt stress,TIR2the cotyledon expansion rate of the gene over-expression homozygous strain is obviously higher than that of the wild type strain and the mutant strain. The above results indicate that salt stress can significantly reduce germination and cotyledon development of Arabidopsis seeds, whileTIR2The gene can obviously improve the salt stress resistance of the plant, further improve the germination rate, the germination quantity and time of the plant and the expansion of the cotyledon, increase the germination of the plant seed and the expansion capability of the strain cotyledon, and relieve the salt stress suffered by the seed.

Example 3: salt-stressed Arabidopsis thalianaTIR2Effect of genes on seedling growth phenotype

1. Preparation of a culture medium: the same as in example 2.

2. Seedling growth phenotype detection

The medium was divided into 2 groups, and 1/2MS (-) medium and high salt medium were added as control group CK and salt stress group, respectively, with 4 replicates per group.

Wild type strain WT, T-DNA mutant strain (tir2-3) And an overexpression homozygous strain (OE 141) is used as a processing material, Arabidopsis thaliana seeds with the same period and the same size and full seeds are selected, sterilized by chlorine for 5 hours, then uniformly and dispersedly paved on 1/2MS (-) culture medium, the culture medium is sealed, the seeds are laminated in dark at 4 ℃ for three days, then are vertically cultured and grown for 3 days at 22 ℃, then seedlings with the same root length are selected, transferred into 1/2MS (-) culture medium and high salt culture medium respectively, are vertically grown for two weeks, the root growth is observed, and the root length data is counted.

The root length phenotype of each strain of Arabidopsis thaliana under different treatments is shown in FIG. 3A, the statistical data is shown in FIG. 3B, and compared with the control group, the root length of the salt stress group is obviously shorter, which indicates that the salt stress can obviously reduce the root of the Arabidopsis thaliana seedlingGrowing the line; whereas in the salt stress group,TIR2the root length of the seedlings of the gene over-expression homozygous lines is obviously higher than that of wild lines and mutant lines, which shows thatTIR2The gene can obviously improve the salt stress resistance of the plant seedling, further improve the growth of the root system of the plant seedling and relieve the salt stress suffered by the seedling.

Example 4: screening and identification of Arabidopsis TIR2 interaction protein

(1) Construction of promoter T7-drivenTIR2The bait protein expression vector (pGBKT 7-TIR2): according to Arabidopsis thalianaTIR2CDS sequence of the gene, gene-specific primers were designed, and the sequences were as follows (5 '-3'):

TIR2-F: ATGGTGAAACTGGAGAACTCGAGG；

TIR2-R: AAGGTCAATGCTTTTAATGAGCTTCATGTTG；

amplification by PCR techniqueTIR2The gene, and the amplified product is recovered and purified and then cloned on a pMD18-T Simple cloning vector to obtain pMD18-T-TIR2, recombinant plasmids;

(2) pMD18-T-TIR2After the recombinant plasmid is confirmed by plasmid PCR sequencing, restriction endonuclease is usedEcoRI andSali vs pMD18-T-TIR2Carrying out double enzyme digestion on the recombinant plasmid and pGBKT7 plasmidTIR2The gene is connected to the rear of a T7 promoter in pGBKT7 to obtain an expression vector pGBKT7-TIR2；

(3) The bait protein expression vector pGBKT7-TIR2Transformed into Yeast strain Gold Yeast competence, coated on DDO and QDO and incubated, the result is shown in FIG. 4A, the Yeast cell only grows on DDO, which shows that it has no self-activating activity of transcription, and can be used in Yeast two-hybrid sieve library experiment;

(4) according to the yeast two-hybrid screening library process, the final product is obtainedTIR2Two proteins that interact with each other: IAA4 and IAA8, the interaction results are shown in fig. 4B.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

<110> Shandong university of agriculture

Application of <120> Arabidopsis TIR2 gene in improving salt stress resistance of plants

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