Application of Glyma. 16G210600 gene in improving mosaic virus resistance of plant

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a Glyma. 16G210600 gene to improvement of resistance of plants to mosaic viruses. According to the application of the Glyma. 16G210600 gene in improving the resistance of plants to mosaic viruses, the nucleotide sequence of the Glyma. 16G210600 gene is as shown in SEQ ID NO: 1. The soybean gene Glyma. 16G210600 is screened from a soybean genome in a homologous comparison manner, and molecular biology and genetic engineering technical means prove that the over-expression of the soybean gene Glyma. 16G210600 can improve the resistance of tobacco to viruses such as tobacco mosaic virus (TMV), soybean mosaic virus (SMV), cabbage leaf roll virus (CaLCuV) and barley stripe mosaic virus (BSMV).

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to Glyma.16G210600 Application of genes in improving plant resistance to mosaic virus. Background Art

[0002] During the growth and development of plants, they are often attacked by various pathogens and pests such as bacteria, fungi, viruses, and oomycetes. In order to resist the harm of pathogens, plants have evolved a very complex immune system during their long-term growth. According to the different locations of the immune response, the plant immune system is considered to be divided into two reactions, namely, the PTI immune response (PAMP-triggered immunity) induced by the recognition of pathogen-associated molecular patterns (PAMPs) by receptor proteins on the cell membrane, also known as nonspecific immunity, and the specific immunity of the ETI immune response (effector-triggered immunity) induced by pathogens secreting effectors (effectors) into cells to escape the PTI response and being recognized by anti-disease proteins in the cells. The anti-disease proteins in the cells are mainly encoded by resistance genes, and most of them are NLR proteins, which have the characteristics of nucleotide binding sites (NBS) and leucine-rich repeat sequences (LRRs). According to the Toll / interleukin-1 receptor (TIR) ​​or coiled coil (CC) domain contained in their N-termini, they are usually divided into two subtypes, TNL and CNL.

[0003] Soybean is an important crop used for both food and oil and feed. At present, there is a large gap between domestic soybean production capacity and actual demand, and it mainly relies on soybean imports. Due to limited arable land resources, soybean planting cannot threaten the production of staple crops, and the increase in soybean production capacity is only limited by the soybean planting area. Therefore, increasing the yield per unit area is of great significance to the development of the soybean industry. Soybean mosaic disease is a soybean virus disease caused by soybean mosaic virus (SMV), which seriously restricts the increase in soybean production. The use of disease-resistant genes to cultivate SMV-resistant varieties is an effective means to increase soybean yields. Therefore, obtaining disease-resistant screening and identification of new disease-resistant genes is the key to achieving this goal.

[0004] Traditional gene mining methods are mainly through map-based cloning, transposon tagging, gene chip technology and RNA-Seq, etc., and these methods mostly require the construction of a certain number of populations and high-density molecular markers, and there are problems such as high cost and low efficiency in isolating and cloning genes. In order to improve cloning efficiency, with the development of sequencing technology and the reduction of sequencing costs, genome-wide association analysis (GWAS) has become the main way to obtain excellent traits. For NLR genes, based on their evolutionary conservation, the development of resistance gene enrichment sequencing (RenSeq) technology has also made their acquisition accurate and efficient. However, in this process, the synthesis of nucleic acid probes needs to rely on professional laboratories, which limits its development.

[0005] Therefore, a more convenient way is needed to clone resistance genes. The homologous sequence method is a method of cloning by designing primers based on the known sequence of the gene to be studied or the homologous sequence to the known gene. Many studies have reported that the N-terminal domain of the NLR gene plays an indispensable role in its function. Using the TIR domain coding sequence to compare and screen with soybean genomic DNA or cDNA library to obtain candidate gene sequences, and then cloning and identifying them using molecular biological methods may be an effective means to obtain soybean SMV resistance genes. Summary of the invention

[0006] The object of the present invention is to provide Glyma.16G210600 Application of genes in improving plant resistance to mosaic virus.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides Glyma.16G210600 Application of genes in improving plant resistance to mosaic virus, the Glyma.16G210600 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0008] Preferably, the plant is soybean, tobacco, cabbage or barley; and the mosaic virus is tobacco mosaic virus, soybean mosaic virus, cabbage leaf curl virus or barley streak mosaic virus.

[0009] The present invention also provides an expression vector, comprising an initial vector and the Glyma.16G210600 Gene.

[0010] Preferably, the initial vector is pCAMBIA1300.

[0011] The present invention also provides a host cell transformed or transfected with the expression vector; the host is a microorganism.

[0012] The present invention also provides the use of the expression vector or the host in improving the resistance of plants to mosaic virus.

[0013] Preferably, the plant is soybean, tobacco, cabbage or barley; and the mosaic virus is tobacco mosaic virus, soybean mosaic virus, cabbage leaf curl virus or barley streak mosaic virus.

[0014] The present invention also provides Glyma.16G210600 Application of genes in plant disease resistance breeding, the Glyma.16G210600 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0015] Preferably, the plant is soybean, tobacco, cabbage or barley.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention screens soybean genes in the soybean genome by homology comparison. Glyma.16G210600 , using molecular biology and genetic engineering techniques to prove that overexpression of soybean genes Glyma.16G210600 The invention can improve the resistance of tobacco to viruses such as tobacco mosaic virus (TMV) and soybean mosaic virus (SMV). The invention provides an effective method for screening and identifying plant resistance genes, which can conveniently and quickly obtain plant disease resistance activity genes; in addition, the genes identified by this method can be Glyma.16G210600 It is an ideal genetic resource for enhancing plant disease resistance and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0018] Figure 1 Soybean gene Glyma.16G210600 Schematic diagram of sequence and domain annotation; Figure 2 Soybean gene Glyma.16G210600 Fragment glue map; Figure 3 For the transient expression verification of antiviral activity, TMV-GFP and soybean genes Glyma.16G210600 Co-infection of Nicotiana benthamiana leaves. Observe the infected area of ​​the leaves under white light and hand-held UV lamp (wavelength = 365nm) respectively ( Figure 3 Upper) and GFP fluorescence ( Figure 3 16G210600 is Glyma.16G210600 Infection group; SRC7 and SRC7 TNAll were used as positive control groups. 09G056400 and 05G165800, which do not have disease resistance, were used as negative control groups, and the EV empty vector was used as a negative control group. Each experiment was repeated three times.

[0019] Figure 4 For the transient expression verification of antiviral activity, SMV-GFP and soybean genes Glyma.16G210600 Co-infection of Nicotiana benthamiana leaves. Observe the infected area of ​​the leaves under white light and hand-held UV lamp (wavelength = 365nm) respectively ( Figure 4 Upper) and GFP fluorescence ( Figure 4 16G210600 is Glyma.16G210600 Infection group; SRC7 and SRC7 TN All were used as positive control groups. 09G056400 and 05G165800, which do not have disease resistance, were used as negative control groups, and the EV empty vector was used as a negative control group. Each experiment was repeated three times.

[0020] Figure 5 Soybean gene Glyma.16G210600 Identification of BSMV pathogen resistance.

[0021] Figure 6 Soybean gene Glyma.16G210600 Identification of CaLCuV pathogen resistance. DETAILED DESCRIPTION

[0022] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0023] Example 1

[0024] The tobacco mosaic virus (TMV-GFP) infectious cloning vector PJL24 can be obtained from "Bao, W.; Yan, T.; Deng, X.; Wuriyanghan, H. Synthesis of Full-Length cDNA Infectious Clones of Soybean Mosaic Virus and Functional Identification of a Key Amino Acid in the Silencing Suppressor Hc-Pro. Viruses 2020, 12, 886.". The expression vector is pCAMBIA1300-Cluc. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. The primers involved in the implementation of the present invention are synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0025] 1. Compare TIR domain homologous sequences with soybean genome database and screen soybeans with TIR domains NLR Gene Based on the results of previous studies, the N-terminal domain of NLR genes is an essential domain for their function (A cluster of atypical resistance genes in soybean confers broad-spectrum antiviral activity). The N-terminal TIR sequences of TNL-type genes were aligned with the soybean genome database (Wm82.a2.v1, https: / / soybase.org / ), and soybean genes were screened based on sequence homology. Glyma.16G210600 .

[0026] 2. Glyma.16G210600 Sequence analysis and structural domain prediction Based on the reference genome, the gene is located on soybean chromosome 16, with a gene fragment size of 4263bp and several exons and introns. Using the Pfam domain prediction website, it is predicted that the encoded protein contains two atypical domains zf-RVT and motA ( Figure 1 ). It encodes an amino acid sequence of 1420, a molecular weight of 161.57 kDa, and an isoelectric point of 6.69.

[0027] 3. Glyma.16G210600 clone Williams soybean varieties were selected. After the seeds were soaked in tap water for 16 hours, they were sown in pots prepared in advance with culture soil (vermiculite: nutrient soil = 1:1) and cultured in a greenhouse (25℃±3℃, light / darkness = 14h / 10h, relative humidity 60%~70%, light intensity 8000lux). After the first true leaf of soybean was fully expanded, the newly grown leaves were taken, and the total RNA was extracted using the Trizol method after quick freezing in liquid nitrogen. The quality of RNA was detected by agarose gel electrophoresis and NanoDrop, and subsequent experiments were carried out after the quality was qualified. The extracted RNA was reverse transcribed into cDNA using the reverse transcription kit of Takara Company, and the specific operation method was carried out according to the instructions.

[0028] Primers (primers shown in SEQ ID NO: 3 and 4) were designed according to the gene sequences.

[0029] SEQ ID NO: 3: atggcttggggatcaagttc SEQ ID NO: 4: ttatctgtattctgaaatataaggttggc The fragments are connected by homologous recombination. The resulting gene sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0030] SEQ ID NO: 1: atggcttggg gatcaagttc ctcttcctct tcctcttcct ccaattacga tgtgttcctc 60 agcttcagag gtgaagacac acgtcatgct tttactggcc cttttactgg agctcttcat 120 gacaagggaa tccacacttt cattgatgat gagaagcttc aaagagggga tcaaagaggg 180 cgagcactta tggaggcaat tcaagattcc agagttgcca tcactgtgct ttctcaaaac 240 ctttctcaaa cctcgttttg tttagatgaa cttgcaacca tccttcactg tcaccagagg 300 aaacggttgt ggaaacggtt ggtcttttat aaggtggatc cttctgatgt gagacaccag 360 aaaggtagtt atgcagaagc ttatgcagaa cttgagacga ggttccagca tgacccggag 420 aagttgcaaa aatggaagat ggctctgaag atggctctga acttgtctgg ctatcatttc 480 aaagagggag atggatatga attcaagttt attgagaaga ttatgagaa ggtatctagg 540 gagattaatc ctcgtacttt acatgttgcg gattacccag ttggactaga agttggacta 600 ctagatgtaa ggagactttt ggatgctgga tctgatgatg gtgtccacat gatagggatt 660 atgataggga gcgggctagg aaaatcaaca cttgctcgag ctgtctacaa tgaattgata 720 attgctgaga taattgctga tttttgcttt cttgcaaatg tgagggaaaa gtcagacaaa 780 aaagacgggt tagaacacct gttagaacac cttctttcag aaatactcgg agagaagaac 840 atcagctaa caagtacgca acaaggaatt caacaaggaa agtctagct caagggaaag 900 aaggttctct tgattctaga tgatgttaac acacatgggc acacacatgg aattggagaa 960 cgtgattggt ttggtcccgg cagcaaaatc atcatcacaa ctcgggacga aactcgggac1020 gcatatcatg aagttaatga aacatatgag atgaaggaat tgaatcagaa agacgctctt1080 aaagacgctc catggaacgc ttttaagaag gaaaaagctg atccaactta cgtggaggtc1140 ttgcatcgtg tcttgcatcg tgcttctggt cttccattgg cgttggaagt aataggttcc1200 cacctggttg gaaaaagtat tggaaaagt gaatctgcca tcaaacaata taaaagaatt1260 1320 cagaaagttt ttcttgacat tgcttgttgc ttgaaaggat gcttgaaagg agaggtcgaa1380 catatacttc cgggtcttta tgatgactgc atgaaacata atattggggt taatattggg1440 aaatctctaa taaagttag ttggggtt gtgtagtta acatgcatga cttgattcag1500 gacttgattc ggcgaataga ccaaaga tcccaaag agccaggga gcgaaggaga1560 ttgtggttaa gattgtggtt aattcaagtt ttagacgaca actcgggaac tagtgaaatt1620 siaatgatat ctctggattt atctctggat gagaagaa siaataga ttggaatgga1680 aacgccttca gaaagataaaaaccttaaaaaacctta ttagaatgg taaatttcc1740 aaaggtccca attattttcc agaagttta agagtactgg taagagtact gtatccttct1800 aattgtttac catctaattt tcctccgaag gaacttgtca tatgcaagtt catatgcaag1860 tacattacgt catttgggtt ccatggctca aggaagt tcaggaagct aaggttctg1920 ctaaaggttc attgtaaaat ttacagag atacctgacg tatctgttct cgtaaatttg1980 gagaacttt tggaact gtgtgggaat ttattacag ttcaccactc aattggtttt2040 ctaataaac ttaaaatatt acttaaaata gggttgcagca agcttaccac atttccacct2100 ctcaacttga cctctttga aggacttcaa gaaggacttc gttccagtct tgagaatttt2160 ccagaaatat taggagagat gaaaaactta ttgatgcttc tattgatgct ccttcttggc2220 gtaaaagaat tgccagtttc atttcaaaat cttgttggac tccaaagtttt actccaaagt2280 gattgtgaaa atttttgtt accaagtaac attattgcca tgatgcctaa actgtcttca2340 aaactgtctt aaagttgcaa ggggttgcaa tgggtgaaat cagaagaggg tgaagaaaa2400 gtgggctcaa aagtgggctc aaatgtagat gactccagtt tcgatggttg caacctgtat2460 gatgattttt tttcaacagg ttttcaaca cttgatcatg tgaaaacttt aagcctaagg2520 gacaataatt tcacattcct tcctgaatgc cttcctgaat tgcaattttt aacaagactt2580 gatgtgagtg gctgcttgcg tcttcaggaa attagagggg aaattagagg cttaaaagaa2640 ttcatggcaa gagaatgtat atccttgtct tcctcgagtt caagcatgtt ttcaagcatg2700 gaattgcatg aggctggaca aactgagttt ttgtttccag gagcaactat tccagagtgg2760 attccagagt aaagtagggg accttcaagt tctttctggt ttcgtaataa attccctgac2820 aatgttcttt acaatgttct agcacgtgtg gaatccatag atctagatga tattcccatg2880 cccaaggtgt tcatcaatgg gttcatcaat aagatttctt ctaggaacta tcaataga2940 aaggtgaaat tggattatac atatctattt acatatctat cagctttata taagcttgat3000 gatccatctg gcctaatatc agctttacat gagcttgatg atgagcttga ggaccatgtg3060 gaaattacat acggaggtat catagagacc tcactgctta aagcaaccgg taagcaacc3120 ttcagacagg atgatattcg atatgatgat ccctatggca agaaaaatt agagcatgat3180 ttagagcatg ctgaatcaca atcactcatt aaaaagccta ggttatcgag gtgggttggc3240 cctgaaagaa gccctgaaag tctggggaat gctgcagatg gagctttatt tactaatccc3300 ctgaggagat ataggcatac atataggcat tcctcttgtc cgcgatgccc cgagttggag3360 gagacatgtc tgcatgctct tcgtgattgt cttcgtgatt ctgcattttg gagaagtgtg3420 ttacctaaga aattagcacc aaagtttttt aatggtgatg ttaatggtga gctggaaaca3480 aatttatctt tttcagaggc ggcttttttt tggccaactt tttttgggat tttttttggg3540 ctgttgtggg aatcccgcaa cgaccttgtg ttctaaag atggcacttg ggactattta3600 tgggactatt atatcacaga tattgtcttt aatcgctata aagattgtat gagagcccat3 gcttctcaca atgcttctca gaggaatctc ttgaagtgga ggaggccttt gcccctttca3 catgggcatt ggttgttgag ttggttgttg agtggtgcct atgatcgctc ttcagatact3780 gctgcatgcg gaggtatatt cagagacaac ttcagagaca tcgtgcttgg gttttcggtt3 aagcttgggg aatgtttgtc aatgatgag ggtgaaatat agggtgaat tcatggtatg3 aagattgcaa ggaggtatga fatherggggt ttatatcgtg fatherctaag tgatattcta3 attccaata gtgctaagat tgcaaggcag catgattttg gtaaataat agtggaatcg4 atagtggaat aagctattgg gtttgtgtta gatggatgcc ctacatctac atcgctggag4080 cattgtttcc agcattgttt tgagttgaaa gctctaacct ctgctacaaa tcacttatac4 ttcgataacg attatgctgc cgattatgct gccaaattcg gtttatccat gaaacggcag4200 cctgtgaaaa tttttcgtgt ttgtccccct gtttgtcccc cttatatttc agaatacaga4260 taa 4263 SEQ ID NO:2: MAWGSSSSSSSSSSNYDVFLSFRGEDTRHAFTGHLYKALHDKGIHTFIDDEKLQRGEQIT RALMEAIQDSRVAITVLSQNYASSSFCLDELATILHCHQRKRLLVIPVFYKVDPSDVRHQ KGSYAEALEKLETRFQHDPEKLQKWKMALKQVADLSGYHFKEGDGYEFKFIEKIVERVSR EINPRTLHVADYPVGLESRVLDVRRLLDAGSDDGVHMIGIHGMGGLGKSTLARAVYNELI IAEKFDGFCFLANVREKSDKKDGLEHLQRILLSEILGEKNISLTSTQQGISIIQSRLKGK KVLLILDDVNTHGQLQAIGRRDWFGPGSKIIITTRDEQLLAYHEVNETYEMKELNQKDAL QLLTWNAFKKEKADPTYVEVLHRVVAYASGLPLALEVIGSHLVGKSIEAWESAIKQYKRI PKKEILDVLTVSFDALEEEEQKVFLDIACCLKGWTLTEVEHILPGLYDDCMKHNIGVLVE KSLIKVSWGDGVVNMHDLIQDMGRRIDQQRSSKEPGKRRRLWLTKDIIQVLDDNSGTSEI QMISLDLSLSEKETTIDWNGNAFRKIKNLKILFIRNGKFSKGPNYFPESLRVLEWHGYPS NCLPSNFPPKELVICKLSQSYITSFGFHGSRKKFRKLKVLKFDYCKILTEIPDVSVLVNL EELSFNRCGNLITVHHSIGFLNKLKILSAYGCSKLTTFPPLNLTSLEGLQLSACSSLENF PEILGEMKNLLmLQLFGLLGVKELPVSFQNLVGLQSLILQDCENFLLPSNIIAMMPKLSS LLAESCKGLQWVKSEEGEEKVGSIVCSNVDDSSFDGCNLYDDFFSTGFMQLDHVKTLSLR DNNFTFLPECLKELQFLTRLDVSGCLRLQEIRGVPPNLKEFMARECISLSSSSSmLSNQ ELHEAGQTEFLFPGATIPEWFNHQSRGPSSSFWFRNKFPDNVLCLLLARVESIDLDDIPM PKVFINGILCKISSRNYQVRKVKLDYTYLFDLKSALYKLDDPSGLISALHELDEKEWDHV EITYGGIIETSLLKATGIHVFRQDDIRYDDPYGKRKLEHDLNSSESQSLIKKPRLSRWVG PERIINLLGNAADGALFTNPLRRYRHTSMDSSCPRCPELEETCLHALRDCPKVAAFWRSV LPKKLAPKFFNGDVAVWLETNLSFSEAAFFWPTFFGIAVELLWESRNDLVFYKDGTWDYL DLSDITDIVFNRYKDCMRAHASHILMPRNLLKWRRPLPLSHGHWLLRLNVSGAYDRSSDT AACGGIFRDNNDRFVLGFSVKLGECLSNDEGEIWGIYHGMKIARRYDIWGLYRDILRQHS IPISAKIARQHDFGKIIVESGSEKAIGFVLDGCPTTSTSLEHCFPLCDELKALTSATNHLY FDNDYAADSFAKFGLSMKRQPVKIFRVCPPFCQPYISEYR 4. Construction of overexpression vector pCAMBIA-Gm16G210600 (1) Expression using the overexpression vector pCAMBIA1300 Glyma.16G210600 The pCAMBIA1300 vector was linearized by double digestion with SacI and KpnI (vector 2.5 μg; Buffer J 2 μL; SacI 0.2 μL; KpnI 0.2 μL; 37°C, 4 h), purified by 1% agarose gel electrophoresis, and recovered using a Thermo gel recovery kit to detect the concentration.

[0031] (2) Recombinant vector construction and E. coli transformation The linearized vector was Glyma.16G210600 The gene fragments were connected under the action of homologous recombinase. The connection system was 5μL (1μL linear vector, 1μL fragment, 1μL Mix, 5μL ddH2O supplemented, 50℃, 30min). After the connection product was placed on ice for 5min, 50μL E. coli DH5α was added and placed on ice for 30min, then cooled on ice for 3min in a 42℃ water bath for 90s, 600μL LB liquid culture medium was added, and cultured at 37℃ shaking table at 180r / min for 1h. The culture was centrifuged at 5000r / min for 3min, and the supernatant was aspirated with a gun. 100μL of mixed precipitate was left in the centrifuge tube and cultured on LB solid culture medium with kanamycin resistance for 14h.

[0032] The cultured single colony was verified by colony PCR (using primers shown in SEQ ID NO: 3 and 4). 10μL system (0.5μL of primers F and R, 5μL of greenMix, supplemented with ddH2O to 10μL. PCR conditions: pre-denaturation at 98℃ for 3min; (denaturation at 95℃ for 15s; annealing at 55℃ for 30s; extension at 72℃ for 4min, repeated for 30 cycles); 72℃ for 10min; 16℃ for 1h).

[0033] Plasmid PCR and restriction enzyme verification. Add 6 mL of LB liquid culture medium with kanamycin resistance (kanamycin 50μg / mL) to the shaking tube, inoculate the positive colonies detected above with a sterile toothpick, place on a shaker (37℃, 180r / min, 12h) for culture, extract the plasmid using a plasmid extraction kit, and perform PCR verification on a 10μL system (plasmid 1μL; Mix 5μL; R, F 0.5μL each; ddH2O to 10μL; use the primers shown in SEQ ID NO: 3 and 4), and perform the PCR under the above conditions. Enzyme digestion verification (plasmid 2.5μg; Buffer J 2μL; SacI 0.2μL; KpnI 0.2μL; 37℃, 4h), followed by gel electrophoresis ( Figure 2 The positive band plasmid was sent to GenScript for sequencing.

[0034] (3) Transformation with Agrobacterium tumefaciens GV3101 Transformation of Agrobacterium GV3101 was performed by freeze-thaw method. 5 μL of recombinant plasmid was added to 50 μL of GV3101 competent Agrobacterium, and gently pipetted to mix. Place on ice for 5 min, place in liquid nitrogen for 5 min, and finally place in a 37°C water bath for 5 min, and place on ice for 3 min. Then add 600 μL of antibiotic-free LB liquid medium and culture on a shaker (28°C, 180 r / min) for 4 h. After the culture is completed, centrifuge the bacteria at 5000 rpm for 5 min. Discard the supernatant, keep the precipitate, pipet and mix, and then apply it on LB triple antibody (kanamycin, rifampicin, gentamicin) solid medium.

[0035] Colony PCR and plasmid PCR verification. After 2 days of culture, single colonies grew on the culture medium for numbered colony PCR verification. The PCR conditions were the same as those described in the colony PCR verification in (2). The positive colonies were inoculated into LB triple-antibody liquid culture medium (6 mL) using a sterile toothpick and cultured on a shaker (28°C, 180r / min) for 2 days. A small amount of bacterial solution was mixed with 50% glycerol at a volume ratio of 1:1 and frozen at -80°C for later use. The remaining bacterial solution was used for plasmid extraction using a plasmid extraction kit and the plasmid PCR conditions and system described in (2) were used. Finally, the positive frozen bacterial solution was retained for verification.

[0036] 5. Glyma.16G210600 Disease resistance research After bacterial activation, the overexpression vector pCAMBIA-Gm16G210600, the silencing suppressor P19 (P19 protein is derived from tomato bushy stunt virus, which can inhibit the RNA silencing effect of the host on foreign genes, improve the stability of heterologous gene transcripts, and promote the expression of heterologous proteins. It is widely used in the transient expression system of transgenic plants and tobacco leaves, Arabidopsis leaves, tomato leaves or protoplasts. It can be purchased from Beijing International Biotechnology Co., Ltd.), TMV-GFP and SMV-GFP, as well as SRC7 and SRC7 TN After thawing the frozen solution of overexpression vector Agrobacterium (reference: Yan T, Zhou Z, Wang R, et al. Acluster of atypical resistance genes in soybean confers broad-spectrumantiviral activity[J]. Plant Physiology, 2022, 188(2):1277-1293.) on ice, 40 μL was aspirated and spread on LB triple antibody solid culture medium and cultured at 28°C for 48 h.

[0037] In a sterile clean bench, 1 mL of infection buffer was added to the culture plate full of colonies, and the colonies were washed and placed in a 5 mL centrifuge tube as the infection mother solution. The concentration of the bacterial solution (OD600 In order to calculate the concentration C of 50 μL infection stock solution contained in 1 mL infection solution, 3 mL infection solution containing a target gene concentration of 0.5 and a virus concentration of 0.1 was prepared. The calculation formulas for the amount of gene and virus infection stock solution added were: 3×0.5×50 / C 基因 μL and 3×0.1×50 / C 病毒 μL, the concentration of P19 infection solution is 0.1. Add the calculated infection stock solution to a 5mL centrifuge tube and add infection buffer to 3mL. Mix well and treat in a dark environment for more than 2 hours.

[0038] Preparation method of infection buffer: Add MES (1M) and MgCl2 (1M) in ddH2O at a ratio of 1:100 respectively, and add Acetosyringone (200mM acetosyringone) at a ratio of 1:1000, mix well and set aside.

[0039] Infection by infiltration method. Select 4-week-old tobacco leaves and mark them with a marker pen. Use the above-prepared infection solution to draw a circle with a sterile syringe (1 mL) and inject it into the marked circles. Continue to culture in the greenhouse for 4 days, use ultraviolet light to irradiate and photograph, and observe the spread of the virus ( Figure 3 , 4 ).

[0040] 4 days after Agrobacterium infection, SMV-GFP ( Figure 4 In the figure below, all treatments show green fluorescence), TMV-GFP ( Figure 3 In the figure below, all treatments showed green fluorescence) and all were expressed normally, with strong green fluorescence and obvious virus accumulation. Glyma.16G210600 In the co-expression area, the green fluorescence weakened or even disappeared, and the expression of SMV-GFP and TMV-GFP was inhibited, which was different from the positive control SRC7 (SMV resistance cluster 7; the 7th gene in the SMV resistance gene cluster) and SRC7 TN (TN (TIR-NBS) domain of SRC7) co-expression results were the same, and the virus could not accumulate successfully. Therefore, the soybean gene was obtained by TIR sequence alignment Glyma.16G210600 It can confer tobacco resistance to SMV and TMV. Among them, EV is the blank control, 09G56400 ( Glyma.09G056400 ) and 05G165800 ( Glyma.05G165800) is a TNL gene identified on soybean chromosome 9 (Chromosome 9: 5,080,364-5,088,515) and chromosome 5 (Chromosome 5: 35,633,857-35,645,843) by homologous sequence alignment. It does not have disease resistance function and serves as a negative control. TN It is a truncated form of SRC7 protein (with partial resistance to SMV and TMV, and its truncated form SRC7 TN The plant with complete resistance (reference Yan T, Zhou Z, WangR, et al. A cluster of atypical resistance genes in soybean confers broad-spectrum antiviral activity[J]. Plant Physiology, 2022, 188(2):1277-1293.) was used as the positive control.

[0041] Experimental Example 2

[0042] Based on Experimental Example 1 Glyma.16G210600 It has strong viral resistance to TMV and SMV, and it is speculated that it may mediate broad-spectrum antiviral activity. Therefore, based on the experiment of Experimental Example 1, according to the literature (references: Bao, W.; Yan, T.; Deng, X.; Wuriyanghan, H. Synthesis of Full-Length cDNA Infectious Clones of Soybean Mosaic Virus and Functional Identification of a Key Amino Acid in the Silencing Suppressor Hc-Pro. Viruses 2020, 12, 886. Shao, W.; Shi, G.; Chu, H.; Du, W.; Zhou, Z.; Wuriyanghan, H. Development of an NLR-ID Toolkit and Identification of Novel Disease-Resistance Genes in Soybean. Plants 2024, 13, 668.), Agrobacterium carrying the infectious clones of CalcuV and BSMV viruses were transiently infected with tobacco, and overexpressed at the same time. Glyma.16G210600 Agrobacterium carrying an empty vector was used as a control, and the virus titer content after 4 dpi was detected to characterize the virus resistance of the target gene.

[0043] To detect Glyma.16G210600 The changes in the titers of cabbage leaf curl virus and barley mosaic virus after expression were analyzed. The total RNA of the virus-infected area samples was extracted using the Plant RNA Rapid Extraction Kit (Coolaber Technology Co., Ltd., Beijing, China) according to the manufacturer's instructions, and the concentration and purity of the RNA were detected by Nanodrop2000 (ThermoScientific, USA). The first-strand cDNA was synthesized using the Hifair®Ⅲ1st Strand cDNA Synthesis Kit (Yeasen Biotechnology Co., Ltd. Shanghai, China). The synthesized cDNA was used as a template and RT-qPCR was performed using the PerfectStart Green qPCR SuperMix Kit (TransGen Biotech, TG-AQ601) in a real-time thermal cycler Applied Biosystems ViiA 7.

[0044] Among them, the CaLCuV detection primers are: F: CAGTTCTTACGAGCAGCGACA, R: TGTCACGCACCAACCAAAAC.

[0045] BSMV detection primers are: F: GAAAGTGTGCTGGCCTTTCG, R: CATCTGAGCGCTGACTCGTA.

[0046] As shown in SEQ ID NO: 5-8 The experiment was repeated three times and the results were based on 2 -ΔΔCT The calculations were performed using GraphPad Prism 8 (La Jolla, CA, USA) and Excel 2019.

[0047] Results: Soybean gene was analyzed by qPCR. Glyma.16G210600 The changes of CaLCuV and BSMV virus titers after co-infection with cabbage leaf curl virus (CaLCuV) and barley mosaic virus (BSMV) compared with single virus infection in soybean Glyma.16G210600 The expression of the gene was combined with infection with pathogens, and the virus titer was significantly reduced after 4 days. This indicates that it can inhibit virus growth and has anti-BSMV and CaLuV activity. It is a potential plant disease resistance gene resource, and it is necessary to further explore its disease resistance mechanism. Glyma.16G210600 The discovery of this invention proves that this invention is an effective way to explore and study the functions of plant disease resistance genes and has practical significance. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Glyma.16G210600 The use of a gene in improving plant resistance to mosaic virus, characterized in that: Said Glyma.16G210600 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The plant is soybean, tobacco, cabbage or barley; the mosaic virus is tobacco mosaic virus, soybean mosaic virus, cabbage leaf curl virus or barley stripe mosaic virus.

3. An expression vector, characterized in that: Comprising an initial carrier and the method according to claim 1 or 2 Glyma.16G210600 Gene.

4. The expression vector according to claim 3, characterized in that The original vector was pCAMBIA1300.

5. A host cell, characterized in that The host is transformed or transfected with the expression vector according to claim 3 or 4; the host is a microorganism.

6. Use of the expression vector according to claim 3 or 4 or the host according to claim 5 in improving the resistance of plants to mosaic virus.

7. The use according to claim 6, characterized in that: The plant is soybean, tobacco, cabbage or barley; the mosaic virus is tobacco mosaic virus, soybean mosaic virus, cabbage leaf curl virus or barley stripe mosaic virus.

8. Glyma.16G210600 The application of the gene in plant disease resistance breeding is characterized in that: Said Glyma.16G210600 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

9. The use according to claim 8, characterized in that: The plant is soybean, tobacco, cabbage or barley.

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