A disease-resistant protein against Rice black-streaked dwarf virus and its application method

Abstract

The present invention discloses a disease-resistant protein ZmGLK36 against Rice black-streaked dwarf virus, which is as follows (a1) or (a2): (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing; (a2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of (a1). The present invention further discloses the gene, vector, expression cassette, transgenic cell line, transgenic plant tissue, transgenic plant organ or recombinant microorganism encoding the above protein. Through transgenic technology, the present invention obtains the disease-resistant gene ZmGLK36 against Rice black-streaked dwarf virus. After introducing this gene into maize, stable genetic transformants can be obtained. In addition, this gene can also be transformed into crops such as rice and wheat to endow them with disease-resistant characteristics, thereby reducing the usage amount of pesticides to reduce environmental pollution, and having important economic value and broad application prospects.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and biological control, and specifically, to a disease-resistant protein against Rice black-streaked dwarf virus and its application method. Background Art

[0002] Rice black-streaked dwarf virus (RBSDV), belonging to the genus Phytoreovirus, damages more than 20 host plants of the Gramineae family, such as rice, barley, wheat, corn, sorghum, millet, barnyard grass, Alopecurus aequalis Sobol., and Setaria viridis. The virus is mainly transmitted by the small brown planthopper (Laodelphax striatellus). Once the vector is infected, it remains viruliferous throughout its life, but does not transmit the virus through eggs. The virus can overwinter on winter wheat, perennial grasses, and the virus-transmitting vectors. In spring, the first-generation adult small brown planthoppers obtain the virus by feeding on the overwintering hosts and then migrate from wheat to corn successively, forming a migration peak during the wheat harvest period. The second-generation, third-generation, and fourth-generation small brown planthoppers mainly over-summer on corn and field weeds. As corn matures, they migrate to grasses. After wheat emerges in autumn, the fourth-generation small brown planthoppers migrate to wheat fields to transmit the virus and cause damage, and then overwinter, forming an annual infection cycle.

[0003] After being infected by Rice black-streaked dwarf virus, rice plants that are infected early show obvious dwarfing and cannot head; those that are infected late have small ears and poor seed setting. After corn seedlings are damaged by black-streaked dwarf virus, the leaf color is dark green, the root system is small, the internodes are thick and short, dwarfed, the heart leaves cannot unfold normally, are clustered, the seedlings are short, similar to ginger leaves. In the adult stage of corn, infected plants have swollen lower parts, shortened internodes, are dwarfed, thick, and have unevenly thick and thin waxy white strip-like protrusions on the leaf veins of the leaf back, leaf sheath, and bract. At the 9-10 leaf stage, the plants are severely dwarfed. Although some male inflorescences can emerge, there are very few branches; the female inflorescence is short, with few silks, deformed, and cannot set seeds severely. On a small number of ears that form cobs, the grains are sparse and not well-developed, uneven. After wheat is infected by black-streaked dwarf virus, the diseased plants are dwarfed, have increased tillering, dark green leaf color, thick and hard leaf texture, are slightly twisted, have late and few heading, the ear stalks are retracted in the leaf sheath, and have poor seed setting.

[0004] Regarding the control of Rice black-streaked dwarf virus, at present, in addition to selecting disease-resistant varieties, it is mainly chemical control. However, the current control methods cannot effectively eradicate Rice black-streaked dwarf virus and there are residue problems. How to ensure the safe production of crops, improve the quality and yield of crops, and conduct effective biological control is an urgent problem to be solved. Summary of the Invention

[0005] The purpose of the present invention is to provide a disease-resistant protein ZmGLK36 against Rice black-streaked dwarf virus, its expression vector, and application.

[0006] Another purpose of the present invention is to provide a method for improving the resistance of crops to black-streaked dwarf virus.

[0007] The object of the present invention can be further achieved by the following technical measures:

[0008] To achieve the object of the present invention, the present invention first provides a disease-resistant protein ZmGLK36 against rice black-streaked dwarf virus, which is as follows (a1) or (a2):

[0009] (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing;

[0010] (a2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of (a1).

[0011] The ZmGLK36 protein can be artificially synthesized, or its coding gene can be synthesized first and then biotically expressed.

[0012] Furthermore, the present invention also provides a nucleic acid molecule encoding the above protein. The nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA. The nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.

[0013] The gene encoding the ZmGLK36 protein is named the ZmGLK36 gene.

[0014] The nucleic acid molecule is a DNA molecule as follows (b1) or (b2) or (b3) or (b4):

[0015] (b1) The nucleotide shown in SEQ ID NO: 1; or

[0016] (b2) The nucleotide shown in SEQ ID NO: 2; or

[0017] (b3) The nucleotide encoding the amino acid shown in SEQ ID NO: 3; or

[0018] (b4) A nucleotide that can hybridize with the complementary sequence of SEQ ID NO: 1 under stringent hybridization conditions, and the protein encoded by the nucleotide has the function of the ZmGLK36 transcription factor.

[0019] Those skilled in the art can easily mutate the ZmGLK36 gene provided by the present invention by known methods, such as directed evolution and point mutation methods. Those nucleic acids that have been artificially modified and have 90% or higher identity with the ZmGLK36 gene provided by the present invention, as long as they encode the ZmGLK36 protein and have the function of the ZmGLK36 protein, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0020] As used herein, the term "identity" refers to sequence similarity to a native nucleic acid sequence. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0021] Furthermore, the present invention also provides recombinant expression vectors, expression cassettes, transgenic cell lines, transgenic plant tissues, transgenic plant organs or recombinant microorganisms containing the ZmGLK36 gene, which all fall within the scope of protection of the present invention.

[0022] Existing plant expression vectors can be used to construct recombinant vectors containing the ZmGLK36 gene. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. The plant expression vector may also contain the 3′ untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3′ end of the mRNA precursor. For example, the nopaline synthase gene Nos of the Agrobacterium tumefaciens Ti plasmid gene and the 3′ untranslated region transcribed by plant genes (such as soybean storage protein genes) have similar functions. When using the gene of the present invention to construct a recombinant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the adjacent region start codon, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene. Specifically, the double-digested vector P3301 or the single-digested CUB vector is selected in the present invention.

[0023] The expression cassette includes a promoter, the ZmGLK36 gene, and a terminator in sequence from upstream to downstream. Further, the expression cassette may also include an enhancer. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters.

[0024] In the examples of the present invention, the promoter that initiates the transcription of the ZmGLK36 gene in the ZmGLK36 gene expression cassette is the ATG promoter.

[0025] The starting microorganism of the recombinant microorganism can be yeast, bacteria, algae, or fungi.

[0026] The transgenic plant tissue or the transgenic plant organ does not include propagation materials.

[0027] The present invention also protects the application of the ZmGLK36 protein, which is as follows (c1) or (c2) or (c3) or (c4):

[0028] (c1) Regulating the disease resistance of plants;

[0029] (c2) Regulating the disease resistance of maize or wheat or rice;

[0030] (c3) Improving the disease resistance of plants;

[0031] (c4) Improving the disease resistance of maize or wheat or rice.

[0032] Among them, the disease resistance refers to the disease resistance against Rice black-streaked dwarf virus.

[0033] Furthermore, the present invention also provides a method for improving the resistance of crops to black-streaked dwarf virus. The method is to obtain the resistance to Rice black-streaked dwarf virus by transferring the disease resistance-related gene ZmGLK36, thereby improving the ability of crops to resist Rice black-streaked dwarf virus.

[0034] In a preferred embodiment, the sequence of the disease resistance-related gene ZmGLK36 referred to in the present invention is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0035] Specifically, the method includes the following steps:

[0036] (1) Designing primers using the sequence of the disease resistance gene ZmGLK36 in the B73 RefGen_V4 reference genome;

[0037] (2) Respectively obtaining the genome and promoter sequences of the maize inbred line Qi 319 resistant to Rice black-streaked dwarf virus by PCR amplification, and then splicing to obtain the full-length sequence of the disease resistance gene;

[0038] (3) Respectively constructing a ZmGLK36 genomic complement vector and an overexpression vector, and performing genetic transformation based on the maize inbred line Zong 31, the rice variety kitaake, and the wheat variety Filed, and verifying the ability of ZmGLK36 to resist black-streaked dwarf virus.

[0039] Advantages and beneficial effects of the present invention:

[0040] Through transgenic technology, the present invention has obtained the disease resistance-related gene ZmGLK36 against Rice black-streaked dwarf virus. After introducing the disease resistance gene into maize, stable genetic transformants can be obtained. In addition, this gene can also be transformed into crops such as rice and wheat to endow them with corresponding disease resistance characteristics, thereby reducing the use of pesticides and reducing environmental pollution, having important economic value and broad application prospects. Brief Description of the Drawings

[0041] Figure 1 5’ Race and 3’ Race analysis of ZmGLK36 in Example 2 of the present invention.

[0042] Figure 2 Genomic complementary vector P3301 in Example 3 of the present invention.

[0043] Figure 3 Identification and double digestion verification of the recombinant plasmid in Example 3 of the present invention. Here, M represents 1Kb DNA ladder, Lane 1 represents the recombinant plasmid DNA, and Lane 2 represents the recombinant plasmid DNA verified by double digestion with EcoRI-HindIII. The upper band is the vector fragment, and the lower band is the DNA fragment ligated with ZmGLK36.

[0044] Figure 4 Identification of genomic complementary transgenic positive plants in Example 3 of the present invention. Here, A) is the detection of transgenic plants by Bar test strip; B) is the PCR detection of transgenic plants.

[0045] Figure 5 Phenotype identification and RBSDV virus detection of genomic transgenic plants in Example 3 of the present invention. Here, A) and C) are the phenotype identification and analysis of genomic complementary transgenic plants after RBSDV inoculation; B) is the detection of the transcription level of coat protein P10 in genomic complementary transgenic plants after RBSDV inoculation.

[0046] Figure 6 Overexpression vector CUB in Example 4 of the present invention.

[0047] Figure 7 Construction and identification of positive plants of the overexpression transgenic vector in Example 4 of the present invention. Here, A) is the schematic diagram of the overexpression vector structure; B) is the PCR detection of transgenic plants; C) is the detection of transgenic plants by Bar test strip.

[0048] Figure 8 Field phenotype identification and RBSDV virus detection of maize transgenic plants in Example 4 of the present invention. Here, A) and C) are the phenotype identification and analysis of overexpression transgenic plants after RBSDV inoculation; B) is the detection of the transcription level of coat protein P10 in complementary transgenic plants after RBSDV inoculation.

[0049] Figure 9 Phenotype identification and RBSDV virus detection of rice transgenic plants in Example 4 of the present invention. Here, A) is the schematic diagram of the overexpression vector structure; B) is the PCR detection of transgenic plants; C) is the detection of the transcription level of P10 after transgenic rice is inoculated with RBSDV; D) is the phenotype identification after transgenic rice is inoculated with RBSDV.

[0050] Figure 10 Phenotypic identification of transgenic wheat plants and detection of RBSDV virus in Example 4 of the present invention. Among them, A): Schematic diagram of the overexpression vector structure; B): PCR detection of transgenic plants; C): Phenotypic identification of transgenic wheat after inoculation with RBSDV; D): Detection of the transcriptional level of ZmGLK36 in transgenic plants; E): Detection of the transcriptional level of P10 in transgenic wheat after inoculation with RBSDV. Specific implementation manners

[0051] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0052] Unless otherwise specified, the examples are carried out under conventional experimental conditions, such as those described in the Molecular Cloning Experiment Manual by Sambrook et al. (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or under the conditions recommended by the manufacturer's instructions.

[0053] Example 1 Obtaining of the ZmGLK36 gene sequence

[0054] According to the sequence of the disease-resistant gene ZmGLK36 in the B73 RefGen_V4 reference genome, NCBI Gene (EntrezGene): LOC100281611, primers were designed to amplify the 319 genome, including 2Kb of the upstream promoter region of ATG, 3Kb of the full-length genome, and 500bp downstream of the 3'UTR. The primers are shown in Table 1 (the primer nucleotide sequences are SEQ ID NO: 5-10) and Table 2 (the primer nucleotide sequences are SEQ ID NO: 11-18) below. Using the Qi 319 genomic DNA as a template, the promoter region and the full-length genomic sequence were amplified, and the PCR products were ligated into a vector and sequenced for comparison.

[0055] The results are as follows;

[0056] The genomic sequence of ZmGLK36 in Qi 319 (3105bp), shown as SEQ ID NO: 1;

[0057] The promoter sequence of ZmGLK36 in Qi 319 (1890bp), as shown in SEQ ID NO: 4;

[0058] Table 1 Primers for amplifying the full length of the ZmGLK36 genome

[0059]

[0060] Table 2 Primers for amplifying the ZmGLK36 promoter

[0061]

[0062]

[0063] Example 2 Obtaining the ZmGLK36 Protein Sequence

[0064] To verify the authenticity of the full-length ZmGLK36 cDNA, based on the 5’Race and 3’Race experiments as Figure 1 shown, the primer sequences are shown in Table 3 (the primer nucleotide sequences are SEQ ID NO: 19-24), and the full-length cDNA of ZmGLK36 in Qi 319 material was obtained. The length of the CDS nucleic acid sequence of ZmGLK36 in Qi 319 is 825 bp (as shown in SEQ ID NO: 2), encoding 274 amino acids (as shown in SEQ ID NO: 3), and having a typical DNA-binding domain and GCT-box.

[0065] Table 3 5'-RACE and 3'-RACE Primers

[0066]

[0067] Example 3 Construction of Genomic Vector and Phenotypic Identification of Transgenic Maize

[0068] The full-length ZmGLK36 sequence in Qi 319 was amplified by PCR, including the 3105 bp genomic sequence and 1890 bp of the promoter upstream of ATG. The vector P3301 was digested with EcoRI-HindIII to obtain a linearized vector as Figure 2 shown, the primer sequences are shown in Table 4 (the primer nucleotide sequences are SEQ ID NO: 25-28). The linearized vector was ligated with the gel recovery product of the genomic full-length by Infusion, and the ligation product was cultured overnight for 12-16 hours. Positive clones and the direction and size of the inserted fragments were identified by PCR and enzyme digestion as Figure 3 . Two monoclonal clones containing the full-length ZmGLK36 genome were obtained. After plasmid extraction and sequencing, they were transferred into Agrobacterium EHA105 by the chemical transformation method and transferred into maize immature embryos by transgenic technology to obtain transgenic plants.

[0069] The complementary vector P3301 contains the Bar gene. Therefore, transgenic plants were first detected with a Bar test strip containing Bar. The Bar protein in positive plants can be rapidly expressed, showing two bands, while negative plants cannot, indicating that the Bar test strip can effectively distinguish positive and negative plants( Figure 4A). Considering the stability of the Bar protein and the fact that high-generation transgenic plants are prone to loss due to methylation modification, histone modification, etc., it is necessary to perform PCR detection on the positive plants screened by the Bar test strip. The P3301 vector contains the GUS reporter gene. Therefore, R primers were designed for the GUS reporter gene, and the F primers were located in the self-region of the connected genome. The primer names are ZmGLK36-F / P3301-GUS-R. The detection results are as Figure 4 B. Single bands could be detected in all positive plants of the three transgenic events, while negative plants showed the opposite. In summary, the PCR identification results were consistent with the contemporary results of the Bar test strip detection.

[0070] Table 4 Primer sequences of genetic transformation vectors

[0071]

[0072] Three independent transgenic events were selected for inoculation identification. 180 seeds were selected from the transgenic receptor Zong 31 and each of the three independent events. Every 60 seeds were grouped for seedling raising. Artificial inoculation was carried out in a container at the V3 stage, and the insect inoculation ratio was 1:1, that is, one maize seedling was inoculated with 1 effective virus-carrying insect. After 48 hours of inoculation, insecticide was sprayed, and the seedlings were hardened for 1-2 days and then transplanted to the field. Normal field management was carried out, and phenotypic identification was carried out 20-30 days later. The field identification results showed that: the disease index of Zong 31 was 89.7%, the disease index of transgenic event #260-1 was 61.4%, #260-2 was 68.3%, and #260-3 was 65.7%. Compared with Zong 31 after inoculation, the disease indexes of transgenic events #260-1, #260-2, and #260-3 reached significant differences (P<0.05)( Figure 5 A). At the same time, 30 positive plants after inoculation of each of the three independent events #260-1, #260-2, and #260-3 were randomly selected to detect the expression abundance of the viral RBSDV coat protein P10. The results showed that the expression abundance of the P10 coat protein in the three complementary events was significantly lower than that of Zong 31 (P<0.05)( Figure 5 B). The ZmGLK36 transgenic complementation experiment showed that compared with non-transgenic offspring, the transgenic offspring carrying the exogenous ZmGLK36 gene could significantly improve the resistance of maize to rice black-streaked dwarf virus.

[0073] Example 4 Construction of overexpression vector and phenotypic identification of transgenic maize, rice and wheat

[0074] Based on RACE amplification, the full-length ZmGLK36 cDNA in Qi 319 was obtained. Using the obtained plasmid as a template for PCR amplification, the CUB vector was digested with BamHI alone to obtain a linearized vector, as Figure 6As shown, the primer sequences are shown in Table 4 (the primer nucleotide sequences are SEQ ID NO: 25 - 28). The promoter of this vector is the Ubi promoter. The linearized vector and the gel - recovered product with restriction enzyme sites and homologous arms CDS are ligated by Infusion recombinase. The ligation product is cultured overnight for 12 - 16 hours, and positive clones and the size of the inserted fragment are identified by PCR. One monoclonal containing the ZmGLK36 CDS is obtained. After extracting the plasmid and sequencing correctly, it is transferred into Agrobacterium tumefaciens EHA105 by the chemical transformation method and then transferred into maize immature embryos by transgenic technology to obtain transgenic plants. At the same time, RBSDV can infect gramineous plants such as rice and wheat, causing plant dwarfing, which has a similar phenotype to maize. Therefore, the constructed over - expression vector is transferred into the immature embryos of rice and wheat by transgenic technology, and the recipients are the diseased - susceptible rice variety Kitaake and the wheat variety Filed.

[0075] The over - expression vector CUB contains the Bar gene ( Figure 6 and Figure 7 A), so the Bar test strip is used for detection first. The detection results show that the Bar protein in positive plants can be rapidly expressed, showing two bands, indicating that the Bar test strip can effectively distinguish positive and negative plants. Figure 7 C). Considering the stability of the Bar protein and the situation that high - generation plants are prone to loss due to methylation modification, histone modification, etc., the positive plants detected by the Bar test strip need to be subjected to secondary PCR detection. Primers are designed for the CUB vector. The F primer falls in the Ubi promoter region, and the R primer falls in the gene CDS region. The primer names are Ubi - F / ZmGLK36 - R, and the detection results are as Figure 7 shown in B. A single band can be detected in all positive plants, and vice versa for negative plants. In summary, the PCR identification results are accurate compared with the contemporary results of the Bar test strip detection.

[0076] 180 seeds are selected from Zhong 31 and 3 independent over - expression transgenic events respectively. Every 60 seeds are taken as a group for artificial inoculation identification. The inoculation method is the same as that of the transgenic complementation event, and the identification is carried out 20 - 30 days after inoculation. The field identification results show that: the disease index of Zhong 31 plants is 87.3%, the disease index of the transgenic event OE - 1 is 57.6%, the disease index of OE - 2 is 48.4%, and the disease index of OE - 3 is 52.1%. Compared with Zhong 31, the disease indexes of the transgenic events OE - 1, OE - 2, and OE - 3 all reach significant differences (P < 0.05), and can effectively reduce the disease index of maize rough dwarf disease by 27.6% - 38.9%. Figure 8A). Meanwhile, 30 positive plants after inoculation of three independent events of OE-1, OE-2 and OE-3 were randomly selected to detect the expression abundance of the viral RBSDV coat protein P10. The results showed that the transcriptional level of the P10 coat protein in the three overexpression events of OE-1, OE-2 and OE-3 was significantly lower than that of the receptor Zong 31( Figure 8 B), reaching a significant difference level (P<0.05). The transgenic overexpression experiment of ZmGLK36 showed that the transgenic offspring with the exogenous ZmGLK36 gene could significantly improve the resistance of maize to rice black-streaked dwarf virus compared with the non-transgenic offspring.

[0077] RBSDV can infect rice and cause the same dwarfing symptoms. To verify whether ZmGLK36 can resist virus infection in rice, the overexpression vector CUB-ZmGLK36 was genetically transformed into the susceptible rice variety Kitaake by Agrobacterium-mediated infection( Figure 9 A), and two overexpression transgenic events, Os-OE-1 and Os-OE-2, were selected for artificial inoculation identification( Figure 9 B). The results showed that the two independent transgenic events could effectively reduce the incidence of the receptor Kitaake, and at the same time, the transcriptional level of the RBSDV coat protein P10 after inoculation of the two overexpression transgenic lines was significantly lower than that of the receptor (P<0.05)( Figure 9 C). In summary, it is shown that the disease-resistant gene ZmGLK36 can effectively resist RBSDV infection in rice.

[0078] To further verify whether ZmGLK36 can resist RBSDV infection in wheat, the overexpression vector CUB-ZmGLK36 was genetically transformed into the susceptible wheat variety Filed by Agrobacterium-mediated infection( Figure 10 A), and two overexpression transgenic events, Ta-OE-1 and Ta-OE-2, were selected for artificial inoculation identification( Figure 10 B). The results showed that the two independent transgenic events could effectively improve the resistance of wheat to rice black-streaked dwarf virus, and at the same time, the transcriptional level of the RBSDV coat protein P10 after inoculation of the two overexpression transgenic lines was significantly lower than that of the receptor (P<0.05)( Figure 10 C). In summary, it is shown that the disease-resistant gene ZmGLK36 can effectively resist RBSDV infection in wheat.

[0079] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed. Sequence Listing <110> Institute of Crop Science, Chinese Academy of Agricultural Sciences <120> Disease-resistant Protein of Rice Black-Streaked Dwarf Virus and Its Application Method <130> P20210100 <160> 28 <170> SIPOSequenceListing 1.0 <210> 1 <211> 3105 <212> DNA <213> Qi 319 Artificial Sequence <400> 1 atgcagggga gctacggcta cgacggggcg gcgtcacggg accccaagcc gcggctgcgc 60 tggacgccgg tcctccacca gcgcttcgtc gacgccgtca ccaagctggg cggaccggac 120 agtgagtcct cgctctcgtt ttccttcctt cctcccgtcc gttccccccc gtgtagatgt 180 agtgctcctc tagtcgtctt gtcttgatga ccccaccccg tccgacacca aggcgattgc 240 ttgatgaccc gacacctctg cagttcgcaa gtatggtgct tgtgctacag agcatgtcgg 300 tctgcattct tttttttttc agtccgaatc cttcgctgcc tggctttgcg cttctgccat 360 tgccaagtct atagcatgca gtcagaggaa aaggcagttc cgttgacatg accaaaaggc 420 agttgaaaaa aaatgcctct ggaaaaaggc cgcgtcgccc tgggaaaacg acaagtgcaa 480 ttcctctcaa atcagagatc tcaagtctga acatccattc tctagctttt cgagctcata 540 taataatcat ctgaatcttg cctcctcgaa cacaccgaca tgcctacgct gtggagtact 600 gaccatttct tgtgcttcct gcgaacagaa gcaacaccca agtcggtgct cagattgatg 660 ggcatgaaag acctcactct ttaccagtta aaaagccacc tccaggtccg tctgacctct 720 ctttcttcac cgcctcagcg ttaccagcag tccagcactc tcctttttct gttgcaccac 780 tgtgccgaaa cactgaggct cgtctgatgt gcgcagaagt atagactggg gatacagggc 840 aagaaaagca caggcctcga acctgccagc ggcggcggta tgagcctcgc actacctcta 900 tgtcaccagg ccttgccgcg caagcgttcc tttcttatct tatcttccgc tctttctgtc 960 atcagtactt cgctcacagg gcggcttcag ttcaaccaca gcgcatcctc ctccaggtgt 1020 tcctgatgaa gggaagaaca caaggtaact ggcaattacc ttgacccgac aaatttctgc 1080 aagtggtaat acccgccttt tgtgcacagc agaaggccag tatttattac aactaatata 1140 tactgcattc agtcaagtag acagctgaag atgctagaaa atgttacctt gaaaaaaaaa 1200 tgaccatacc gttacaagca cgtgtatggg gatttcctct ctgaacctta agctgcaact 1260 caaatatttc ttgatctcaa ctcatatgct caagtcataa tgactgagac atgagtcctt 1320 aattggttta ttatcagacc atgtattatt cgactgcgag tatgggactg tagaggtgta 1380 accaaaagca ggaaaaataa aaaaaaattc caaaaccagg attctgtcta tcagtagata 1440 ttaccataca aacggcgtcg aagtgctaaa gaatacaagt tgcctaaagt tctcctaagc 1500 atacttagcg ctccacctgt tacaagcaag tattcagggg ctctcctgtt acaaacatgt 1560 actccttccg tttttttatt tgtcgtgttt tagttcaaaa atgaactagc gggtgacaaa 1620 tattcgagaa cagagcgggt attaacttac actaaatgca agcaatttat attccatttt 1680 atttccaaaa ctttgtatgc aaacttccat atatagaagc ccaggaatgt gaatcctatt 1740 gaatgcctgt taattgactg cagtgggccc attttatttg gttgaacttt tctgcattga 1800 ttgagctaac catttgtatc acatttctta tgtgaatcag agaaatagca cttagcgatg 1860 cactaaggta tcaaattcaa gtccaaagga aactacaaga acaaactgag gcaagacaga 1920 tttacatcat tgtcatgaga tttgcactcc aaaactttct tccataaacg ctacaagtgc 1980 ttatgtctgt aatcaggtgc aaaagaagct gcagatgcga attgaggccc aagggaagta 2040 cttaaagaca atactagaga aagctcagac aaatatttcc tttcacacaa atgcatccaa 2100 tggcatagaa tcaaccagat cacagctcat ggacttcaac ctagctctat caggtttcat 2160 gaacaatgca actcaagtat gcaaagaaca tagggagcaa ttggtgaaag ctatgtcgga 2220 tgaaaacgat aaagatagtc taggccttca gctctaccat ctaggaagtc aggaggctaa 2280 ggaagtcaaa tgcacaccaa aaactgaaga ctcgctccta ctagacttaa atattagagg 2340 gaggatatga cctctcttct agaggaatgc aagcatgtga attagaactg aaaataaacc 2400 agcagatatt ataaatttgt tgaaaactca tagttttggg aagttcactc tttgctatta 2460 gattattaac ttttccctgt ggctgctgag cgtccttgtc caatgcccac atctcacaag 2520 ttttgtttac tctctgctca tgttatattt gctgaaagaa acttttgaat aaatgtgggc 2580 attgttttaa ttcttgaatt ttacacatat gtagaatgtg gataggagta agagaatgaa 2640 agagctgcga cagtctagac tcaaggtgta tgcatatcta gacatttagt tacttatctt 2700 aatgtgcacc aaatatgttc agttttcccc gtttccctaa ccaaacaata tacatacatc 2760 gaaactaaaa cttgtatgag tgccaatagc aggacaaccg aagcactgaa atgttgagtt 2820 tgaattatct gcaaataagt ccgtggtgtt aacatgccag ccatatgcac taaaatttct 2880 gtcattagtt ctgattgtta ttgaagcatt ttttccatag ttcacattat cgaactatta 2940 acttagttgt aatgtattaa taccgagtta cccacatttt ttttgaataa taacattaat 3000 caagtcaaga tgtccatatt tattatgaca taagaatgtc caaatatttt tctggtagct 3060 tggtcaaact gtacctcatg gcctttgcaa cactcaaaac ccctc 3105 <210> 2 <211> 825 <212> DNA <213> Qi 319 Artificial sequence <400> 2 atgcagggga gctacggcta cgacggggcg gcgtcacggg accccaagcc gcggctgcgc 60 tggacgccgg tcctccacca gcgcttcgtc gacgccgtca ccaagctggg cggaccggac 120 aaagcaacac ccaagtcggt gctcagattg atgggcatga aagacctcac tctttaccag 180 ttaaaaagcc acctccagaa gtatagactg gggatacagg gcaagaaaag cacaggcctc 240 gaacctgcca gcggcggcgt acttcgctca cagggcggct tcagttcaac cacagcgcat 300 cctcctccag gtgttcctga tgaagggaag aacacaagag aaatagcact tagcgatgca 360 ctaaggtatc aaattcaagt ccaaaggaaa ctacaagaac aaactgaggt gcaaaagaag 420 ctgcagatgc gaattgaggc ccaagggaag tacttaaaga caatactaga gaaagctcag 480 acaaatattt cctttcacac aaatgcatcc aatggcatag aatcaaccag atcacagctc 540 atggacttca acctagctct atcaggtttc atgaacaatg caactcaagt atgcaaagaa 600 catagggagc aattggtgaa agctatgtcg gatgaaaacg ataaagatag tctaggcctt 660 cagctctacc atctaggaag tcaggaggct aaggaagtca aatgcacacc aaaaactgaa 720 gactcgctcc tactagactt aaatattaga ggaggatatg acctctcttc tagaggaatg 780 caagcatgtg aattagaact gaaaataaac cagcagatat tataa 825 <210> 3 <211> 274 <212> PRT <213> Qi 319 Artificial sequence <400> 3 Met Gln Gly Ser Tyr Gly Tyr Asp Gly Ala Ala Ser Arg Asp Pro Lys 1 5 10 15 Pro Arg Leu Arg Trp Thr Pro Val Leu His Gln Arg Phe Val Asp Ala 20 25 30 Val Thr Lys Leu Gly Gly Pro Asp Lys Ala Thr Pro Lys Ser Val Leu 35 40 45 Arg Leu Met Gly Met Lys Asp Leu Thr Leu Tyr Gln Leu Lys Ser His 50 55 60 Leu Gln Lys Tyr Arg Leu Gly Ile Gln Gly Lys Lys Ser Thr Gly Leu 65 70 75 80 Glu Pro Ala Ser Gly Gly Val Leu Arg Ser Gln Gly Gly Phe Ser Ser 85 90 95 Thr Thr Ala His Pro Pro Pro Gly Val Pro Asp Glu Gly Lys Asn Thr 100 105 110 Arg Glu Ile Ala Leu Ser Asp Ala Leu Arg Tyr Gln Ile Gln Val Gln 115 120 125 Arg Lys Leu Gln Glu Gln Thr Glu Val Gln Lys Lys Leu Gln Met Arg 130 135 140 Ile Glu Ala Gln Gly Lys Tyr Leu Lys Thr Ile Leu Glu Lys Ala Gln 145 150 155 160 Thr Asn Ile Ser Phe His Thr Asn Ala Ser Asn Gly Ile Glu Ser Thr 165 170 175 Arg Ser Gln Leu Met Asp Phe Asn Leu Ala Leu Ser Gly Phe Met Asn 180 185 190 Asn Ala Thr Gln Val Cys Lys Glu His Arg Glu Gln Leu Val Lys Ala 195 200 205 Met Ser Asp Glu Asn Asp Lys Asp Ser Leu Gly Leu Gln Leu Tyr His 210 215 220 Leu Gly Ser Gln Glu Ala Lys Glu Val Lys Cys Thr Pro Lys Thr Glu 225 230 235 240 Asp Ser Leu Leu Leu Asp Leu Asn Ile Arg Gly Gly Tyr Asp Leu Ser 245 250 255 Ser Arg Gly Met Gln Ala Cys Glu Leu Glu Leu Lys Ile Asn Gln Gln 260 265 270 Ile Leu <210> 4 <211> 1890 <212> DNA <213> Qi 319 Artificial sequence <400> 4 ttcaggtcaa gtccggcata tgggcagact cgctgagccg gggattgatt gatctgaaca 60 ttcaggtcaa gtccggcata tgggcagact cgctgagccg gggattgatt gatctgaaca 60 taactgcacg tatgttccta gttcctgttt cccctgttgc catggcatct atcagcttgg 120 taactgcacg tatgttccta gttcctgttt cccctgttgc catggcatct atcagcttgg 12OP tgtaaatttg tttatggttc agacatgttc atggttctcc ttgtttctga caagctgcag 180 tgtaaatttg tttatggttc agacatgttc atggttctcc ttgtttctga caagctgcag 180 cgaagaaggt gaacctagct gatattggca tcgtcggtgg ccttggcgat gggtccgatg 240 cgaagaaggt gaacctagct gatattggca tcgtcggtgg ccttggcgat gggtccgatg 240 agaaggcact gccctcttgg accatgggcg ccggatccgg cctaggaatg tctggtattc 300 agaaggcact gccctcttgg accatgggcg ccggatccgg cctaggaatg tctggtattc 300 caccgtcaac acaacaagct ggtggcatcg agagcttggc caactacaac aagcatcatt 360 caccgtcaac acaacaagct ggtggcatcg agagcttggc caactacaac aagcatcatt 360 tcggcttcaa ataggcctcg atctttcata ctggaaaata cccgtcatct gcggtttcct 420 tcggcttcaa ataggcctcg atctttcata ctggaaaata cccgtcatct gcggtttcct 420 cctctgtcgg cctgcttctt acatgtgctg ccctattgat ttaatcacct tttgttttgt 480 cctctgtcgg cctgcttctt acatgtgctg ccctattgat ttaatcacct tttgttttgt 480 tttgtttttt gttttggtga ttacattaca tggtatcgac caatcttggc cccgtcttgt 540 tttgtttttt gttttggtga ttacattaca tggtatcgac caatcttggc cccgtcttgt 540 cacgcgtgta tgttatttgt cgggtttgtg ggtaagcatg caactacata cacatcacac 600 cacgcgtgta tgttatttgt cgggtttgtg ggtaagcatg caactacata cacatcacac 600 cccctgtgtt ccagctcgat gataggtggt atgttggcca tgcagtttgt gtaaattccg 660 cccctgtgtt ccagctcgat gataggtggt atgttggcca tgcagtttgt gtaaattccg 660 tggcctgcct tctctacttc atacagtact ctacgcagcg atttgcagtg cttgagttta 720 tggcctgcct tctctacttc atacagtact ctacgcagcg atttgcagtg cttgagttta 720 tattgaataa taagagaacc tatgcccttt atgtcttgaa tcgtggctga tgctgtttga 780 tgtaacactg aagtggcaaa gtgaagcggt tttttttatc aacggatgtt gtaaatggag 840 ccatcacatc acccgttcgc cgatcagtat cggcggctga tagtggtaga gttgcgtttc 900 tgagggacta atcaggaaag tgacgctaca ggaactccca aatccatcgt caagtcgcat 960 cgtacgagaa gagccaaagt gagtcacgca gatgctcgaa tctagcaggc ttagcattct 1020 gcacgcacgg ttttaaaagt gttgagaatg gcgattagtc tcggctaatc gtccgccaat 1080 ctgtgatgga gaggcggtgg ccagtggcca ctgccactgg tcagcaggtc acaccggcag 1140 ccacagctgg cgcgaatatt ccagcgacat cagcgccgaa catcttcgcc gcatctcccg 1200 ctcgccccgg gtcccaccgg aggccgccgt cgttctcccc gcgcttccgc cggaatattc 1260 cgcagccaca gctggactcg cggtcgagtc cgctccggac gaggatatgt catccgactc 1320 gtgcgattcg cgttcagttt ggttcccacg caaagatttc cctcctcttg tccagtctga 1380 atgctgctgc tgctaggttt tgccgttcac gtagcatagc atctgacgaa acgccgacgg 1440 tattttctgg tgaaaaattc gacgcgtccc aaccccagcc gtgtgcttct tgttgccaat 1500 tgtgatgtgg gtggagccgc cgtcttggcg cctgggcctc aatgagcggc ggccgagtgg 1560 cgcccccggg ttggacctgg aggcgtgatg cccgtccgct ttgttcttgc attgcatctc 1620 gcacgcaccc cggcccagct gccaacaagt cgtcctagat tcctcgcaca catgacgccc 1680 tccttccccg ccacgctctg cgctcctcct ctcgctcctc ctcttgtcct cacatacaca 1740 cacccctgtg cttgctaacg acgctcgcgt tacaacttat tacacctacg accaggaggc 1800 ggtaacgtta cattcattct ctcttcagca ctagccgtgt ctttggccac cggatagcta 1860 gggagcggcg atcgccagga aggaacgacg 1890 <210> 5 <211> 19 <212> DNA <213> Artificial sequence <400> 5 tatgagcctc gcactacct 19 <210> 6 <211> 21 <212> DNA <213> Artificial sequence <400> 6 ccatccctat ccacattcta c 21 <210> 7 <211> 19 <212> DNA <213> Artificial sequence <400> 7 cggtgctcag attgatggg 19 <210> 8 <211> 21 <212> DNA <213> Artificial sequence <400> 8 cgtgctccct atgttctttg c 21 <210> 9 <211> 22 <212> DNA <213> Artificial sequence <400> 9 gccaaagtag agtcacgcag at 22 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <400> 10 ggcacagtgg tgcaacagaa 20 <210> 11 <211> 21 <212> DNA <213> Artificial sequence <400> 11 ttcaatcggc aaccgcaagt a 21 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <400> 12 acccagcctg tggtggaaat 20 <210> 13 <211> 25 <212> DNA <213> Artificial sequence <400> 13 acatgccatt aaataaataa gcagt 25 <210> 14 <211> 21 <212> DNA <213> Artificial sequence <400> 14 atttgaagcc gaaatgatgc t 21 <210> 15 <211> 22 <212> DNA <213> Artificial sequence <400> 15 ttggcgatgg gtccgatgag aa 22 <210> 16 <211> 22 <212> DNA <213> Artificial sequence <400> 16 ttaaaaccgt gcgtgcagaa tg 22 <210> 17 <211> 22 <212> DNA <213> Artificial sequence <400> 17 tatttgtcgg gtttgtgggt aa 22 <210> 18 <211> 22 <212> DNA <213> Artificial sequence <400> 18 cgagatgcaa tgcaagaaca aa 22 <210> 19 <211> 25 <212> DNA <213> Artificial sequence <400> 19 gcatgctcag gagccacagg ggaaa 25 <210> 20 <211> 27 <212> DNA <213> Artificial sequence <400> 20 aggagcgagt cttcagtttt tggtgtg 27 <210> 21 <211> 26 <212> DNA <213> Artificial sequence <400> 21 cttcccttgg gcctcaattc gcatct 26 <210> 22 <211> 24 <212> DNA <213> Artificial sequence <400> 22 ccgtgtcttt ggccaccgga tagc 24 <210> 23 <211> 24 <212> DNA <213> Artificial sequence <400> 23 gcaggggagc tacggctacg acgg 24 <210> 24 <211> 27 <212> DNA <213> Artificial sequence <400> 24 agatgcgaat tgaggcccaa gggaagt 27 <210> 25 <211> 36 <212> DNA <213> Artificial sequence <400> 25 tcgactctag aggatccatg caggggagct acggct 36 <210> 26 <211> 44 <212> DNA <213> Artificial sequence <400> 26 ctcggtaccc ggggatccct aatagcaaag agtgaacttc ccaa 44 <210> 27 <211> 45 <212> DNA <213> Artificial sequence <400> 27 cagctatgac catgattacg aattcttcag gtcaagtccg gcata 45 <210> 28 <211> 46 <212> DNA <213> Artificial sequence <400> 28 gtaaaacgac ggccagtgcc aagcttgagg ggttttgagt gttgca 46

Claims

1. Use of the disease-resistant protein ZmGLK36 against Rice black-streaked dwarf virus in resisting Rice black-streaked dwarf virus of maize, wheat or rice, wherein the protein ZmGLK36 is as follows (a1) or (a2): (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing; (a2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of (a1).

2. The application according to claim 1, characterized in that The nucleic acid molecule of the protein ZmGLK36 is a DNA molecule as follows (b1) or (b2) or (b3) or (b4): (b1) The nucleotide shown in SEQ ID NO: 1; or (b2) The nucleotide shown in SEQ ID NO: 2; or (b3) The nucleotide encoding the amino acid shown in SEQ ID NO:

3.

3. A method for improving the resistance of corn, wheat or rice to black-streaked dwarf virus, characterized in that, The method is to obtain the resistance to Rice black-streaked dwarf virus by transferring the disease-resistant gene ZmGLK36, thereby improving the ability of maize, wheat or rice to resist Rice black-streaked dwarf virus. The amino acid sequence of the gene ZmGLK36 is as follows (a1) or (a2): (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing; (a2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of (a1).

4. The method according to claim 3, wherein The method comprises the following steps: (1) Design primers using the sequence of the disease-resistant gene ZmGLK36 in the B73 RefGen_V4 reference genome; (2) Respectively obtain the genome and promoter sequence of the maize inbred line Qi 319 resistant to Rice black-streaked dwarf virus by PCR amplification, and then splice to obtain the full-length sequence of the disease-resistant gene; (3) Respectively construct the ZmGLK36 genome complementary vector and overexpression vector, and perform genetic transformation based on the maize inbred line Zong 31, the rice variety kitaake and the wheat variety Filed, and verify the ability of ZmGLK36 to resist Rice black-streaked dwarf virus.

Images