CN116121297A

CN116121297A - Rice OsNPR1 gene and application thereof in resisting rice stripe disease

Info

Publication number: CN116121297A
Application number: CN202310055817.8A
Authority: CN
Inventors: 孙宗涛; 张合红; 魏中艳; 李雁军; 谢凯丽; 陈剑平
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2023-01-17
Filing date: 2023-01-17
Publication date: 2023-05-16

Abstract

The invention relates to the technical field of transgenosis and the field of plant virus disease control, in particular to application of an important regulatory factor OsNPR1 gene of a rice salicylic acid pathway in rice stripe disease resistance of plants.

Description

Rice OsNPR1 gene and application thereof in resisting rice stripe disease

Technical Field

The invention relates to the technical field of transgenosis and the field of plant virus disease control, in particular to the application field of important regulation factor OsNPR1 genes of a rice salicylic acid pathway in rice stripe disease resistance of plants.

Background

Rice stripe virus (Rice stripe virus, RSV), which is a multi-split negative sense single stranded RNA virus, is transmitted and prevalent in rice populations mediated by Laodelphax striatellus, which belongs to the genus Tenuivirus. Under natural conditions, RSV infects rice to cause rice stripe leaf blight, and typical symptoms of plant infection RSV include plant wilt dwarf, mottled and mottled leaf vein, discontinuous yellow stripes, twisted and drooping heart leaves, slow growth and development, no or less heading of the whole plant, and death of the plant seriously. The RSV shows different symptoms at different periods of plant infection, and in the example of rice, after the rice is infected with the RSV at the seedling stage, the plant is dwarfed and withered, and the death of the whole plant can be caused when the plant is serious; the dwarfing symptom of the RSV infected in the adult stage is not obvious, the veins have discontinuous yellow stripes, and the tillers are increased. In recent years, a great deal of research has been conducted by students at home and abroad on the biological functions of RSV molecules, including genomics, proteomics, interactions of viral proteins with host factors, and the like. The genome of RSV was found to consist of four single-stranded RNA fragments, which were approximately 17kb in total, and were named RNA1, RNA2, RNA3 and RNA4, respectively, according to their size, encoding a total of 7 proteins. Each viral protein has a different function to promote the replication of the infection by the virus. Wherein, RNA1 encodes a protein replicase RdRp, which mainly assists in viral replication; the P2 protein was identified as a weak inhibitor of silencing; the P3 protein is reported to be a silencing inhibitor of RSV; p4 is a disease-associated protein that can interact with the host PsbP (photosystem II subunit P), altering the localization of PsbP to recruit it into the cytoplasm to enhance RSV symptoms. The reduction of the insect population density of the mediator insects by the use of a large amount of insecticide causes serious environmental pollution. In addition, the rice virus disease has the characteristics of strong recessive nature, serious hazard and difficult prevention and control, and the possibility of outbreak at any time exists.

The rice stripe disease is a viral disease transmitted by the Laodelphax striatellus, and once the rice is infected, the rice has great influence on the yield, and the prevention and the control are very difficult. The disease is called "cancer" of rice by rice farmers. Typical yield losses of diseased fields are 20% -30% and severe fields can cause harvest failure. It was investigated that the Jiangsu province, like the Tong county, the city of the river shared more than 7000 hectares of rice, 500 hectares of disease occurred in 2004, rising to 4500 hectares in 2005, the dead area reached more than 20 hectares, and the direct economic loss was 2000 kiloyuan. Therefore, the prevention and treatment of rice stripe disease is related to national folk life, and effective prevention and treatment measures are very necessary; so far, the most economical and effective measure for preventing and treating rice stripe disease is to cultivate disease-resistant varieties, so that the cultivation of transgenic rice resistant materials can provide important theoretical and technical guidance for crop disease-resistant breeding.

Salicylic Acid (SA) is an important plant resistance hormone that plays an important role in the host's resistance to pathogen infection. NPR1 has the function of transcriptional activation, positively regulates the expression of downstream defense related genes, and NPR3/4 has the function of transcriptional co-inhibitor, negatively regulates the expression of downstream related genes. When the SA content in the plant body is increased, SA can promote the combination of NPR1 and a transcription factor TGA to start PR gene transcription, and meanwhile, salicylic acid inhibits the combination of NPR3/4 and the transcription factor TGA, so that the inhibition of NPR3/4 on gene transcription reaction is relieved. The homology of the OsNPR1 and the NPR1 in the Arabidopsis thaliana is highest, but the OsNPR1 and the Arabidopsis thaliana play different roles in the process of coping with infection of different types of pathogenic bacteria. For example: studies have shown that NPR1 positively regulates plant infection with bacteria in Arabidopsis, while negatively regulates plant resistance to fungi. However, no research report is available on the relationship between rice viruses and OsNPR 1. These studies indicate that OsNPR1 plays an important role in the process of rice pathogen infection resistance. However, no detailed study has been reported concerning the relationship between rice virus and OsNPR 1.

The invention provides a salicylic acid pathway important regulatory factor OsNPR1 gene and application thereof in rice stripe disease resistance. The full-length OsNPR1 gene is introduced into rice by utilizing a plant transgenic technology, a rice plant line which can be inherited stably is further identified, and an RSV virus inoculation experiment is carried out on the rice plant line, so that an RSV-resistant rice plant is obtained. Has important scientific significance for further understanding the pathogenic mechanism of viruses and the disease-resistant way of rice, and simultaneously provides theoretical basis and new strategy for rice antiviral breeding.

Disclosure of Invention

The invention relates to an important regulation factor OsNPR1 gene of a rice salicylic acid pathway and a coding protein thereof;

the nucleotide sequence of the OsNPR1 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the protein corresponding to the OsNPR1 gene coded by the rice regulatory factor is the amino acid coded by SEQ ID NO. 2.

In some specific embodiments, the nucleotide sequence of the osppr 1 gene is as follows:

OsNPR1(SEQ ID NO:1)

ATGGAGCCGCCGACCAGCCACGTCACCAACGCGTTCTCCGACTCGGA

CAGCGCGTCCGTGGAGGAGGGGGGCGCCGACGCGGACGCCGACGTGGAG

GCGCTCCGCCGCCTCTCCGACAACCTCGCCGCGGCGTTCCGCTCGCCCGA

GGACTTCGCGTTCCTCGCCGACGCGCGCATCGCCGTCCCGGGCGGCGGCG

GCGGCGGCGGCGACCTGCTGGTGCACCGCTGCGTGCTCTCCGCGCGGAGC

CCCTTCCTGCGCGGCGTCTTCGCGCGCCGCGCCGCCGCCGCCGCAGGCGG

CGGCGGCGAGGATGGCGGCGAGAGGCTGGAGCTCCGGGAACTCCTCGGC

GGCGGCGGCGAGGAGGTGGAGGTCGGGTACGAGGCGCTGCGGCTGGTGC

TCGACTACCTCTACAGCGGCCGCGTCGGCGACCTGCCCAAGGCGGCGTGC

CTCTGCGTCGACGAGGACTGCGCCCACGTCGGGTGCCACCCCGCCGTCGC

GTTCATGGCGCAGGTCCTCTTCGCCGCCTCCACCTTCCAGGTCGCCGAGCT

CACCAACCTCTTCCAGCGGCGTCTCCTTGATGTCCTTGATAAGGTTGAGGT

AGATAACCTTCTATTGATCTTATCTGTTGCCAACTTATGCAACAAATCTTGCA

TGAAACTGCTTGAAAGATGCCTTGATATGGTAGTCCGGTCAAACCTTGACA

TGATTACTCTTGAGAAGTCATTGCCTCCAGATGTTATCAAGCAGATTATTGA

TGCACGCCTAAGCCTCGGATTAATTTCACCAGAAAACAAGGGATTTCCTAA

CAAACATGTGAGGAGGATACACAGAGCCCTTGACTCTGACGATGTAGAGCT

AGTCAGGATGCTGCTCACTGAAGGACAGACAAATCTTGATGATGCGTTTGC

ACTGCACTACGCCGTCGAACATTGTGACTCCAAAATTACAACCGAGCTTTT

GGATCTCGCACTTGCAGATGTTAATCATAGAAACCCAAGAGGTTATACTGTT

CTTCACATTGCTGCGAGGCGAAGAGAGCCTAAAATCATTGTCTCCCTTTTA

ACCAAGGGGGCTCGGCCAGCAGATGTTACATTCGATGGGAGAAAAGCGGT

TCAAATCTCAAAAAGACTAACAAAACAAGGGGATTACTTTGGGGTTACCG

AAGAAGGAAAACCTTCTCCAAAAGATAGGTTATGTATTGAAATACTGGAGC

AAGCTGAAAGAAGGGACCCACAACTCGGAGAAGCATCAGTTTCTCTTGCA

ATGGCAGGTGAGAGTCTACGAGGAAGGTTGCTGTATCTTGAAAACCGAGT

TGCTTTGGCGAGGATTATGTTTCCGATGGAGGCAAGAGTAGCAATGGATATT

GCTCAAGTGGATGGAACTTTGGAATTTAACCTGGGTTCTGGTGCAAATCCA

CCTCCTGAAAGACAACGGACAACTGTTGATCTAAATGAAAGTCCTTTCATA

ATGAAAGAAGAACACTTAGCTCGGATGACGGCACTCTCCAAAACAGTGGA

GCTCGGGAAACGCTTTTTCCCGCGATGTTCGAACGTGCTCGACAAGATCAT

GGATGATGAAACTGATCCGGTTTCCCTCGGAAGAGACACGTCCGCGGAGA

AGAGGAAGAGGTTTCATGACCTGCAGGATGTTCTTCAGAAGGCATTCCACG

AGGACAAGGAGGAGAATGACAGGTCGGGGCTCTCGTCGTCGTCGTCATCG

ACATCGATCGGGGCCATTCGACCAAGGAGATGA

in some specific embodiments, the protein sequence encoded by the osppr 1 gene is as follows:OsNPR1(SEQ ID NO: 2)

MEPPTSHVTNAFSDSDSASVEEGGADADADVEALRRLSDNLAAAFRSPEDFA

FLADARIAVPGGGGGGGDLLVHRCVLSARSPFLRGVFARRAAAAAGGGGED

GGERLELRELLGGGGEEVEVGYEALRLVLDYLYSGRVGDLPKAACLCVDEDC

AHVGCHPAVAFMAQVLFAASTFQVAELTNLFQRRLLDVLDKVEVDNLLLILSV

ANLCNKSCMKLLERCLDMVVRSNLDMITLEKSLPPDVIKQIIDARLSLGLISPE

NKGFPNKHVRRIHRALDSDDVELVRMLLTEGQTNLDDAFALHYAVEHCDSKI

TTELLDLALADVNHRNPRGYTVLHIAARRREPKIIVSLLTKGARPADVTFDGR

KAVQISKRLTKQGDYFGVTEEGKPSPKDRLCIEILEQAERRDPQLGEASVSLA

MAGESLRGRLLYLENRVALARIMFPMEARVAMDIAQVDGTLEFNLGSGANPP

PERQRTTVDLNESPFIMKEEHLARMTALSKTVELGKRFFPRCSNVLDKIMDDE

TDPVSLGRDTSAEKRKRFHDLQDVLQKAFHEDKEENDRSGLSSSSSSTSIGAIR

PRR

on the other hand, the invention relates to the application of the important regulation factor OsNPR1 gene of the salicylic acid pathway of rice in the breeding of anti-graminaceous virus crops, especially gramineous food crops;

in some embodiments, the tenuiviridae virus (tenuivirens) comprises barnyard grass white leaf virus (Echinochloa hoja blanca virus, EHBV), corn stripe virus (Maize stripe virus, MSpV), rice grass dwarf virus (Rice grassy stunt virus, RGSV), rice white leaf virus (Rice hoja blanca virus, RHBV), rice stripe virus (Rice stripe virus, RSV), tail grass white leaf virus (Urochloa hoja blanca virus, UHBV).

In some embodiments, the leptospirovirus is preferably a rice stripe virus.

In some embodiments, the gramineous food crop is preferably rice, maize, wheat, oats, and barley; more preferably rice, most preferably Nippon Temminck;

on the other hand, the invention relates to a construction method of a rice OsNPR1 over-expression vector, which comprises the following steps:

primers with BamHI and SacI cleavage sites were designed to amplify OsNPR1, and the template was sequenced to identify the correct pMD18-T-OsNPR1 recombinant plasmid. The pCV1300 vector was double digested with BamHI and SacI enzymes.

The primer of the enzyme cutting site is used for constructing an OsNPR1 binary expression vector PCV1300, and the primer sequence is as follows:

PCV-OsNPR1-F:

GTTCCAGATTACGCTGGATCCATGGAGCCGCCGACCAGCCA

SEQ ID NO:5

PCV-OsNPR1-R:

ATCGGGGAAATTCGAGCTCTCATCTCCTTGGTCGAATGGC

SEQ ID NO:6

PCR amplification system: the reaction system (50. Mu.L) was as follows: 2X PCR KOD FX Neo Buffer

25. Mu.L, 2mM dNTPs,KOD FX Neo 1. Mu.L, 1.5. Mu.L upstream primer, 1.5. Mu.L downstream primer, 1. Mu.L cDNA, H ₂ O15. Mu.L, total volume 50. Mu.L.

PCR procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, renaturation at 60℃for 30s, extension at 72℃for 2min,35 cycles; final extension at 72℃for 10min.

After the PCV1300 vector is digested, the PCR products are recovered, and the PCR products are respectively connected with the vector; positive clones were selected and sequenced to confirm that the PCV1300-OsNPR1 expression vector was successfully constructed.

In another aspect, the present invention relates to a method for preparing a resistant transgenic plant comprising the steps of:

(1) Culturing EHA105 agrobacterium and rice callus induction culture: transforming agrobacterium tumefaciens EHA105 with a plasmid which is preserved at the temperature of minus 80 ℃ and contains a target gene OsNPR1 expression vector;

(2) Agrobacterium transfected callus: monoclone of EHA105 is inoculated into LB liquid culture medium and OD ₆₀₀ =0.6. Collecting thallus, re-suspending the thallus with AS agrobacterium suspension culture liquid and OD ₆₀₀ =0.1. And adding the induced callus into an agrobacterium suspension culture solution, and soaking for 5min. Taking out the callus, and placingThe cells were incubated in a co-culture medium at 25℃in an illumination incubator for 2.5d.

(3) Screening and culturing of resistant calli: placing the callus on a culture medium containing hygromycin B, screening after 30-45d, placing the callus obtained by screening into a rooting culture medium for culture until a green plant with roots is produced, and culturing for 2 weeks to obtain the transgenic rice.

In some embodiments, the specific steps of culturing the EHA105 agrobacterium include: firstly, 1 mu L of plasmid is sucked into 100 mu L of competent cells, blown and uniformly mixed, then the mixture is added into an electrode cup which is cooled in advance at 4 ℃, voltage conversion is carried out at 2200V, 500 mu L of non-resistant LB liquid culture medium is added, the culture is carried out for 2-3h at 28 ℃ and 200rpm, the mixture is coated on a plate containing 50 mu g/ml Kan and Rif, and the culture is carried out for 3d in a culture box at 28 ℃. Removing the shell of mature rice seeds, soaking in 75% alcohol for 10min, and cleaning with sterile water for 3 times; 30% sodium hypochlorite solution, soaking for 30min; cleaning with sterile water, and soaking for 30min. The seeds were placed in mature embryo induction medium with sterilized forceps, the incubator was illuminated at 28 ℃ for 3 weeks, and the grown calli were transferred to subculture medium with sterilized forceps, and subcultured for 1 week in the incubator illuminated at 28 ℃.

In some embodiments, the induction medium comprises: 24.1g/L of N6 culture medium, 2.5mg/L2,4-D, PH =5.8;

in some embodiments, the secondary medium comprises: 24.1g/L of N6 culture medium, 2.5mg/L of 2,4-D, 50mg/L of hygromycin, 300mg/mL of cephalosporin, and PH=5.8;

in some embodiments, the rooting medium comprises: 1/2MS 39.45g/L, 0.5mg/L NAA, 50mg/L hygromycin, pH=5.8;

in some embodiments, the co-culture medium comprises: 24.1g/L of N6 medium, 2.5mg/L of 2,4-D, 200 mu mol/L of acetosyringone and PH=5.8;

on the other hand, the invention also relates to a method for detecting rice stripe virus resistance of rice, which comprises the following steps:

genomic DNA of transgenic rice was extracted by SDS method. PCR amplification was performed using 0.2. Mu.L of DNA template, 10. Mu.L of amplification system, and the detection primers were as follows:

OsNPR1-detect-F:ATGGAGCCGCCGACCAGCCA SEQ ID NO:3

OsNPR1-detect-R:TCATCTCCTTGGTCGAATGGC SEQ ID NO:4

and (3) sequencing and comparing the PCR products to confirm whether the OsNPR1 gene exists.

Description of the drawings:

fig. 1: relative expression level of ONPR1 in osppr 1 overexpressing transgenic rice.

Fig. 2: osNPR1 over-expression transgene after RSV infection and control symptoms of japan, scale represent 5cm (top) and 1cm (bottom), respectively.

Fig. 3: qRT-PCR after RSV infection detects the virus content of the OsNPR1 over-expression transgene and the control Japanese sunny.

Fig. 4: western blotting after RSV infection detects the virus content of the OsNPR1 over-expression transgene and the control Japanese sunny.

The following examples are given for better illustration of the present invention, but are not intended to limit the scope of the present invention to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible and can be made without departing from the scope of the invention. Examples:

Detailed Description

The rice varieties selected in the series of experiments are as follows: nippon sunny day

The relevant medium components are as follows:

induction medium: n6 Medium (manufacturer: haibo Biotechnology Co., ltd., product number: HBZ 0601) 24.1g/L, 2.5mg/L2,4-D, PH =5.8.

Subculture medium: n6 Medium (manufacturer: haibo Biotechnology Co., ltd., product number: 15HBZ 0601) 24.1g/L, 2,4-D, 50mg/L hygromycin, 300mg/mL cephalosporin, pH=5.8.

Co-culture medium: n6 Medium (manufacturer: haibo Biotechnology Co., ltd., product number: HBZ 0601) 24.1g/L, 2.5mg/L2,4-D, 200. Mu. Mol/L acetosyringone, pH=5.8.

Rooting medium: :1/2MS (manufacturer: haibo biotechnology Co., ltd., product number: HB 8469-6)

39.45g/L, 0.5mg/L NAA, 50mg/L hygromycin, pH=5.8.

Example 1: construction of rice OsNPR1 plant expression vector

(1) Cloning of Rice OsNPR1 Gene

Primers OsNPR1-F and OsNPR1-R were designed based on the full-length sequence SEQ ID NO:1 of OsNPR1, and the primer sequences used were as follows:

OsNPR1-F：5’-ATGGAGCCGCCGACCAGCCA-3’；SEQ ID NO:3

OsNPR1-R：5’-TCATCTCCTTGGTCGAATGGC-3’；SEQ ID NO:4

PCR amplification system: the total volume was 50. Mu.L, including 5. Mu.L of 10 XPCR buffer, upstream and downstream primers (10. Mu.M)

1.5. Mu.L, 5. Mu.L dNTP Mix (2.5 mM), 1. Mu.L cDNA template, 1. Mu.L Taq enzyme (5U/. Mu.L), 35. Mu.L ddH2O, respectively.

And (3) recovering the PCR product, connecting the PCR product with a pMD18-T vector, picking and cloning, carrying out measurement to obtain the correct pMD18-T-OsNPR1 recombinant plasmid, and carrying out sequencing confirmation by Hangzhou Kangshengmbh. The obtained gene has a length of 1749bp and codes 582 amino acids as shown in SEQ ID No. 1.

(2) Construction of an overexpression vector

PCV-OsNPR1-F:

GTTCCAGATTACGCTGGATCCATGGAGCCGCCGACCAGCCA

SEQ ID NO:5

PCV-OsNPR1-R:

ATCGGGGAAATTCGAGCTCTCATCTCCTTGGTCGAATGGC

SEQ ID NO:6

25. Mu.L, 2mM dNTPs,KOD FX Neo 1. Mu.L, 1.5. Mu.L of the upstream primer, 1.5. Mu.L of the downstream primer, 1. Mu.L of cDNA, 15. Mu.L of H2O, and a total volume of 50. Mu.L.

After the PCV1300 vector is digested, the PCR products are recovered, and the PCR products are respectively connected with the vector; positive clones were selected and sequenced to confirm that the PCV1300-OsNPR1 expression vector was successfully constructed. Sequencing clearly and successfully constructs the PCV1300-OsNPR1 expression vector.

Example 2: genetic transformation of rice

(1) Culturing EHA105 agrobacterium and rice callus induction culture: the method comprises the steps of taking plasmid transformed Agrobacterium tumefaciens EHA105 (Digeable organism, CAT#: AE 1010) containing a target vector stored in a refrigerator at-80 ℃, firstly sucking 1 mu L of plasmid, adding the plasmid into 100 mu L of competent cells, blowing and mixing uniformly, adding the mixture into an electrode cup which is cooled in advance at 4 ℃, transforming at 2200V, adding 500 mu L of non-resistant LB liquid medium, culturing for 2-3h at 28 ℃ at 200rpm, coating the mixture on a Kan and Rif flat plate containing 50 mu g/ml, and culturing for 3d in a 28 ℃ incubator. Removing the shell of mature rice seeds, soaking in 75% alcohol for 10min, and cleaning with sterile water for 3 times; 30% sodium hypochlorite solution, soaking for 30min; cleaning with sterile water, and soaking for 30min. The seeds were placed in mature embryo induction medium with sterilized forceps, the incubator was illuminated at 28 ℃ for 3 weeks, and the grown calli were transferred to subculture medium with sterilized forceps, and subcultured for 1 week in the incubator illuminated at 28 ℃.

(2) Agrobacterium transfected callus: the monoclonal EHA105 was selected and inoculated into LB liquid medium, od600=0.6. The cells were collected and resuspended in AS agrobacteria suspension medium, od600=0.1. And adding the induced callus into an agrobacterium suspension culture solution, and soaking for 5min. The callus was taken out and placed in a co-culture medium and cultured in an illumination incubator at 25℃for 2.5d.

Example 3: identification of transgenic Rice positivity

(1) RT-PCR detection

Genomic DNA of transgenic rice was extracted by SDS method. And taking 0.2 mu L of DNA template, carrying out PCR amplification, wherein the amplification system is 10 mu L, and the size of a PCR product is 1000bp. The detection primers were as follows:

OsNPR1-detect-F:CTGCTGGTGCACCGCTG SEQ ID NO:7

OsNPR1-detect-R:GCTCCAGTATTTCAATACATAA SEQ ID NO:8

(2) Transgenic plant RT-qPCR detection

The positive transgenic plants are subjected to leaf total RNA extraction and reverse transcription to form cDNA, the quantitative primers are qRT-OsNPR1, the relative expression level of the OsNPR1 gene is shown in a figure 1, and the relative expression level of the OsNPR1 gene in the over-expression lines (OsNPR-2# and OsNPR-7#) is obviously higher than that of the control Japanese rice. The quantitative primer sequences were as follows:

qRT-OsNPR1-F:CTGGGTTCTGGTGCAAATCC SEQ ID NO:9

qRT-OsNPR1-R:AACCTCTTCCTCTTCTCCGC SEQ ID NO:10

example 4: transgenic rice inoculation RSV

(1) Soaking the OsNPR1 transgenic rice and the control Nippon Rice material for accelerating germination for 2-3d, sowing the soaked seeds in a 1L beaker after the seeds are whitened, biologically repeating about 3 seedlings per cup, and culturing under the dark condition of 16h illumination at 30 ℃ for 8 h.

(2) Virus inoculation experiments are carried out by 3-4-year-old Laodelphax striatellus carrying RSV and healthy Laodelphax striatellus, the RSV carrying and the Laodelphax striatellus without toxicity are inoculated on 3-4-leaf-stage rice plants according to the proportion of 2-3 seedlings, and all insects are swept out after 3d feeding.

(3) After 30d, symptoms of the diseased rice were observed and the virulence status of the rice was determined by qRT-PCR.

Example 5: resistance analysis of Rice after RSV inoculation

(1) qRT-PCR detection of virus content of OsNPR1 transgene and control Japanese sunny plant

After transplanting the rice for 30 days, it was found that RSV-infected rice exhibited symptoms of flowers and leaves, and that two overexpressed transgenic lines of OsNPR1 (OsNPR-2# and OsNPR-7#) exhibited slight symptoms of flowers and leaves, as compared with control NiP, which exhibited obvious symptoms of flowers and leaves, as shown in FIG. 2. The diseased rice plants are mixed and sampled, 3 biological repeats of each group are carried out, and the expression quantity of the virus RSV CP gene is detected by qRT-PCR, as shown in figure 3, the expression quantity of the transgenic plant CP gene is obviously lower than that of the control by 0.37 times and 0.40 times respectively. The result shows that the resistance of the transgenic plant of the OsNPR1 to the RSV infection is influenced by the expression of the gene of the OsNPR1, and the overexpression of the OsNPR1 in rice can obviously enhance the resistance of the rice to the RSV infection.

The quantitative primer sequences were as follows:

qRSV-CP-F AGGCAATCAATGACATCTCC；SEQ ID NO:11

qRSV-CP-R ATCTCTCACAAAGCCAGTGC；SEQ ID NO:12

(2) Western blotting detection of virus content of OsNPR1 transgene and control Japanese sunny plant

After 30d of transplanting, mixing and sampling samples with obvious symptoms, taking down the samples, quick-freezing the samples by liquid nitrogen, grinding the samples until the samples are in powder form, weighing 0.1g of the ground samples by a 2mL centrifuge tube, adding 300 mu L of protein lysate Lysis buffer (100 mm Tris-HCl, pH 6.8 and 10% SDS) into the centrifuge tube, and uniformly mixing the samples on ice for 10min; after full lysis, placing the sample in a pre-cooled centrifuge at 4 ℃ for 15min at 12000rpm/min, sucking 100 mu L of supernatant into a new centrifuge tube at 1.5mL, adding 25 mu L of 5×loading buffer (1 m Tris-HCl, pH 6.8, 10% SDS,1% bromophenol blue, 50% glycine and 2% beta-mercaptoethanol), mixing uniformly, and boiling in boiling water at 100 ℃ for 10min; taking out, centrifuging at room temperature for 1min, and storing in a refrigerator at-40deg.C; SDS-PAGE gel electrophoresis is carried out, gel containing the target band is transferred onto PVDF film, and a proper amount of primary antibody incubation liquid (RSV CP antibody, 1:5000) prepared by 5% skimmed milk powder is added; then incubating the rabbit secondary antibody for about 1 h; finally, developing: the secondary antibody incubation reaction was discarded, and the solution was rinsed with an appropriate amount of TBST Buffer (1 XTBS Buffer 3L, 1.5mL of Tween-20 was added, i.e., the final concentration of Tween-20 was 0.5%) for 10 min/time and three times. And uniformly mixing ECL developer, and uniformly coating the ECL developer on a film to perform chemiluminescence and color development. Finally, the virus content of the sample is determined according to the size of the RSV CP 35 KD. CBB stands for coomassie brilliant blue staining, with the aim of ensuring consistent loading of samples. As shown in fig. 4, the accumulation of CP protein was significantly lower in transgenic plants than in controls. The above results demonstrate that overexpression of OsNPR1 in rice can significantly enhance the resistance of rice to RSV infection.

The comprehensive experimental results show that:

(1) According to the invention, the important regulation factor OsNPR1 gene of the salicylic acid pathway is over-expressed to obtain transgenic rice with stable inheritance, and the rice resistance to rice stripe virus can be enhanced after the over-expression of the OsNPR1 gene is found, so that the research result further enriches a germplasm resource pool for resisting rice stripe disease.

Claims

Application of OsNPR1 gene in crop breeding of anti-gracilaria viruses, wherein the gene sequence is as follows: SEQ ID NO. 1.
2. The use as claimed in claim 1, wherein the protein sequence of the gene encoding OsNPR1 is shown as SEQ ID NO. 2.
3. The use of any one of claims 1-2, wherein the tenuiviridae virus (tenuivirens) comprises barnyard grass white leaf virus (Echinochloa hoja blanca virus, EHBV), corn stripe virus (Maize stripe virus, MSpV), rice grass dwarf virus (Rice grassy stunt virus, RGSV), rice white leaf virus (Rice hoja blanca virus, RHBV), rice stripe virus (Rice stripe virus, RSV), tail grass white leaf virus (Urochloa hoja blanca virus, UHBV).
4. The use according to claim 3, wherein the crop is preferably rice, maize, wheat, oats and barley; more preferably rice, most preferably Nippon Temminck.
5. A method for preparing Rice Stripe Virus (RSV) transgenic rice, comprising the steps of:

(1) Construction of Rice overexpression OsNPR1 vector

Designing a primer with BamHI and SacI cleavage sites to amplify the OsNPR1 gene in claim 1, carrying out double cleavage on the pCV1300 vector by BamHI and SacI enzymes, and constructing an OsNPR1 binary expression vector PCV1300 by using the primer with the cleavage sites, wherein the primer sequence is as follows:

PCV-OsNPR1-F:

GTTCCAGATTACGCTGGATCCATGGAGCCGCCGACCAGCCA

SEQ ID NO:5

PCV-OsNPR1-R:

ATCGGGGAAATTCGAGCTCTCATCTCCTTGGTCGAATGGC

SEQ ID NO:6

after the PCV1300 vector is digested, the primer amplification PCR products are respectively connected with the vector; positive clones were selected and sequenced to confirm that the PCV1300-OsNPR1 expression vector was successfully constructed.

(2) Culturing EHA105 agrobacterium and rice callus induction culture: taking a plasmid PCV1300-OsNPR1 which is preserved in a refrigerator at the temperature of minus 80 ℃ and contains a target gene vector with the sequence shown as SEQ ID NO. 1 to transform agrobacterium tumefaciens EHA105; the detailed operation steps are as follows, firstly, 1 mu L of plasmid is sucked into 100 mu L of competent cells, blown and mixed uniformly, then the mixture is added into an electrode cup which is cooled in advance at 4 ℃,2200V voltage conversion is carried out, 500 mu L of non-resistant LB liquid culture medium is added, the mixture is cultured for 2 to 3 hours at 28 ℃ and 200rpm, the mixture is coated on a plate containing 50 mu g/ml Kan and Rif, and the mixture is cultured for 3 days in a 28 ℃ incubator; removing the shell of mature rice seeds, soaking in 75% alcohol for 10min, and cleaning with sterile water for 3 times; 30% sodium hypochlorite solution, soaking for 30min; cleaning with sterile water, and soaking for 30min; placing the seeds into a mature embryo induction culture medium by using sterilized forceps, illuminating an incubator at 28 ℃ for 3 weeks, transferring the grown calli into a secondary culture medium by using sterilized forceps, and subculturing the calli in the incubator at 28 ℃ for 1 week;

(3) Agrobacterium transfected callus: monoclone of EHA105 is inoculated into LB liquid culture medium and OD ₆₀₀ =0.6; collecting thallus, re-suspending the thallus with AS agrobacterium suspension culture liquid and OD ₆₀₀ =0.1; adding the induced callus into agrobacterium suspension culture solution, and soaking for 5min; taking out the callus, placing the callus into a co-culture medium, and culturing in an illumination incubator at 25 ℃ for 2.5d;

(4) Screening and culturing of resistant calli: placing the callus on a culture medium containing hygromycin B, screening after 30-45d, placing the callus obtained by screening into a rooting culture medium for culture until a green plant with roots is produced, and culturing for 2 weeks to obtain the transgenic rice.
6. The method of preparation of claim 5, the induction medium comprising: n6 medium 24.1g/L, 2.5mg/L2,4-D, PH =5.8.
7. The method of preparation of claim 5, the secondary medium comprising: n6 medium 24.1g/L, 2.5mg/L2,4-D, 50mg/L hygromycin, 300mg/mL cephalosporin, pH=5.8.
8. The method of preparation of claim 5, wherein the rooting medium comprises: 1/2MS 39.45g/L, 0.5mg/L NAA, 50mg/L hygromycin, pH=5.8.
9. The method of preparation of claim 5, the co-culture medium comprising: n6 medium 24.1g/L, 2.5mg/L2,4-D, 200. Mu. Mol/L acetosyringone, pH=5.8.
10. A method for detecting rice stripe virus resistance of rice comprises the following steps:

extracting genome DNA of transgenic rice to be detected by SDS method, taking 0.2 mu L of DNA template, carrying out PCR amplification, and amplifying 10 mu L of system, wherein the detection primers are as follows:

OsNPR1-detect-F:ATGGAGCCGCCGACCAGCCA SEQ ID NO:3

OsNPR1-detect-R:TCATCTCCTTGGTCGAATGGC SEQ ID NO:4

and (3) sequencing and comparing the PCR products to confirm whether the OsNPR1 gene exists.