CN112608370A - Brachypodium Bsr1 protein and coding gene and application thereof - Google Patents

Abstract

The invention discloses a brachypodium japonicum Bsr1 protein, and a coding gene and application thereof. The brachypodium Bsr1 protein is the protein of a) or b) or c) or d) as follows: a) the amino acid sequence is a protein shown in a sequence 3; b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 3; c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 3; d) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the sequence 3 and having the same function. The brachypodium Bsr1 protein has the following functions: B1) regulating and controlling the disease resistance of plants; B2) preparing a product for regulating and controlling the disease resistance of the plants; B3) cultivating disease-resistant plants; B4) preparing a product for cultivating disease-resistant plants; B5) and (5) plant breeding.

Description

Brachypodium Bsr1 protein and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to brachypodium japonicum Bsr1 protein and a coding gene and application thereof.

Background

Viral diseases are important diseases of crops and can cause serious yield loss. The discovery and cloning of antiviral genes is the basis for crop antiviral molecule breeding and for studying host and virus interaction. The plant dominant antiviral gene cloned at present is mainly derived from dicotyledonous plants. Therefore, the cloning of dominant plant virus resistant genes from monocotyledons, especially gramineae, is very important, and has important significance for constructing a research model of monocotyledon and virus interaction, analyzing the difference of virus resistance mechanisms of monocotyledons and dicotyledons and analyzing the common mechanism of disease resistant gene evolution.

The Barley Streak Mosaic Virus (BSMV) is a representative member of the genus Barley virus (Hordeivirus), consists of 3 positive sense single-stranded RNAs and coat protein subunits, is widely distributed, is a model virus in gramineous plant virus research, and in production, the BSMV mainly harms Barley and wheat and can cause Barley and wheat streak mosaic diseases, and the typical infection symptoms are that irregular stripes with alternate chlorosis and faint yellow appear at the base of a new leaf in early stage, the stripe appears brown necrosis when the stripe is serious, then the stripe can expand towards the direction, and the whole leaf shows a chlorosis stripe-shaped lesion spot which is further diffused to the whole plant to generate systemic floral leaves. The infected plants are obviously shorter than healthy plants, batch ears are produced, the yield of a single plant is reduced by 30-70%, the thousand seed weight is reduced by 30-60%, and the agricultural production is seriously influenced. BSMV has a plurality of isolates and strains, and the research focuses on virus structure, pathogenicity and the like, but the research on host resistance is less.

The brachypodium distachyon is a new warm-zone gramineae model plant, has small volume, short first-generation breeding time and simpler genome (the whole genome sequencing shows that the genome size of the brachypodium distachyon is 272Mb, which is one of grass plants with the smallest genome, only 15 percent of repeated sequences and compact gene structure, predicts that 25532 coding protein genes are contained, are similar to rice and sorghum of gramineae, have the capability of self-pollination and have low requirements on the growth environment, the characteristics are very similar to arabidopsis thaliana, the germplasm resources of the brachypodium distachyon are very rich, a large number of naturally-existing ecotypes exist, the polymorphism exists in disease resistance, in addition, the systematic evolution status of the brachypodium distachyon shows that the brachypodium distachyon is very close to the warm-zone cereal crops, and has very outstanding genome application breadth, so the brachypodium distachyon is widely applied to comparative gene omics of barley, wheat and other, Functional genomics and host-pathogen interaction.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the disease resistance of plants.

In order to solve the above technical problems, the present invention firstly provides a protein.

The protein provided by the invention is derived from Brachypodium distachyon (L.) Beauv, named Bsr1, and is a) or b) or c) or d) as follows:

a) the amino acid sequence is a protein shown in a sequence 3;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 3;

c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 3;

d) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the sequence 3 and having the same function.

Wherein, the sequence 3 consists of 809 amino acid residues.

In order to facilitate the purification of the protein in a), the amino terminal or the carboxyl terminal of the protein shown in the sequence 3 in the sequence table can be connected with a label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein Bsr1 in c) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The Bsr1 protein in c) can be synthesized artificially, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein Bsr1 of c) above can be obtained by deleting one or several amino acid residues of codons in the DNA sequence shown in sequence No. 2, and/or by performing missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in table 1 above at its 5 'end and/or 3' end.

In order to solve the technical problems, the invention also provides a biological material related to the Bsr1 protein.

The biomaterial related to Bsr1 protein provided by the invention is any one of the following A1) to A12):

A1) nucleic acid molecules encoding the Bsr1 protein;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising a4) said recombinant vector;

A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);

A10) a transgenic plant cell line comprising the expression cassette of a 2);

A11) a transgenic plant cell line comprising the recombinant vector of a 3);

A12) a transgenic plant cell line comprising the recombinant vector of a 4).

In the above biological material, the nucleic acid molecule of A1) is a gene represented by the following 1) or 2) or 3):

1) the coding sequence is a cDNA molecule shown in a sequence 2 or a genome DNA molecule shown in a sequence 1;

2) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes Bsr1 protein;

3) a cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence limited by 1) or 2) under strict conditions and codes for Bsr1 protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, the sequence 1 consists of 7282 nucleotides, the sequence 2 consists of 2430 nucleotides, and the amino acid sequences are shown as the coding sequence 3.

The nucleotide sequence of the present invention encoding Bsr1 can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified to have 75% or more identity to the nucleotide sequence of Bsr1 isolated in the present invention are derived from and identical to the nucleotide sequence of the present invention as long as they encode Bsr1 and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 3 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above-mentioned biomaterials, the expression cassette containing a nucleic acid molecule encoding Bsr1 (Bsr1 gene expression cassette) described in a2) refers to a DNA capable of expressing Bsr1 in host cells, which may include not only a promoter for initiating transcription of Bsr1 but also a terminator for terminating transcription of Bsr 1. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: a constitutive promoter; tissue, organ and development specific promoters and inducible promoters. Suitable transcription terminators include, but are not limited to: the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator and the pea rbcS E9 terminator.

The recombinant vector containing the Bsr1 gene expression cassette can be constructed using existing expression vectors. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corp.) and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector. In the invention, the recombinant expression vector is obtained by inserting the Bsr1 gene into a pCAMBIA1300 vector or a pMDC32 vector.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium. The agrobacterium may specifically be agrobacterium strain LBA4404 and EHA 105. In the present invention, the recombinant bacteria are LBA4404 and EHA105 containing the recombinant vector described above.

In the above biological material, none of the transgenic plant cell lines comprises propagation material.

In order to solve the technical problems, the invention also provides a new application of the Bsr1 protein or biological material.

The invention provides the application of the Bsr1 protein or the biological material in any one of the following B1) -B5):

B1) regulating and controlling the disease resistance of plants;

B2) preparing a product for regulating and controlling the disease resistance of the plants;

B3) cultivating disease-resistant plants;

B4) preparing a product for cultivating disease-resistant plants;

B5) and (5) plant breeding.

In the above application, the regulation is an improvement.

In the above application, the disease resistance is stripe mosaic disease resistance. The streak mosaic disease can be streak mosaic disease induced by Barley Streak Mosaic Virus (BSMV) ND18 strain.

The regulation and control of the disease resistance of the plants are specifically characterized in that: when the expression level and/or activity of the Bsr1 protein in a plant is increased, the disease resistance of the plant is increased; when the expression amount and/or activity of the Bsr1 protein in a plant is decreased, the disease resistance of the plant is decreased.

In order to solve the technical problems, the invention also provides a method for cultivating disease-resistant plants.

The method for cultivating disease-resistant plants comprises the steps of improving the expression quantity and/or activity of Bsr1 protein in receptor plants to obtain transgenic plants; the transgenic plant has higher disease resistance than the recipient plant.

In the above method, the disease resistance is stripe mosaic disease resistance. The streak mosaic disease can be streak mosaic disease induced by Barley Streak Mosaic Virus (BSMV) ND18 strain.

In the above method, the disease resistance of the transgenic plant is higher than that of the recipient plant specifically by: transgenic plants inoculated with the strain BSMV ND18 showed no signs of spotting, streaking, chlorosis and plant dwarfing compared to recipient plants.

In the above method, the method for increasing the expression level and/or activity of Bsr1 protein in a recipient plant is to overexpress Bsr1 protein as defined in claim 1 in the recipient plant.

Further, the overexpression is performed by introducing a gene encoding the Bsr1 protein into a recipient plant.

The nucleotide sequence of the coding gene of the Bsr1 protein is a DNA molecule shown in a sequence 2.

The gene encoding the Bsr1 protein can be introduced into plant cells by using conventional biotechnological methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, and the like.

The encoding gene of the Bsr1 protein can be modified as follows and then introduced into a target plant to achieve better expression effect:

C1) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

C2) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

C3) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

C4) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

Further, the gene encoding the Bsr1 protein was introduced into recipient plants via overexpression vectors pMBsr1 or pCBsr 1.

In the above methods, the transgenic plant is understood to include not only the first to second generation transgenic plants but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

In the above application or method, the plant may be a monocotyledon or a dicotyledon; the monocotyledonous plant may be a graminaceous plant, such as Brachypodium distachyon (L.) Beauv or barley Hordeum vulgare L or wheat Triticum aestivum L. The brachypodium distachyon can be brachypodium distachyon BD 21-3; the barley can be specifically a barley susceptible strain Golden Promise; the wheat can be specifically wheat susceptible strain Kenong 199.

The invention clones a BSMV-resistant Bsr1 gene from brachypodium distachyon, and the Bsr1 gene is the first reported dominant BSMV-resistant gene. The invention researches whether the Bsr1 gene has the function of resisting BSMV in wheat crops by transforming the Bsr1 gene into wheat and barley varieties which are infected with ND 18. The experimental results show that: the gene Bsr1 can improve the BSMV resistance of wheat crops, and has important value in the genetic improvement of the wheat crops.

Drawings

FIG. 1 shows the genotype and phenotype identification of Bsr 1-transferred brachypodium. FIG. 1A shows the results of genotyping. FIG. 1B shows the results of phenotypic identification. Wherein, L2-14 and L2-20 are trans-Bsr 1 brachypodium strain, Bd3-1 is a disease-resistant strain, and Bd21-3 is an infectious strain.

FIG. 2 shows the genotyping and phenotyping of Bsr 1-transferred barley and Bsr 1-transferred wheat. FIGS. 2A and 2C are the genotyping and phenotyping of Bsr1 transgenic barley. FIGS. 2B and 2D show the genotyping and phenotyping of Bsr1 transgenic wheat. Wherein, BSWV_NDInoculating BSMV strain ND 18; BSWV_NWInoculating BSMV strain to Norwich; mock is an unvaccinated virus control; non-transgenic control; CBB3-211, CBB6-11 and CBB8-99 are transgenic barley progeny of Bsr 1; CBW119-1, CBW94-18 and CBW94-3 are wheat progeny transformed with Bsr 1.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The pMDC32 vector in the following examples is described in the literature "Curtis MD and Grossniklaus U.A Gateway cloning vector set for high-throughput functional analysis of genes in plant Physiology,2003,133(2): 462-469". The pMDC32 vector carries attR sequence which can be used as Gateway target vector, and simultaneously carries kanamycin and hygromycin resistance genes, and the full length is 11752 bp.

The pCAMBIA1300 vector in the following examples is a product of Beijing Huabode billion Biotechnology Ltd, having a product number of VT 3011. The pCAMBIA1300 vector carries both kanamycin and hygromycin resistance genes, and has a full length of 8958 bp.

Agrobacterium strain LBA4404 in the following examples is a product of Beijing Tiannzze Gene technology, Inc., having a product number of 12-96.

Agrobacterium strain EHA105 in the examples described below is a product of Beijing Tiannzze Gene technology, Inc., having a product number of 12-153.

The susceptible strain Bd21-3 in the examples described below is described in the literature "Yu Cui, Mi Yeon Lee, Naxin Huo, Jennifer Bragg, Lijie Yan, Cheng Yuan, Cui Li, Sara J.Holdach, Jingtzhong Xie, Ming-Cheng Luo, Dawei Li, Jianlin Yu, Joel Martin, Wendy Schackwitz, Yong Qiang Gu, John P.Vogel, Andrew O.Jackson, Zhiyon Liu, David F.Garvin, Fine mapping of the 1 ley strain mos.

The disease-resistant line Bd3-1 in the examples described below is described in the literature "Yu Cui, Mi Yeon Lee, Naxin Huo, Jennifer Bragg, Lijie Yan, Cheng Yuan, Cui Li, Sara J. Holdach, Jingngzhong Xie, Ming-Cheng Luo, Dawei Li, Jianlin Yu, Joel Martin, Wendy Schackwitz, Yong Qiang Gu, John P. Vogel, Andrew O. Jackson, Zhiyong Liu, David F. Garvin, Fine mapping of the 1 ley strain mos resistant gene in the strain outlet.P. Jackson, PLoS E,7 Bsjon 6, 333, this invention is not applicable to other biological materials of interest (for which the applicant can only use this invention repeatedly.

BSMV ND18 in the following examples is described in the literature "Yu Cui, Mi Yeon Lee, Naxin Huo, Jennifer Bragg, Lijie Yan, Cheng Yuan, Cui Li, Sara J.Holdach, Jingzhong Xie, Ming-Cheng Luo, Dawei Li, Jianlin Yu, Jovin Martin, Wendy Schackwitz, Yong Qiang Gu, John P.Vogel, Andrew O.Jackson, Zhiyong Liu, David F.Garvin, Fine mapping of the Bs 1 barley strain said resistant gene in model strain Brachyyo type, 2012, PLoS, 7 J.386," the invention is not applicable to other biological sources, but is not applicable to this application.

BSMV Norwich in the following examples is described in the documents "Mi Yeon Lee, Lijie Yan, Florin A. Gorter, Brian Y.T.Kim, Yu Cui, Yue Hu, Cheng Yuan, Jessian Grindheim, Uma Ganesan, Zhiyong Liu, Chenggui Han, Jianlin Yu, Dawei Li and Andrew O.Jackson, Brachydium discodyn line Bd3-1resistance is elastic by the barrel mobile virus gene block 1 expression protein.2012, Journal of General Virology,93: 2729-.

Example 1 cloning of Bsr1 Gene

The inventor clones a Bsr1 gene related to BSMV resistance from brachypodium distachyon, the cDNA sequence of the gene is shown as sequence 2 in a sequence table, the protein coded by the gene is named as Bsr1 protein, and the amino acid sequence of the Bsr1 protein is shown as sequence 3 in the sequence table. The specific cloning method is as follows:

extracting the total RNA OF brachypodium distachyon, carrying out reverse transcription on the extracted RNA sample according to the procedure OF a first strand cDNA synthesis kit OF Tiangen biochemistry company to synthesize first strand cDNA serving as a template OF gene cloning, and carrying out reverse transcription on the RNA sample by using Bsr1-OF 1: 5'-ATGGCTGGGTTAATTGTGAGCGCTTC-3' and Bsr1-OR 1: 5'-TGTTCGTATTAAATTGAAAACGCGGACTAGGACG-3' is used as a primer for PCR amplification. After the PCR reaction is finished, the agarose gel electrophoresis detection is carried out, and a target PCR band is recovered and sequenced.

The sequencing result shows that: and carrying out PCR amplification to obtain the gene shown in the sequence 2 in the sequence table. The gene shown in the sequence 2 in the sequence table is named as Bsr1 gene, the protein coded by the Bsr1 gene is named as Bsr1 protein, and the amino acid sequence of the Bsr1 protein is shown as the sequence 3 in the sequence table.

Example 2 acquisition of transgenic Brettanomyces brachypus with Bsr1 and analysis of BSMV resistance

First, obtaining of the transformed Bsr1 brachypodium

1. Construction of overexpression vector pMBsr1

An overexpression vector pMBsr1 of the brachypodium Bsr1 gene is constructed by using Gateway technology. The method comprises the following specific steps:

the Bsr1 gene shown in the sequence 2 cloned in example 1 was ligated into pDONR221 vector (Tiandz, cat # 60908-2426) by Gateway method to obtain a recombinant vector, and sequencing was performed.

And (3) uniformly mixing the recombinant vector with correct sequencing verification, the pMDC32 vector and the recombinase, carrying out overnight reaction at 22 ℃, recombining the Bsr1 gene shown in the sequence 2 into the pMDC32 vector to obtain an over-expression vector pMBsr1, and carrying out sequencing verification on the over-expression vector pMBsr 1. The sequencing result shows that: the overexpression vector pMBsr1 contained the Bsr1 gene shown in sequence 2 (Bsr1 protein shown in sequence 3).

2. Construction of recombinant bacterium

And (3) transferring the overexpression vector pMBsr1 constructed in the step (1) into the agrobacterium strain LBA4404 to obtain a recombinant bacterium pMBsr1/LBA 4404.

3. Obtaining of transgenic Bsr1 brachypodium

Transforming the recombinant bacterium pMBsr1/LBA4404 constructed in the step 2 into a susceptible brachypodium strain Bd21-3 by utilizing an agrobacterium-mediated method to obtain the Bsr1 brachypodium. The specific transformation steps are as follows:

genetic transformation is carried out on the embryogenic callus of Bd21-3 by utilizing pMBsr1/LBA4404, and then a plant is obtained by culturing, namely the T0 generation plant; selfing the T0 generation plants to obtain grains, and culturing the grains into plants, namely T1 generation plants.

4. Identification of Scorzonera fasciata Bsr1

And (3) carrying out genotype identification on the T1 generation of the brachypodium through Bsr1 by using a primer pair 5'-AACCATTGACTTCTAGTTCC-3' and 5'-TTTCCAACCAAGAGGACAGG-3'. The T1 generation of Bsr1 short stalk grass with 1kb band obtained by PCR amplification is positive Bsr1 generation of T1 generation of short stalk grass (figure 1A), and the T1 generation of Bsr1 short stalk grass with 1kb band obtained by PCR amplification is negative plant.

Second, BSMV resistance detection of Bsr 1-transferred brachypodium

Inoculating a barley stripe mosaic virus strain ND18 (BSMV ND18) into the positive T1 generation trans-Bsr 1 brachypodium, detecting the BSMV ND18 resistance of the trans-Bsr 1 brachypodium, and taking a disease-resistant strain Bd3-1 and a disease-susceptible strain Bd21-3 as controls. Specific detection procedures are described in references "Mi Yeon Lee, Lijie Yan, Florin A. Gorter, Brian Y. T. Kim, Yu Cui, Yue Hu, Cheng Yuan, Jessian Grindheim, Uma Ganesan, Zhiyong Liu, Chenggui Han, Jianlin Yu, Dawei Li and Andrew O. Jackson, Brachydium dischoin line Bd3-1resistance and is said tubular strain gene block 1movement protein.2012, Journal of General Virology,93: 2729-.

The results are shown in FIG. 1B. As can be seen from the figure: after 50 days of virus inoculation, positive Bsr1 brachypodium strains L2-14 and L2-20 of T1 generation are consistent with the phenotype of a disease-resistant strain Bd3-1, symptoms such as scab and plant dwarfing do not appear, while negative plants are consistent with the phenotype of a disease-sensitive strain Bd21-3, and obvious plant dwarfing appears. The Bsr 1-transferred brachypodium positive strain obtains the resistance to the BSMV ND18, and the Bsr1 can improve the BSMV resistance of brachypodium.

Example 3 acquisition of transgenic barley of Bsr1 and analysis of its BSMV resistance

First, obtaining of Bsr1 transferred barley

1. Construction of overexpression vector pCBsr1

The DNA fragment between the Eco53KI and XbaI enzyme cutting sites of the pCAMBIA1300 vector is replaced by the Bsr1 gene shown in the sequence 2, and other sequences of the pCAMBIA1300 vector are kept unchanged to obtain an over-expression vector pCBsr1, and sequencing verification is carried out on the over-expression vector pCBsr 1. The sequencing result shows that: the overexpression vector pCBsr1 contains the Bsr1 gene shown in sequence 2 (Bsr1 protein shown in sequence 3).

2. Construction of recombinant bacterium

And (3) transforming the overexpression vector pCBsr1 constructed in the step (1) into an agrobacterium strain EHA105 to obtain a recombinant bacterium pCBsr1/EHA 105.

3. Obtaining of Bsr1 transferred barley

And (3) transforming the recombinant bacteria constructed in the step (2) into young embryo callus of a barley variety Golden Promise by using an agrobacterium infection method to obtain Bsr1 transformed barley. The specific transformation steps are as follows:

carrying out genetic transformation on the embryogenic callus of the Golden Promise by utilizing pCBsr1/EHA105, and then culturing to obtain a plant, namely a T0 generation plant; selfing the T0 generation plants to obtain grains, and culturing the grains into plants, namely T1 generation plants.

4. Identification of Bsr 1-transferred barley

The T1 generation Bsr1 barley obtained in step 3 was genotyped using primer pairs 5'-AACCATTGACTTCTAGTTCC-3' and 5'-TTTCCAACCAAGAGGACAGG-3'. Bsr1 barley transformed from T1 and obtained 1kb band by PCR amplification is Bsr1 barley transformed from T1 (FIG. 2C), and Bsr1 barley transformed from T1 and obtained 1kb band by PCR amplification is negative plant. And selecting positive Bsr1 barley strains CBB3-211, CBB6-11 and CBB8-99 of the T1 generation to carry out BSMV resistance detection.

Second, BSMV resistance detection of Bsr1 transferred barley

The positive T1-generation trans-Bsr 1 barley obtained in the first step was inoculated with barley streak mosaic virus strain ND18 (BSMV ND18) and barley streak mosaic virus Norwich strain (BSMV Norwich), and BSMV ND18 and BSMV Norwich resistance of trans-Bsr 1 barley were examined while virus-inoculated barley variety Golden Promise (non-transgenic) and Mock-treated (non-inoculated) barley variety Golden Promise (Mock) were used as controls. Specific detection procedures are described in references "Mi Yeon Lee, Lijie Yan, Florin A. Gorter, Brian Y. T. Kim, Yu Cui, Yue Hu, Cheng Yuan, Jessian Grindheim, Uma Ganesan, Zhiyong Liu, Chenggui Han, Jianlin Yu, Dawei Li and Andrew O. Jackson, Brachydium dischoin line Bd3-1resistance and is said tubular strain gene block 1movement protein.2012, Journal of General Virology,93: 2729-.

The results are shown in FIG. 2A. As can be seen from the figure: 14 days after inoculation with the virus, the Bsr 1-transferred barley positive plants showed disease resistance to BSMV ND18, no streaking and chlorosis in the leaves, and no accumulation of the virus was detected with primers BSMV10-32 (5'-GGTGCTTGATGCTTTGGATAAGG-3' and 5'-TGGTCTTCCCTTGGGGGAC-3'). While the negative control plants and leaves of wild-type barley of Golden Promise (non-transgenic) showed marked streaking and chlorosis, and accumulation of virus was detected with the primer BSMV 10-32. The Bsr1 transferred barley positive plants are infected by BSMV Norwich, the leaves have streaks and chlorosis, and the accumulation of viruses can be detected by using the primer BSMV 10-32. The above results indicate that the Bsr 1-transgenic barley-positive plants acquired resistance to BSMV ND18, but not to BSMV Norwich.

Example 4 acquisition of Bsr1 transgenic wheat and analysis of its BSMV resistance

One, Bsr1 wheat

1. Construction of overexpression vector pCBsr1

The DNA fragment between the Eco53KI and XbaI enzyme cutting sites of the pCAMBIA1300 vector is replaced by the Bsr1 gene shown in the sequence 2, and other sequences of the pCAMBIA1300 vector are kept unchanged to obtain an over-expression vector pCBsr1, and sequencing verification is carried out on the over-expression vector pCBsr 1. The sequencing result shows that: the overexpression vector pCBsr1 contains the Bsr1 gene shown in sequence 2 (Bsr1 protein shown in sequence 3).

2. Bsr1 wheat transfer

The overexpression vector pCBsr1 constructed in the step 1 is co-transformed into the young embryo callus of wheat variety Kenong 199 by using a gene gun bombardment method. The specific transformation steps are as follows:

carrying out genetic transformation on the embryogenic callus of Kenong 199 by using pCBsr1/EHA105, and then culturing to obtain a plant, namely a T0 generation plant; selfing the T0 generation plants to obtain grains, and culturing the grains into plants, namely T1 generation plants.

4. Identification of Bsr 1-transferred wheat

And (3) carrying out genotype identification on the T1 generation Bsr1 wheat obtained in the step 3 by adopting a primer pair 5'-AACCATTGACTTCTAGTTCC-3' and 5'-TTTCCAACCAAGAGGACAGG-3'. The Bsr1 wheat transformed from T1 with 1kb band obtained by PCR amplification is T1 positive Bsr1 wheat (figure 2D), and the Bsr1 wheat transformed from T1 with 1kb band obtained by PCR amplification is negative plant. And selecting positive Bsr1 wheat strains CBW119-1, CBW94-18 and CBW94-3 of the T1 generation to carry out BSMV resistance detection.

Second, BSMV resistance detection of Bsr 1-transferred wheat

The positive Bsr1 wheat of T1 generation obtained in the first step was inoculated with barley streak mosaic virus strain ND18 (BSMV ND18) and barley streak mosaic virus Norwich strain (BSMV Norwich), and BSMV ND18 and BSMV Norwich resistance of the Bsr1 wheat were examined while virus-inoculated wheat cultivar farmer 199(non-transgenic) and Mock-treated wheat cultivar farmer 199(Mock) were used as controls. Specific detection procedures are described in references "Mi Yeon Lee, Lijie Yan, Florin A. Gorter, Brian Y. T. Kim, Yu Cui, Yue Hu, Cheng Yuan, Jessian Grindheim, Uma Ganesan, Zhiyong Liu, Chenggui Han, Jianlin Yu, Dawei Li and Andrew O. Jackson, Brachydium dischoin line Bd3-1resistance and is said tubular strain gene block 1movement protein.2012, Journal of General Virology,93: 2729-.

The results are shown in FIG. 2B. As can be seen from the figure: 14 days after inoculation of the virus, the Bsr 1-transferred wheat positive plants showed disease resistance to BSMV ND18, no streaks and chlorosis appeared in leaves, and no accumulation of the virus was detected by primers BSMV10-32 (5'-GGTGCTTGATGCTTTGGATAAGG-3' and 5'-TGGTCTTCCCTTGGGGGAC-3'). While the negative control plant and wild type wheat variety Ke nong 199(non-transgenic) leaf had obvious streaks and chlorosis, and the accumulation of virus was detected by using the primer BSMV 10-32. The Bsr 1-transferred wheat positive plants are infected by BSMV Norwich, the leaves have streaks and chlorosis, and the accumulation of viruses can be detected by using the primer BSMV 10-32. The above results indicate that Bsr 1-transgenic wheat positive plants acquired resistance to BSMV ND18, but not to BSMV Norwich.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of genetics and developmental biology of Chinese academy of sciences

<120> brachypodium japonicum Bsr1 protein and coding gene and application thereof

<160>3

<170>PatentIn version 3.5

<210>1

<211>7282

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

caacatgcca cgtactccac ttgtattggc tcctgctagt agctgcccca cacctctctc 60

tctcctttcc tcgttggctg cctgcatatg cctctctctc tctagatctc tctctcatta 120

ttgggctcca caaaagtgga aaactcaagc tgccatagga cttttctcta tatggtttga 180

tactttgatt tatttttggg gagaggagct gcatgctacc aagtcatcaa attagattat 240

ttttttaatt agataaggga agatttagat agaccattag ttttttttta aaataaaata 300

aatggttgcc tccggacgaa gagtgaacgg aggaccaata taaaagtcgg ttatatatag 360

cactagagat ggcccgtgga gacttaggtt tctcgacacg tgcctgctcg atcttagtgg 420

ctagctagct gggggactac gacatatctg gcgctttgtt ttttttaaga ttcgcgtgcg 480

tggtcctggt aacatacggg ccagaaactg ttcacaagat aagattccgc acatgcatct 540

ttttagtctc gttaattata tattgtcgcg tgagttctac gggcgtgtgc ctgtagcgtt 600

tgggcctatg cacaattgcg gtgcaaattt tgaccggcga gaccatctat tgtagaagac 660

tattccttct cagcctagct aggccaagtg agcacgcgcc acttggtcct caacaggacc 720

tgagcctctc agtggcgaac aaaacctcaa gcttctggtt tgtctaaggg cgggtctttt 780

aggaccataa gacttgtcgg acccttaaat ttgtataacc cattgcgtgt atagcacaat 840

gttttcacat tgtcatctct aattaagtga gacgaatggc catgagaagg ctgatatcac 900

aacatctgga tttctataac caaggatgtg tgtctgatgg accgaataaa tatatttttc 960

ccatgcttgc ttatgtatgc gtgccaaatt actctgcatg ccaataggat aggcatacat 1020

atatatgccg ccaagccaat cgttccatgc acccacccct gatctggatc agacgcatgc 1080

aatggcaatg catcatctca aaagaaagaa gaatggacta attaatgaaa ccagattaag 1140

tgcatgtgtt tgttgcagca tcagctaagc tgagctgggt agcctcaaaa ggcaaacaag 1200

gaatctccct atccagtgtg catctcttgt gttgttttca ccactaccga aatagacaag 1260

tggagcctta cctctttgga ccgcccaaaa gacgactatt cttccgttcc tcgtctccac 1320

gtgtcgaccg tccattgctg gcggcccagg tgggcctgga gtccgtacga tgggcgcgtg 1380

cgggccgggt cgtacggagc gaacgggctg tcgacacgtg tactatgatg gaccaccacg 1440

aaacgacgcg gcccaagtgg gctttctctt catgctgcgc tttggcagcg gcacgactct 1500

cgacgcaccg gtggtccagt tcatttcttt tttcaaagaa gcaaattcca gggagtagag 1560

cccaagtggt gtctgggtaa caaatcgagg cccagcacag cccaatccgg accacacgag 1620

gttagcaaac cgatttattc ttttatcgtt cagtagcaaa tggacacagc aaaatagaaa 1680

caaagaagat aattcagagg ctggaaagtc aaactcccag aggcaacata tatgcagctc 1740

aatctgattc agaattgacg aaatgaggta taacacgaat caattaaagc tgtagctcca 1800

tcttttctag ctgggacatc catctccatg tctgttaatc gtactgacat gctacatgtt 1860

gtcccaaata acacaagttt gcatccactc cttaatttcc tgaaaaccaa tatcatgttc 1920

gtattaaatt gaaaacgcgg actaggacac gaggatggct gggatgcatg ttgatagcac 1980

tcctcaaggc tgactctgcg cttgccatct cggattccgt agcgtacctg caccagataa 2040

atgcggacaa ttcttcaagt gctgacaagt gctccatgcc ggcaggcccg gccccgttct 2100

gctccgatcc gctgccattg tagctcagca cgagcctctg gagcttgggc atcgcaccgg 2160

cctggaacgt caggtaccac gggctgctta aaccattgac ttctagttcc tcgaggttac 2220

aaagtcttcc caaggaggag catagggcat ccatgcgttt ctccttgtca tccccaaggt 2280

tgtcgcagtc gagacgtaga tacctcagat tggtcagccc cccaaggccc ttaacattat 2340

ctagcgtgtt tagcgctaaa ttaaatggtc ttagatatcg tagggacttt aagttgccaa 2400

tctcatcagg tagcctacca taagcatcca gatgcatcaa gtgtggtaga tgaactgtat 2460

ctgatggtat actgtcacaa ccgggcacct caagtgactg caagtgtcgt agcactttaa 2520

tctgtgttgg cagttggtat gaacaatctt gactaatcga tagatacctc agctgataga 2580

gtttacacag tccagtgagg tcaactctag cacccagttc gtcatcaaca aaaagaactc 2640

ggaggaactt gaactctgac agagaaggtg tatttcgaga gctcccaaag aacataactg 2700

accgaacctg tgacacacta gtgttcttcg gtagtattgt ctgaccacta cttgcaccat 2760

ccaagcggat agacagccgg cggaccttgt aatccagtcc tgcaaaatct tgtgggtcat 2820

ccactacagt gagaaaattc tcttctgcag acttaagcaa gataagatcg agcatcatat 2880

cgtgaactct gtaatgtgtc cctatatgat taaattctac gggctggata aggctcctat 2940

tcacaagctc gttgaaataa tttcttgcgg tcttctccac atcctttcca ttttccttac 3000

cgataaaacc ttcggccatc cattgacgtt ccaaagcaat cctgtctatt aggtggtcct 3060

ccggatacat accgagatac aataaacatg tcttgagatg aggaggaaga tctttgtaac 3120

tgaggtttaa gattttcctc accccttcca atgtgaggtt tgttcctgag ccgagagaat 3180

catgtacatg cttccacctg tcctcttggt tggaaacttc cctggctagc atgctagata 3240

tactaatgat tgcaagaggc aaaccgccac acttcttcag aatttcaatc gaaacatctc 3300

tgagctgttg agggcaagct tgttccgagc caaatatcct ttcaaaaaac aattttgttg 3360

aatcttcatt gctaaggggc ttcatttgta aaatgtaatc acgacgacga tcagagcgac 3420

atgccgcggc cacttcctga attcttgtag ttactattac tctgctgcca agatcacttt 3480

ctgggaatgc acatttaatt atttcccagg cagatacagt ccatatgtca tcaattataa 3540

tcaagtacct gccgaacatt aagattacag tcacacgagc agcagaaatt agcaaataat 3600

aaagtcgaaa caaataaagt taactttatc cattcaaata tagttgcgat tgaataacaa 3660

agtagactaa tttattaact ggattgctaa taatcgaaat tatgaaaaat aaaaatatat 3720

aaactgaaat taattttaat ttaagaaatg ttgttcagtt acaagaaact cagcagtcaa 3780

ttcaaactga gctgattttc tcatacaagc tttttagatg caatgaatag atgaatattc 3840

ttttcagaat ttttcttttt tgtgtttatt atccttatct aatggttttc tcccaagaat 3900

ttactcatga aagtgcaaca tatttccttt ccattttttg ggactgaact tgacatatat 3960

acaatactat aatattcgag aaaatttatg atcaaagaca ataacaaaca acagcacatg 4020

aagcaagcaa gaatatgaac ctcttatttt gtagatgttc tctgaggtcg tcgagaagaa 4080

tatttaactc gcaatcatga ggaaatggtg tacccccaag ttgggataat aaagtacgga 4140

gaaggtttgg gatgtttggt ttttgagata ctggtacaaa agcacaacag tcatatttgc 4200

ccttgtcctt gagcgtagca taaaccgcat tggcaagtgt tgttttgcct agacctccaa 4260

atccaacaat tgataccacc ttcaactgct tctcctcatc aattagccaa tcgacaatct 4320

cattctttgg cccttccata ccaacaagat tagccgcttt ttcatagagt gctttaactc 4380

gtgggtccat ggccacatcg cttgacggcg ggatatcaag cttgtatctt tggcgccgtg 4440

cacttgtctc aagcacaagt gccttgatct ctttaatctg gctggcaatc tgatggcggg 4500

atctcaactt tctgagtagt cgagcagtct tccttccaag accatcggtt tcacttttct 4560

cctcaatgtg gtgcatgaag tcatcaatga tgtcctcaat atcataagac atctctctca 4620

cttggtctct ccatcccttt gtttgcttgt ctagctcgtc caaatctgaa agcctctcaa 4680

gggcatcctt catactggta agctcctcac tgataaactt gacctctttc cgcaagttct 4740

tgagtttggc aaactcctcc gccatgaggg tggctagctt aaccaagagt ggtttcaaca 4800

cgcctgtcga agcgctcaca attaacccag ccatggatct cttgctggac aattgtgtat 4860

cttggatcct agttatgctt gtaaggtacc tgcaacgtgg gcgttaaata gaccagagct 4920

ttgagctttc gatctatatc tcatattata aaacgacata cttctatgca tgcatgtatc 4980

cctagcaagt ttaaatgaga tgatacaacc aaaatcaaca taggccgggg tagatatact 5040

tggattaccc catatgtttt tcttaaattt ggaattttta tgtaatactc tcttctcttc 5100

atttagaaat gataaagagt attttaacta gactccaagt tcagtaagca gctttgacaa 5160

caatactttt tgcacaatag tgttagtcct agactcgtag taacaagata ctccctcgtc 5220

caggtttgta aggcctacgc ggaattttat gtcaaacttt gaccatttcc gcgcctggtc 5280

ggcgagggag cacaagaagg agtaagccga acgcacgcgc tgccttggcc tcaccagcga 5340

gctcgacaag gacggggatg gggaggagtg ggacgacgac gagtagatta gttgtttttt 5400

tagaattttg tataaatttc gccccaacta gttaaattta tctcttttta aatttattta 5460

tccctttttg gatttattta ttttagattt ggtattgggg gcagggctgc ggatccccaa 5520

attaaaatat ttgtgccccc catccggcct acaaaacggc agcgccagac gccgaatggg 5580

gggacgcgag tggagatgct ctacctgtac ggaagtgttt tgaatacgga atcccaatgg 5640

ttttgtgatt ttagatgaca cattgcctac tatacatgtg ttgtcattca tggctggctt 5700

tagaaatttg tgctacaaag gcaagaagag accgagagac gaaattaaga tttgaggtct 5760

gcttctgtat aaaccccctc ccaatttgaa aagggtatta tgaacttttc acgttggtaa 5820

attttttaaa agaaaatcag attttttttt ctttctcatc actgccgcag ccttccctcg 5880

gcagttggcg tgtgctgtgc catggcagat ctggatggaa ttggaaagag agcaaggcct 5940

gcagcctgcg attgccttgc ccatggaatg gaagatagta aatcgatcca tgcatcaagc 6000

attttctttt acagtgaatc gatccatcat ggactcttca tactggagct tattgccatt 6060

cgtctaacca tgtgaacagg aacagccatg caacttgaat caattatcac tacacgatta 6120

gacgcgcgag atctgacgcc gggcgcgaaa gaaggcaggg gtggtggggg tttgggtaga 6180

cagaggaaga ggtactgtaa atttaatact ctcctgatgg atttagatag ccatagcctc 6240

ttggatcgaa tgaaatagta tgtaggaaag cacttgaagg tccatacata cactctagga 6300

catacccata cagttttaaa aagagactgc tgagattcat acactctaaa aacaacttga 6360

aaggctgaag aaactgctga gattcatacg ctctaaaaac aacttgaaag gctgaagaaa 6420

ctgctgagat tcatacgctc taaaaacaac ttgaaggctg agaaaacctc tgagatccat 6480

acgctctaac gtaacatcgt acgtacctca agtctgaatc tttgtcgcgc aggcaggatc 6540

acaacagatc tctcaagtct gaatctctgt tcctcatgac ggcaggagga gaatggaacg 6600

aagaagggaa gaggatggtt aaggtccgga tgcggtggag gaaacggccg ggtgagattg 6660

tgacaaggaa cgaacagaga agctgctgaa ttattggctt ctacagtagc ttactagctc 6720

ccagtacagc ctgctgaatt attggcttcc acagtgagct ggctgtgcac atgcctggta 6780

ccatcactcc ttaattgcgc attgtcacta ctcactgtgc atgcgccgga gtagaggaca 6840

gaggacgcag gcgctgagac aagtagacag aaccacaatc aattttcttc aggaccgtcg 6900

aatatctccc ggtagaatat ctcctcctcc tcgtcatgta gaatatctcg aaaaagtgaa 6960

atcagcttat atatggtcgc catcaaaatg tgtgatatga aaacattggc gcgaggtctt 7020

tttgttttcg gagatttttc gtctgttttt tcacatgttc cacaacccaa aacatgtgct 7080

cctcgtttga acacttaaat tgaatccgaa ggcatttttc cttttgcatc gttccaagac 7140

atttccattt tcggcctcga tctcctccac tagttgccgg gcataaactc tccgctatag 7200

cttatggaac acgtcaagtc tgtcaaccgt gttgcctcgt tgaacgaata cgttttcact 7260

tgatgaaagc ggaatcatgg aa 7282

<210>2

<211>2430

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

atggctgggt taattgtgag cgcttcgaca ggcgtgttga aaccactctt ggttaagcta 60

gccaccctca tggcggagga gtttgccaaa ctcaagaact tgcggaaaga ggtcaagttt 120

atcagtgagg agcttaccag tatgaaggat gcccttgaga ggctttcaga tttggacgag 180

ctagacaagc aaacaaaggg atggagagac caagtgagag agatgtctta tgatattgag 240

gacatcattg atgacttcat gcaccacatt gaggagaaaa gtgaaaccga tggtcttgga 300

aggaagactg ctcgactact cagaaagttg agatcccgcc atcagattgc cagccagatt 360

aaagagatca aggcacttgt gcttgagaca agtgcacggc gccaaagata caagcttgat 420

atcccgccgt caagcgatgt ggccatggac ccacgagtta aagcactcta tgaaaaagcg 480

gctaatcttg ttggtatgga agggccaaag aatgagattg tcgattggct aattgatgag 540

gagaagcagt tgaaggtggt atcaattgtt ggatttggag gtctaggcaa aacaacactt 600

gccaatgcgg tttatgctac gctcaaggac aagggcaaat atgactgttg tgcttttgta 660

ccagtatctc aaaaaccaaa catcccaaac cttctccgta ctttattatc ccaacttggg 720

ggtacaccat ttcctcatga ttgcgagtta aatattcttc tcgacgacct cagagaacat 780

ctacaaaata agaggtactt gattataatt gatgacatat ggactgtatc tgcctgggaa 840

ataattaaat gtgcattccc agaaagtgat cttggcagca gagtaatagt aactacaaga 900

attcaggaag tggccgcggc atgtcgctct gatcgtcgtc gtgattacat tttacaaatg 960

aagcccctta gcaatgaaga ttcaacaaaa ttgttttttg aaaggatatt tggctcggaa 1020

caagcttgcc ctcaacagct cagagatgtt tcgattgaaa ttctgaagaa gtgtggcggt 1080

ttgcctcttg caatcattag tatatctagc atgctagcca gggaagtttc caaccaagag 1140

gacaggtgga agcatgtaca tgattctctc ggctcaggaa caaacctcac attggaaggg 1200

gtgaggaaaa tcttaaacct cagttacaaa gatcttcctc ctcatctcaa gacatgttta 1260

ttgtatctcg gtatgtatcc ggaggaccac ctaatagaca ggattgcttt ggaacgtcaa 1320

tggatggccg aaggttttat cggtaaggaa aatggaaagg atgtggagaa gaccgcaaga 1380

aattatttca acgagcttgt gaataggagc cttatccagc ccgtagaatt taatcatata 1440

gggacacatt acagagttca cgatatgatg ctcgatctta tcttgcttaa gtctgcagaa 1500

gagaattttc tcactgtagt ggatgaccca caagattttg caggactgga ttacaaggtc 1560

cgccggctgt ctatccgctt ggatggtgca agtagtggtc agacaatact accgaagaac 1620

actagtgtgt cacaggttcg gtcagttatg ttctttggga gctctcgaaa tacaccttct 1680

ctgtcagagt tcaagttcct ccgagttctt tttgttgatg acgaactggg tgctagagtt 1740

gacctcactg gactgtgtaa actctatcag ctgaggtatc tatcgattag tcaagattgt 1800

tcataccaac tgccaacaca gattaaagtg ctacgacact tgcagtcact tgaggtgccc 1860

ggttgtgaca gtataccatc agatacagtt catctaccac acttgatgca tctggatgct 1920

tatggtaggc tacctgatga gattggcaac ttaaagtccc tacgatatct aagaccattt 1980

aatttagcgc taaacacgct agataatgtt aagggccttg gggggctgac caatctgagg 2040

tatctacgtc tcgactgcga caaccttggg gatgacaagg agaaacgcat ggatgcccta 2100

tgctcctcct tgggaagact ttgtaacctc gaggaactag aagtcaatgg tttaagcagc 2160

ccgtggtacc tgacgttcca ggccggtgcg atgcccaagc tccagaggct cgtgctgagc 2220

tacaatggca gcggatcgga gcagaacggg gccgggcctg ccggcatgga gcacttgtca 2280

gcacttgaag aattgtccgc atttatctgg tgcaggtacg ctacggaatc cgagatggca 2340

agcgcagagt cagccttgag gagtgctatc aacatgcatc ccagccatcc tcgtgtccta 2400

gtccgcgttt tcaatttaat acgaacatga 2430

<210>3

<211>809

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Met Ala Gly Leu Ile Val Ser Ala Ser Thr Gly Val Leu Lys Pro Leu

1 5 10 15

Leu Val Lys Leu Ala Thr Leu Met Ala Glu Glu Phe Ala Lys Leu Lys

20 25 30

Asn Leu Arg Lys Glu Val Lys Phe Ile Ser Glu Glu Leu Thr Ser Met

35 40 45

Lys Asp Ala Leu Glu Arg Leu Ser Asp Leu Asp Glu Leu Asp Lys Gln

50 55 60

Thr Lys Gly Trp Arg Asp Gln Val Arg Glu Met Ser Tyr Asp Ile Glu

65 70 75 80

Asp Ile Ile Asp Asp Phe Met His His Ile Glu Glu Lys Ser Glu Thr

85 90 95

Asp Gly Leu Gly Arg Lys Thr Ala Arg Leu Leu Arg Lys Leu Arg Ser

100 105 110

Arg His Gln Ile Ala Ser Gln Ile Lys Glu Ile Lys Ala Leu Val Leu

115 120 125

Glu Thr Ser Ala Arg Arg Gln Arg Tyr Lys Leu Asp Ile Pro Pro Ser

130 135 140

Ser Asp Val Ala Met Asp Pro Arg Val Lys Ala Leu Tyr Glu Lys Ala

145 150 155 160

Ala Asn Leu Val Gly Met Glu Gly Pro Lys Asn Glu Ile Val Asp Trp

165 170 175

Leu Ile Asp Glu Glu Lys Gln Leu Lys Val Val Ser Ile Val Gly Phe

180 185 190

Gly Gly Leu Gly Lys Thr Thr Leu Ala Asn Ala Val Tyr Ala Thr Leu

195 200 205

Lys Asp Lys Gly Lys Tyr Asp Cys Cys Ala Phe Val Pro Val Ser Gln

210 215 220

Lys Pro Asn Ile Pro Asn Leu Leu Arg Thr Leu Leu Ser Gln Leu Gly

225 230 235 240

Gly Thr Pro Phe Pro His Asp Cys Glu Leu Asn Ile Leu Leu Asp Asp

245 250 255

Leu Arg Glu His Leu Gln Asn Lys Arg Tyr Leu Ile Ile Ile Asp Asp

260 265 270

Ile Trp Thr Val Ser Ala Trp Glu Ile Ile Lys Cys Ala Phe Pro Glu

275 280 285

Ser Asp Leu Gly Ser Arg Val Ile Val Thr Thr Arg Ile Gln Glu Val

290 295 300

Ala Ala Ala Cys Arg Ser Asp Arg Arg Arg Asp Tyr Ile Leu Gln Met

305 310 315 320

Lys Pro Leu Ser Asn Glu Asp Ser Thr Lys Leu Phe Phe Glu Arg Ile

325 330 335

Phe Gly Ser Glu Gln Ala Cys Pro Gln Gln Leu Arg Asp Val Ser Ile

340 345 350

Glu Ile Leu Lys Lys Cys Gly Gly Leu Pro Leu Ala Ile Ile Ser Ile

355 360 365

Ser Ser Met Leu Ala Arg Glu Val Ser Asn Gln Glu Asp Arg Trp Lys

370 375 380

His Val His Asp Ser Leu Gly Ser Gly Thr Asn Leu Thr Leu Glu Gly

385 390 395 400

Val Arg Lys Ile Leu Asn Leu Ser Tyr Lys Asp Leu Pro Pro His Leu

405 410 415

Lys Thr Cys Leu Leu Tyr Leu Gly Met Tyr Pro Glu Asp His Leu Ile

420 425 430

Asp Arg Ile Ala Leu Glu Arg Gln Trp Met Ala Glu Gly Phe Ile Gly

435 440 445

Lys Glu Asn Gly Lys Asp Val Glu Lys Thr Ala Arg Asn Tyr Phe Asn

450 455 460

Glu Leu Val Asn Arg Ser Leu Ile Gln Pro Val Glu Phe Asn His Ile

465 470 475 480

Gly Thr His Tyr Arg Val His Asp Met Met Leu Asp Leu Ile Leu Leu

485 490 495

Lys Ser Ala Glu Glu Asn Phe Leu Thr Val Val Asp Asp Pro Gln Asp

500 505 510

Phe Ala Gly Leu Asp Tyr Lys Val Arg Arg Leu Ser Ile Arg Leu Asp

515 520 525

Gly Ala Ser Ser Gly Gln Thr Ile Leu Pro Lys Asn Thr Ser Val Ser

530 535 540

Gln Val Arg Ser Val Met Phe Phe Gly Ser Ser Arg Asn Thr Pro Ser

545 550 555 560

Leu Ser Glu Phe Lys Phe Leu Arg Val Leu Phe Val Asp Asp Glu Leu

565 570 575

Gly Ala Arg Val Asp Leu Thr Gly Leu Cys Lys Leu Tyr Gln Leu Arg

580 585 590

Tyr Leu Ser Ile Ser Gln Asp Cys Ser Tyr Gln Leu Pro Thr Gln Ile

595 600 605

Lys Val Leu Arg His Leu Gln Ser Leu Glu Val Pro Gly Cys Asp Ser

610 615 620

Ile Pro Ser Asp Thr Val His Leu Pro His Leu Met His Leu Asp Ala

625 630 635 640

Tyr Gly Arg Leu Pro Asp Glu Ile Gly Asn Leu Lys Ser Leu Arg Tyr

645 650 655

Leu Arg Pro Phe Asn Leu Ala Leu Asn Thr Leu Asp Asn Val Lys Gly

660 665 670

Leu Gly Gly Leu Thr Asn Leu Arg Tyr Leu Arg Leu Asp Cys Asp Asn

675 680 685

Leu Gly Asp Asp Lys Glu Lys Arg Met Asp Ala Leu Cys Ser Ser Leu

690 695 700

Gly Arg Leu Cys Asn Leu Glu Glu Leu Glu Val Asn Gly Leu Ser Ser

705 710 715 720

Pro Trp Tyr Leu Thr Phe Gln Ala Gly Ala Met Pro Lys Leu Gln Arg

725 730 735

Leu Val Leu Ser Tyr Asn Gly Ser Gly Ser Glu Gln Asn Gly Ala Gly

740 745 750

Pro Ala Gly Met Glu His Leu Ser Ala Leu Glu Glu Leu Ser Ala Phe

755 760 765

Ile Trp Cys Arg Tyr Ala Thr Glu Ser Glu Met Ala Ser Ala Glu Ser

770 775 780

Ala Leu Arg Ser Ala Ile Asn Met His Pro Ser His Pro Arg Val Leu

785 790 795 800

Val Arg Val Phe Asn Leu Ile Arg Thr

805

Claims (10)

1. The protein is the protein of a) or b) or c) or d) as follows:

a) the amino acid sequence is a protein shown in a sequence 3;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 3;

c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 3;

d) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the sequence 3 and having the same function.

2. The protein-related biomaterial according to claim 1, which is any one of the following a1) to a 12):

A1) a nucleic acid molecule encoding the protein of claim 1;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising a4) said recombinant vector;

A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);

A10) a transgenic plant cell line comprising the expression cassette of a 2);

A11) a transgenic plant cell line comprising the recombinant vector of a 3);

A12) a transgenic plant cell line comprising the recombinant vector of a 4).

3. The related biological material according to claim 2, wherein: A1) the nucleic acid molecule is a gene shown in the following 1) or 2) or 3):

1) the coding sequence is a cDNA molecule shown in a sequence 2 or a genome DNA molecule shown in a sequence 1;

2) a cDNA molecule or genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) and encoding the protein of claim 1;

3) a cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions with a nucleotide sequence defined in 1) or 2) and encodes a protein according to claim 1.

4. Use of the protein of claim 1 or the related biomaterial of claim 2 or 3) in any one of the following B1) -B5):

B1) regulating and controlling the disease resistance of plants;

B2) preparing a product for regulating and controlling the disease resistance of the plants;

B3) cultivating disease-resistant plants;

B4) preparing a product for cultivating disease-resistant plants;

B5) and (5) plant breeding.

5. Use according to claim 4, characterized in that: the disease resistance is stripe mosaic disease resistance.

6. A method for producing a disease-resistant plant, comprising the steps of increasing the expression level and/or activity of the protein of claim 1 in a recipient plant to obtain a transgenic plant; the transgenic plant has higher disease resistance than the recipient plant.

7. The method of claim 6, wherein: the disease resistance is stripe mosaic disease resistance.

8. The method according to claim 6 or 7, characterized in that: the method for increasing the expression level and/or activity of the protein of claim 1 in a recipient plant comprises overexpressing the protein of claim 1 in the recipient plant.

9. The method according to any one of claims 6-8, wherein: the method of overexpression comprises introducing a gene encoding the protein of claim 1 into a recipient plant;

or, the nucleotide sequence of the coding gene of the protein is a DNA molecule shown in sequence 2.

10. Use according to claim 4 or 5 or a method according to any of claims 6-9, wherein: the plant is a monocotyledon or a dicotyledon;

alternatively, the monocotyledonous plant may be brachypodium distachyon or barley or wheat.

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