Rice osnpr1 gene and its application in resistance to rice stripe disease

Abstract

The application relates to the field of transgenic technology and the field of plant virus disease prevention, in particular to application of a rice salicylic acid pathway important regulating factor OsNPR1 gene in plant resistance to rice stripe disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of transgenic technology and the field of plant viral disease prevention, in particular to the application of the important regulatory factor of the salicylic acid pathway in rice, OsNPR1 gene, in the field of plant resistance to rice stripe disease. BACKGROUND

[0002] Rice stripe virus (RSV) is transmitted by the planthopper in rice populations. The virus belongs to the Tenuivirus genus and is a multipartite negative-sense single-stranded RNA virus. In natural conditions, RSV infection of rice causes rice stripe disease. The typical symptoms of plant infection with RSV include plant dwarfing, discolored mottling of leaf veins, and the appearance of discontinuous yellow stripes, twisted and drooping of the heart leaf, and growth retardation, resulting in no or reduced heading of the whole plant, and even death. RSV shows different symptoms at different stages of infection of the plant. For example, in the case of rice, after the rice seedlings are infected with RSV, the plants show dwarfing and heart-leaf wilting, and in severe cases, the whole plant dies. In the case of adult plants, the dwarfing symptom is not obvious, the leaf veins show discontinuous yellow stripes, and the tillering is increased. In recent years, scholars at home and abroad have carried out a large number of researches on the molecular biology functions of RSV, including genomics, proteomics, and the interaction between viral proteins and host factors. It is found that the genome of RSV is composed of four single-stranded RNA fragments, with a total size of about 17 kb, and is named RNA1, RNA2, RNA3 and RNA4 according to its size, which encodes 7 proteins. Each viral protein has different functions to promote the infection and replication of the virus. Among them, RNA1 encodes a protein replicase RdRp, which mainly assists viral replication; P2 protein is identified as a weak silencing suppressor; P3 protein is reported to be a silencing suppressor of RSV; P4 is a pathogenesis-related protein that can interact with host PsbP (photosystem II subunit P) to change the localization of PsbP and recruit it to the cytoplasm to enhance RSV symptoms. The use of a large amount of pesticides to reduce the insect population density of the vector insects will cause serious environmental pollution. In addition, rice viral diseases have the characteristics of strong latency, serious damage, and difficult prevention and control, and there is a possibility of outbreak at any time.

[0003] Rice stripe disease is a viral disease caused by the spread of planthoppers, once infected with rice yield impact is great. And prevention is very difficult. The disease is called by rice farmers as rice "cancer". Generally, the yield loss of the field is 20%-30%, and the severe field will cause absolute loss. According to the survey, Rudong County, Jiangsu Province, has more than 7000 hectares of rice. More than 500 hectares were infected in 2004, and 4500 hectares in 2005. More than 20 hectares were absolutely lost. The direct economic loss is 20 million yuan. Therefore, the prevention and control of rice stripe disease is related to the national economy and people's livelihood, and it is necessary to take effective prevention and control measures; so far, the most economical and effective measure to prevent and control rice stripe disease is to cultivate disease-resistant varieties, therefore, cultivating rice transgenic resistance material can provide important theoretical and technical guidance for crop disease-resistant breeding.

[0004] Salicylic acid (SA) as an important plant resistance hormone, plays an important role in the process of host resistance to pathogen infection. NPR1 has the function of transcription activation, positively regulates the expression of downstream defense-related genes, and NPR3 / 4 has the function of transcription co-suppressor, negatively regulates the expression of downstream related genes. When the SA content in the plant body increases, SA promotes the combination of NPR1 and transcription factor TGA to start PR gene transcription, at the same time, salicylic acid inhibits the combination of NPR3 / 4 and transcription factor TGA, and then releases the inhibition of NPR3 / 4 on gene transcription reaction. OsNPR1 has the highest homology with NPR1 in Arabidopsis, but they play different roles in response to different types of pathogenic bacteria infection. For example: studies have shown that NPR1 in Arabidopsis positively regulates the infection of plants to bacteria, and negatively regulates the resistance of plants to fungi. However, there is no research report on the relationship between rice virus and OsNPR1. These studies show that OsNPR1 plays an important role in the process of rice resistance to pathogenic bacteria infection. However, there is no detailed research report on the relationship between rice virus and OsNPR1.

[0005] The application provides an important regulatory factor OsNPR1 gene in a salicylic acid pathway and application of the important regulatory factor OsNPR1 gene in resisting rice stripe disease. The full-length OsNPR1 gene is introduced into rice by using plant transgenic technology, and a rice strain with stable heredity is further identified, and an RSV infection experiment is conducted on the rice strain, and an RSV-resistant rice plant is obtained. The application has important scientific significance for further understanding of virus pathogenesis and rice self-resistance pathway, and provides a theoretical basis and a new strategy for rice virus-resistant breeding. SUMMARY

[0006] The application relates to an important regulatory factor OsNPR1 gene in a rice salicylic acid pathway and coded protein thereof.

[0007] The OsNPR1 gene, the nucleotide sequence of which is shown as SEQ ID NO: 1, the protein encoded by the rice regulatory factor coding gene OsNPR1 of this type, the amino acid sequence of which is encoded by SEQ ID NO. 2.

[0008] In some specific embodiments, the nucleotide sequence of the OsNPR1 gene is as follows:

[0009] OsNPR1 (SEQ ID NO: 1)

[0010] ATGGAGCCGCCGACCAGCCACGTCACCAACGCGTTCTCCGACTCGGA

[0011] CAGCGCGTCCGTGGAGGAGGGGGGCGCCGACGCGGACGCCGACGTGGAG

[0012] GCGCTCCGCCGCCTCTCCGACAACCTCGCCGCGGCGTTCCGCTCGCCCGA

[0013] GGACTTCGCGTTCCTCGCCGACGCGCGCATCGCCGTCCCGGGCGGCGGCG

[0014] GCGGCGGCGGCGACCTGCTGGTGCACCGCTGCGTGCTCTCCGCGCGGAGC

[0015] CCCTTCCTGCGCGGCGTCTTCGCGCGCCGCGCCGCCGCCGCCGCAGGCGG

[0016] CGGCGGCGAGGATGGCGGCGAGAGGCTGGAGCTCCGGGAACTCCTCGGC

[0017] GGCGGCGGCGAGGAGGTGGAGGTCGGGTACGAGGCGCTGCGGCTGGTGC

[0018] TCGACTACCTCTACAGCGGCCGCGTCGGCGACCTGCCCAAGGCGGCGTGC

[0019] CTCTGCGTCGACGAGGACTGCGCCCACGTCGGGTGCCACCCCGCCGTCGC

[0020] GTTCTTGGCGCAGGTCCTCTTCGCCGCCTCCACCTTCCAGGTCGCCGAGCT

[0021] CACCAACCTCTTCCAGCGGCGTCTCCTTGATGTCCTTGATAAGGTTGAGGT

[0022] AGATAACCTTCTATTGATCTTATCTGTTGCCAACTTATGCAACAAATCTTGCA

[0023] TGAAACTGCTTGAAAGATGCCTTGATATGGTAGTCCGGTCAAACCTTGACA

[0024] TGATTACTCTTGAGAAGTCATTGCCTCCAGATGTTATCAAGCAGATTATTGA

[0025] TGCACGCCTAAGCCTCGGATTAATTTCACCAGAAAACAAGGGATTTCCTAA

[0026] CAAACATGTGAGGAGGATACACAGAGCCCTTGACTCTGACGATGTAGAGCT

[0027] AGTCAGGATGCTGCTCACTGAAGGACAGACAAATCTTGATGATGCGTTTGC

[0028] ACTGCACTACGCCGTCGAACATTGTGACTCCAAAATTACAACCGAGCTTTT

[0029] GGATCTCGCACTTGCAGATGTTAATCATAGAAACCCAAGAGGTTATACTGTT

[0030] CTTCACATTGCTGCGAGGCGAAGAGAGCCTAAAATCATTGTCTCCCTTTTA

[0031] ACCAAGGGGGCTCGGCCAGCAGATGTTACATTCGATGGGAGAAAAGCGGT

[0032] TCAAATCTCAAAAAGACTAACAAAACAAGGGGATTACTTTGGGGTTACCG

[0033] AAGAAGGAAAACCTTCTCCAAAAGATAGGTTATGTATTGAAATACTGGAGC

[0034] AAGCTGAAAGAAGGGACCCACAACTCGGAGAAGCATCAGTTTCTCTTGCA

[0035] ATGGCAGGTGAGAGTCTACGAGGAAGGTTGCTGTATCTTGAAAACCGAGT

[0036] TGCTTTGGCGAGGATTATGTTTCCGATGGAGGCAAGAGTAGCAATGGATATT

[0037] GCTCAAGTGGATGGAACTTTGGAATTTAACCTGGGTTCTGGTGCAAATCCA

[0038] CCTCCTGAAAGACAACGGACAACTGTTGATCTAAATGAAAGTCCTTTCATA

[0039] ATGAAAGAAGAACACTTAGCTCGGATGACGGCACTCTCCAAAACAGTGGA

[0040] GCTCGGGAAACGCTTTTTCCCGCGATGTTCGAACGTGCTCGACAAGATCAT

[0041] GGATGATGAAACTGATCCGGTTTCCCTCGGAAGAGACACGTCCGCGGAGA

[0042] AGAGGAAGAGGTTTCATGACCTGCAGGATGTTCTTCAGAAGGCATTCCACG

[0043] AGGACAAGGAGGAGAATGACAGGTCGGGGCTCTCGTCGTCGTCGTCATCG

[0044] ACATCGATCGGGGCCATTCGACCAAGGAGATGA

[0045] In some specific embodiments, the OsNPR1 gene encodes a protein sequence as follows: OsNPR1 (SEQ ID NO: 2)

[0046] MEPPTSHVTNAFSDSDSASVEEGGADADADVEALRRLSDNLAAAFRSPEDFA

[0047] FLADARIAVPGGGGGGGDLLVHRCVLSARSPFLRGVFARRAAAAAGGGGED

[0048] GGERLELRELLGGGGEEVEVGYEALRLVLDYLYSGRVGDLPKAACLCVDEDC

[0049] AHVGCHPAVAFMAQVLFAASTFQVAELTNLFQRRLLDVLDKVEVDNLLLILSV

[0050] ANLCNKSCMKLLERCLDMVVRSNLDMITLEKSLPPDVIKQIIDARLSLGLISPE

[0051] NKGFPNKHVRRIHRALDSDDVELVRMLLTEGQTNLDDAFALHYAVEHCDSKI

[0052] TTELLDLALADVNHRNPRGYTVLHIAARRREPKIIVSLLTKGARPADVTFDGR

[0053] KAVQISKRLTKQGDYFGVTEEGKPSPKDRLCIEILEQAERRDPQLGEASVSLA

[0054] MAGESLRGRLLYLENRVALARIMFPMEARVAMDIAQVDGTLEFNLGSGANPP

[0055] PERQRTTVDLNESPFIMKEEHLARMTALSKTVELGKRFFPRCSNVLDKIMDDE

[0056] TDPVSLGRDTSAEKRKRFHDLQDVLQKAFHEDKEENDRSGLSSSSSSTSIGAIR

[0057] PRR

[0058] In another aspect, the present application relates to the application of the important regulatory factor of the salicylic acid pathway, OsNPR1 gene in rice, in breeding crops against viruses of the genus Nanovirus, especially in breeding crops of the family Poaceae.

[0059] In some embodiments, the Tenuiviruses include Echinochloa hoja blanca virus (EHBV), Maize stripe virus (MSpV), Rice grassy stunt virus (RGSV), Rice hoja blanca virus (RHBV), Rice stripe virus (RSV), and Urochloa hojablanca virus (UHBV).

[0060] In some embodiments, the Tenuiviruses are preferably Rice stripe virus.

[0061] In some embodiments, the Poaceae food crops are preferably rice, corn, wheat, oat and barley; more preferably rice, and most preferably Nipponbare.

[0062] In another aspect, the present application relates to a method for constructing a rice OsNPR1 overexpression vector, comprising the following steps:

[0063] The primers with BamHI and SacI enzyme cutting sites are designed to amplify OsNPR1, and the template is the pMD18-T-OsNPR1 recombinant plasmid which has been identified correctly by sequencing. The pCV1300 vector is double digested with BamHI and SacI.

[0064] The primers with enzyme cutting sites are used for constructing the OsNPR1 binary expression vector PCV1300, and the primer sequences are as follows:

[0065] PCV-OsNPR1-F:

[0066] GTTCCAGATTACGCTGGATCCATGGAGCCGCCGACCAGCCA

[0067] SEQ ID NO: 5

[0068] PCV-OsNPR1-R:

[0069] ATCGGGGAAATTCGAGCTCTCATCTCCTTGGTCGAATGGC

[0070] SEQ ID NO: 6

[0071] PCR amplification system: the reaction system (50 μL) is as follows: 2×PCR KOD FX Neo Buffer

[0072] 25 μL, 2 mM dNTPs, KOD FX Neo 1 μL, upstream primer 1.5 μL, downstream primer 1.5 μL, cDNA 1 μL, H2O 15 μL, total volume 50 μL.

[0073] PCR program: 95℃ pre-denaturation 3 min; 95℃ denaturation 30 s, 60℃ recombination 30 s, 72℃ extension 2 min, 35 cycles; 72℃ final extension 10 min.

[0074] After the PCV1300 vector is digested, the PCR product is recovered, and the PCR product is connected with the vector; positive clones are selected, and sequencing is performed to confirm that the PCV1300-OsNPR1 expression vector has been successfully constructed.

[0075] On the other hand, the present application relates to a method for preparing a resistant transgenic plant, the steps of which include:

[0076] (1) Culture of EHA105 Agrobacterium and induction of rice callus culture: the plasmid containing the OsNPR1 expression vector of the target gene stored in the -80℃ refrigerator is transformed into the root nodule Agrobacterium EHA105;

[0077] (2) Agrobacterium transfection of callus: the single colony of EHA105 is inoculated into LB liquid medium, and the OD 600 = 0.6. The bacterial cells are collected, and the bacterial cells are resuspended with the Agrobacterium suspension culture solution of AS, and the OD 600 = 0.1. The induced callus is soaked in the Agrobacterium suspension culture solution for 5 min. The callus is taken out and placed in the co-culture medium, and cultured in a 25℃ light incubator for 2.5 d.

[0078] (3) Screening and culture of resistant callus: the callus is placed on the medium containing hygromycin B, and screening is performed after 30-45 d. The callus obtained by screening is placed in the rooting medium, and green plants with roots are generated after 2 weeks of culture, and transgenic rice is obtained.

[0079] In some embodiments, the specific steps of the culture of the EHA105 Agrobacterium include: first, 1 μL plasmid is taken and added to 100 μL competent cells, mixed by blowing, added to a pre-cooled electrode cup at 4°C, transformed by 2200V voltage, added with 500 μL LB liquid medium without resistance, cultured at 28°C and 200 rpm for 2-3 h, coated on a plate containing 50 μg / ml Kan and Rif, and cultured in a 28°C incubator for 3 d. Then the mature rice seeds are shelled, soaked with 75% alcohol for 10 min, washed with sterile water for 3 times, soaked with 30% sodium hypochlorite solution for 30 min, washed with sterile water and then soaked for another 30 min. The seeds are placed into a mature embryo induction medium using sterilized tweezers, cultured in a 28°C light incubator for 3 weeks, and the callus grown is transferred to a subculture medium using sterilized tweezers and subcultured in a 28°C light incubator for 1 week.

[0080] In some embodiments, the induction medium comprises: N6 medium 24.1 g / L, 2.5 mg / L 2,4-D, PH = 5.8.

[0081] In some embodiments, the subculture medium comprises: N6 medium 24.1 g / L, 2.5 mg / L 2,4-D, 50 mg / L hygromycin, 300 mg / mL cefotaxime, PH = 5.8.

[0082] In some embodiments, the rooting medium comprises: 1 / 2MS 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, PH = 5.8.

[0083] In some embodiments, the co-culture medium comprises: N6 medium 24.1 g / L, 2.5 mg / L 2,4-D, 200 μmol / L acetosyringone, PH = 5.8.

[0084] In another aspect, the present application also relates to a method for detecting the resistance of rice stripe virus of rice, comprising the following steps:

[0085] The genomic DNA of the transgenic rice is extracted by SDS method. 0.2 μL DNA template is taken and subjected to PCR amplification, and the amplification system is 10 μL. The detection primers are as follows:

[0086] OsNPR1-detect-F: ATGGAGCCGCCGACCAGCCA SEQ ID NO: 3

[0087] OsNPR1-detect-R: TCATCTCCTTGGTCGAATGGC SEQ ID NO: 4

[0088] The PCR products were sequenced and compared to confirm the presence of the OsNPR1 gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 : Relative expression level of ONPR1 in OsNPR1 overexpressing transgenic rice.

[0090] Figure 2 : Pathogenesis symptoms of OsNPR1 overexpressing transgenic and control Nipponbare after RSV infection. The scale bars represent 5 cm (upper) and 1 cm (lower), respectively.

[0091] Figure 3 : qRT-PCR detection of viral content in OsNPR1 overexpressing transgenic and control Nipponbare after RSV infection.

[0092] Figure 4 :After RSV infection, the viral content of OsNPR1 overexpressing transgenic cells and control Nipponbare was detected by Western blotting.

[0093] The following examples are provided to better illustrate the present invention, but are not intended to limit the present invention to these examples. Non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention. DETAILED DESCRIPTION

[0094] The rice varieties used in this series of experiments are: Nipponbare

[0095] The relevant culture medium components are as follows:

[0096] Induction medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1 g / L, 2.5 mg / L 2,4-D, pH = 5.8.

[0097] Subculture medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: 15HBZ0601) 24.1 g / L, 2,4-D, 50 mg / L hygromycin, 300 mg / mL cephalosporin, pH = 5.8.

[0098] Co-culture medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1 g / L, 2.5 mg / L 2,4-D, 200 μmol / L acetosyringone, pH = 5.8.

[0099] Rooting medium: 1 / 2MS (Manufacturer: Haibo Biotechnology Co., Ltd., Product No.: HB8469-6)

[0100] 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, PH=5.8.

[0101] Example 1: Construction of a rice OsNPR1 plant expression vector

[0102] (1) Cloning of rice OsNPR1 gene

[0103] Primers OsNPR1-F and OsNPR1-R were designed according to the full-length sequence SEQ ID NO: 1 of OsNPR1, and the primer sequences used are as follows:

[0104] OsNPR1-F: 5'-ATGGAGCCGCCGACCAGCCA-3'; SEQ ID NO: 3

[0105] OsNPR1-R: 5'-TCATCTCCTTGGTCGAATGGC-3'; SEQ ID NO: 4

[0106] PCR amplification system: total volume 50 μL, including 5 μL 10×PCR buffer, upstream and downstream primers (10 μM)

[0107] 1.5 μL, 5 μL dNTP Mix (2.5 mM), 1 μL cDNA template, 1 μL Taq enzyme (5 U / μL), 35 μL ddH2O, respectively.

[0108] PCR program: 95°C pre-denaturation 3 min; 95°C denaturation 30 s, 60°C annealing 30 s, 72°C extension 2 min, 35 cycles; 72°C final extension 10 min.

[0109] The PCR product was recovered, ligated with pMD18-T vector, and the clones were picked and sent for testing. The correct pMD18-T-OsNPR1 recombinant plasmid was obtained and sent to Hangzhou Yikang Biotechnology Co., Ltd. for sequencing confirmation. The obtained gene sequence is shown in SEQ ID No: 1, with a length of 1749 bp, encoding 582 amino acids.

[0110] (2) Construction of overexpression vector

[0111] Primers with BamHI and SacI restriction sites were designed to amplify OsNPR1, and the template was the correctly identified pMD18-T-OsNPR1 recombinant plasmid. The pCV1300 vector was double-digested with BamHI and SacI enzymes.

[0112] Primers for OsNPR1 binary expression vector PCV1300 construction, primer sequences are as follows:

[0113] PCV-OsNPR1-F:

[0114] GTTCCAGATTACGCTGGATCCATGGAGCCGCCGACCAGCCA

[0115] SEQ ID NO:5

[0116] PCV-OsNPR1-R:

[0117] ATCGGGGAAATTCGAGCTCTCATCTCCTTGGTCGAATGGC

[0118] SEQ ID NO:6

[0119] PCR amplification system: the reaction system (50 μL) is as follows: 2x PCR KOD FX Neo Buffer

[0120] 25 μL, 2 mM dNTPs, KOD FX Neo 1 μL, upstream primer 1.5 μL, downstream primer 1.5 μL, cDNA 1 μL, H2O 15 μL, total volume 50 μL.

[0121] PCR program: 95°C pre-denaturation 3 min; 95°C denaturation 30 s, 60°C annealing 30 s, 72°C extension 2 min, 35 cycles; 72°C final extension 10 min.

[0122] After the PCV1300 vector is digested, the PCR product is recovered, and the PCR product is connected with the vector; positive clones are selected, and sequencing is performed to confirm that the PCV1300-OsNPR1 expression vector has been successfully constructed.

[0123] Example 2: Genetic transformation of rice

[0124] (1) EHA105 Agrobacterium culture and rice callus induction culture: Take the plasmid containing the target vector stored in the -80°C refrigerator to transform Rhizobium Agrobacterium EHA105 (Eubios, CAT#: AE1010), the detailed operation steps are as follows: first, take 1 μL plasmid and add it to 100 μL competent cells, mix well by blowing, add to the electrode cup cooled in advance at 4°C, transform at 2200V voltage, add 500 μL of antibiotic-free LB liquid medium, cultivate at 28°C, 200 rpm for 2-3 h, spread on a plate containing 50 μg / ml Kan and Rif, and cultivate in a 28°C incubator for 3 d. Then, the mature rice seeds are shelled, soaked in 75% alcohol for 10 min, washed with sterile water for 3 times, soaked in 30% sodium hypochlorite solution for 30 min, washed with sterile water and then soaked for another 30 min. Use sterile forceps to put the seeds into the mature embryo induction medium, cultivate in a 28°C light incubator for 3 weeks, and transfer the callus grown to the subculture medium with sterile forceps and cultivate in a 28°C light incubator for 1 week.

[0125] (2) Agrobacterium transfection of callus: inoculate the single colony of EHA105 into LB liquid medium, OD600 = 0.6. Collect the bacterial cells, resuspend the bacteria with the Agrobacterium suspension culture solution of AS, OD600 = 0.1. Add the induced callus to the Agrobacterium suspension culture solution and soak for 5 min. Take out the callus and put it into the co-culture medium, cultivate in a 25°C light incubator for 2.5 d.

[0126] (3) Screening and culture of resistant callus: place the callus on the medium containing hygromycin B, screen after 30-45 d, and place the callus obtained by screening into the rooting medium to cultivate until green plants with roots are produced, cultivate for 2 weeks, and obtain transgenic rice.

[0127] Example 3: Identification of transgenic rice positives

[0128] (1) RT-PCR detection

[0129] Extract the genomic DNA of transgenic rice by SDS method. Take 0.2 μL DNA template, perform PCR amplification, the amplification system is 10 μL, and the PCR product size is 1000 bp. The detection primers are as follows:

[0130] OsNPR1-detect-F: CTGCTGGTGCACCGCTG SEQ ID NO: 7

[0131] OsNPR1-detect-R: GCTCCAGTATTTCAATACATAA SEQ ID NO: 8

[0132] (2) RT-qPCR detection of transgenic plants

[0133] The positive transgenic plants were subjected to total RNA extraction from leaves, reverse transcription into cDNA, and quantitative primers were used for qRT-OsNPR1. The relative expression of the OsNPR1 gene is shown in Figure 1 , wherein the relative expression of the OsNPR1 gene in the overexpression lines (OsNPR-2# and OsNPR-7#) was significantly higher than that of the control Nipponbare rice. The quantitative primer sequences are as follows:

[0134] qRT-OsNPR1-F: CTGGGTTCTGGTGCAAATCC SEQ ID NO: 9

[0135] qRT-OsNPR1-R: AACCTCTTCCTCTTCTCCGC SEQ ID NO: 10

[0136] Example 4: Inoculation of transgenic rice with RSV

[0137] (1) After the seeds of OsNPR1 transgenic rice and control Nipponbare rice materials were soaked and germinated for 2-3 days, the seeds were sown in 1L beakers after germination, about 30 seedlings per cup, with 3 biological replicates. The culture was carried out under the conditions of 30°C, 16h light and 8h darkness.

[0138] (2) Virus inoculation experiment was carried out with 3-4 age stage RSV-carrying and healthy small brown planthoppers. According to the ratio of 2-3 insects per plant, RSV-carrying and non-virus-carrying small brown planthoppers were inoculated on 3-4 leaf stage rice plants, and after 3 days of feeding, all the insects were swept out.

[0139] (3) After 30 days, the symptoms of diseased rice were observed and the virus-carrying condition of the rice was determined by qRT-PCR.

[0140] Example 5: Analysis of resistance in rice after inoculation with RSV

[0141] (1) qRT-PCR detection of virus content of OsNPR1 transgenic and control Nipponbare plants

[0142] After 30 days of transplanting rice, it was found that RSV-infected rice would show symptoms of leaf mottling. Compared with the control Nipponbare NIP, the two overexpression transgenic lines of OsNPR1 (OsNPR-2# and OsNPR-7#) showed slight leaf mottling symptoms, while the control NIP showed obvious leaf mottling symptoms, as shown in Figure 2 . Mixed sampling was carried out on the diseased rice plants, with 3 biological replicates in each group. The expression amount of the virus RSV CP gene was detected by qRT-PCR, as shown in Figure 3As shown, the expression of transgenic plants CP gene was significantly lower than the control, respectively 0.37, 0.40 times. The above results show that the resistance of OsNPR1 transgenic plants to RSV infection is affected by the expression of OsNPR1 gene, and overexpression of OsNPR1 in rice can significantly enhance the resistance of rice to RSV infection.

[0143] The quantitative primer sequence is as follows:

[0144] qRSV-CP-F AGGCAATCAATGACATCTCC; SEQ ID NO: 11

[0145] qRSV-CP-R ATCTCTCACAAAGCCAGTGC; SEQ ID NO: 12

[0146] (2) Western blotting detection of virus content of OsNPR1 transgenic and control Nipponbare plants

[0147] After 30 days of transplanting, the samples with obvious symptoms were mixed and sampled. The samples were first frozen with liquid nitrogen, then ground until powder, 0.1 g of the ground sample was weighed into a 2 mL centrifuge tube, 300 μL of protein lysis buffer (100 mm Tris-HCl, pH 6.8, 10% SDS) was added, mixed well and placed on ice for 10 min; after complete lysis, the sample was placed in a pre-cooled 4°C centrifuge, centrifuged at 12000 rpm / min for 15 min, 100 μL of supernatant was taken into a new 1.5 mL centrifuge tube, 25 μL of 5xLoading buffer (1 m Tris-HCl, pH 6.8, 10% SDS, 1% bromophenol blue, 50% glycine, 2% β-mercaptoethanol) was added, mixed well and boiled in 100°C boiling water for 10 min; after taking out, centrifuged at room temperature for 1 min, and stored in -40°C refrigerator for standby; SDS-PAGE gel electrophoresis was carried out, the gel containing the target band was transferred to a PVDF membrane, and an appropriate amount of 5% skim milk powder was added to prepare an anti-antibody incubation solution (RSV CP antibody, 1:5000); then rabbit secondary antibody was incubated for about 1 h; finally, development: discard the secondary antibody incubation reaction solution, rinse with appropriate amount of TBST Buffer (3L of 1xTBS Buffer plus 1.5 mL of Tween-20, i.e. the final concentration of Tween-20 is 0.5%) for 10 min / time, rinse three times. After mixing the ECL developing solution, evenly cover the membrane for chemical luminescence color development. Finally, according to the size of RSV CP 35KD, the virus content of the sample was determined. CBB represents Coomassie blue staining, the purpose is to ensure the consistency of sample loading amount. Figure 4As shown, the accumulation of CP protein in the transgenic plants was significantly lower than that in the control. The above results show that overexpression of OsNPR1 in rice can significantly enhance the resistance of rice to RSV infection.

[0148] In summary, the above experimental results show that:

[0149] (1) The present application overexpresses the important regulatory factor OsNPR1 gene in the salicylic acid pathway to obtain transgenic rice with stable inheritance, and finds that overexpression of OsNPR1 can enhance the resistance of rice to rice stripe virus. The research results further enrich the germplasm resource library of resistance to rice stripe disease.

Claims

1. Application of the OsNPR1 gene in rice breeding for resistance to rice stripe disease caused by rice stripe virus. The nucleotide sequence of the gene is shown in SEQ ID NO:

1. The application is achieved by overexpressing the OsNPR1 gene in rice.

2. The use according to claim 1, wherein the protein sequence encoding the OsNPR1 gene is shown in SEQ ID NO:

2.

3. A method for preparing transgenic rice resistant to rice stripe disease caused by rice stripe virus, comprising the following steps: (1) Construction of rice OsNPR1 overexpression vector Primers with BamHI and SacI restriction sites were designed to amplify the OsNPR1 gene described in claim 1. The pCV1300 vector was double-digested with BamHI and SacI enzymes. The primers with restriction sites were used to construct the OsNPR1 binary expression vector PCV1300. The primer sequences are as follows: PCV-OsNPR1-F: GTTCCAGATTACGCTGGATCCATGGAGCCGCCGACCAGCCA SEQ ID NO:5PCV-OsNPR1-R: ATCGGGGAAATTCGAGCTCTCATCTCCTTGGTCGAATGGC SEQ ID NO:6 After digesting the PCV1300 vector with enzymes, the PCR products amplified with the above primers were ligated to the vectors respectively; positive clones were selected and sequenced to confirm the successful construction of the PCV1300-OsNPR1 expression vector; (2) Culture of EHA105 Agrobacterium and induction of rice callus: The plasmid PCV1300-OsNPR1 containing the target gene vector shown in SEQ ID NO: 1 stored in a -80°C refrigerator was used to transform Agrobacterium tumefaciens EHA105; the detailed operation steps were as follows: first, 1 μL of the plasmid was added to 100 μL of competent cells, pipetted to mix, added to an electrode cup pre-cooled at 4°C, transformed at a voltage of 2200 V, added 500 μL of LB liquid medium without resistance, cultured at 28°C, 200 rpm for 2-3 h, and spread on a plate containing 50 μg / ml The seeds were placed on Kan and Rif plates and cultured in a 28°C incubator for 3 days. The mature rice seeds were then shelled, soaked in 75% alcohol for 10 minutes, washed three times with sterile water, soaked in 30% sodium hypochlorite solution for 30 minutes, washed with sterile water, and soaked again for 30 minutes. The seeds were placed in a mature embryo induction medium using sterile tweezers and cultured in a 28°C light incubator for 3 weeks. The grown callus tissue was transferred to a subculture medium using sterile tweezers and subcultured in a 28°C light incubator for 1 week. (3) Agrobacterium transfection of callus: A single clone of EHA105 was selected and inoculated into LB liquid medium, with an OD600 of 0.6; the cells were collected and resuspended in AS Agrobacterium suspension culture medium, with an OD600 of 0.1; the induced callus was added to the Agrobacterium suspension culture medium and soaked for 5 min; the callus was removed and placed in a co-culture medium, and cultured in a light incubator at 25°C for 2.5 days; (4) Screening and culture of resistant calli: The callus tissue was placed on a culture medium containing hygromycin B and screened after 30-45 days. The screened callus tissue was placed in a rooting culture medium and cultured until green plants with roots were produced. The culture was carried out for 2 weeks to obtain transgenic rice. The induction culture medium contained: 24.1 g / L N6 culture medium, 2.5 mg / L 2,4-D, pH = 5.8; the subculture culture medium contained: 24.1 g / L N6 culture medium, 2.5 mg / L 2,4-D, 50 mg / L hygromycin, 300 mg / mL cephalosporin, pH = 5.8; the rooting culture medium contained: 39.45 g / L 1 / 2MS, 0.5 mg / L NAA, 50 mg / L hygromycin, pH = 5.8; the co-culture medium contained: 24.1 g / L N6 culture medium, 2.5 mg / L 2,4-D, 200 μmol / L acetosyringone, pH = 5.8.

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