Application of rice flowering-related transcription factor OsMADS1 in rice virus resistance

Abstract

The invention relates to a transcription factor OsMADS1 related to rice flowering and application thereof.An RNAi interference technology and an agrobacterium-mediated genetic transformation technology are used for remarkably reducing expression of an OsMADS1 gene in rice, a homozygous strain is obtained through screening, the homozygous strain is artificially inoculated with a rice stripe virus (RSV) and a rice stripe mosaic virus (RSMV), and the transcription factor OsMADS1 related to rice flowering is obtained. It is found that an OsMADS1 interference plant with stable genetic expression can obviously resist infection of two viruses, and the obtained transgenic interference material has a wide application prospect in the aspect of virus resistance.

Description

Technical Field

[0001] The invention relates to the field of rice virus disease prevention and control and rice transgenic technology, and mainly to the transgenic technology of rice OsMADS1 gene and its application in plant resistance to different types of virus diseases. Background Art

[0002] Rice is an important food crop in my country, and its high and stable yield is of great significance to my country's food security. However, rice is often attacked by various diseases and pests during its growth and development stage. Rice virus disease, as an important type of rice disease, seriously affects the quality and yield of rice. So far, more than a dozen rice viruses have been reported in my country, which are transmitted by different vector insects such as planthoppers, leafhoppers and aphids.

[0003] Rice stripe virus (RSV) belongs to the genus Tenuivirus and is a multipartite negative-sense single-stranded RNA virus with a genome containing four single-stranded RNAs. RSV is transmitted through eggs by the Laodelphax striatellus in a persistent multiplication mode. Rice infected with RSV shows chlorotic stripes at the base of the heart leaves, and the leaves droop, curl, or even die. The virus has spread to rice-growing areas in 18 provinces in my country, causing serious losses to rice production across the country. Similarly, Rice stripe mosaic virus (RSMV) is a member of the genus Cytorhabdovirus, a negative-sense single-stranded RNA virus with a genome consisting of only one strand. RSMV was first discovered in Guangdong Province in 2017. The virus is transmitted through the electric leafhopper in a persistent multiplication mode. After infection, rice leaves show striped mosaics, some diseased leaves are twisted, and growth is stunted and heading is delayed. In my country, the outbreak of these rice virus diseases is related to many factors, such as rice resistance immunity, the immune barrier of vector insects, and the virus transmission mechanism of vector insects, which has caused great difficulties for the prevention and control of rice virus diseases in my country.

[0004] For the prevention and control of rice virus diseases, the most economical and effective way is still to discover resistance genes and cultivate resistant varieties. However, the resistance of currently planted rice varieties is single, and there is a lack of broad-spectrum resistant varieties that can resist multiple viral diseases at the same time. Therefore, analyzing the interaction mechanism between different rice viruses and hosts and analyzing the common characteristics of different types of rice viruses in pathogenic mechanisms can provide genetic resources for discovering broad-spectrum resistance genes and creating excellent resistant varieties.

[0005] There are 75 MADS-box genes in rice, which are divided into 5 categories according to phylogenetic analysis, namely MIKCc, MIKC*, Mα, Mβ and Mγ. Among them, 38 MIKCc-type genes are divided into 14 types, all of which contain M, I, K and C conserved domains. Among them, OsMADS1 belongs to the AGL2-like member of the MIKCc type. It is reported that OsMADS1 plays an important role in the development of rice floral organs and the formation of meristems. It can regulate the gene expression of different types of OsMADSs in rice and affect the flowering development of rice. It is reported that rice brassinosteroid (BR) can negatively regulate the spatial accumulation of OsMADS1, leading to the specific expression of OsRCN2, so that rice produces more secondary branches and grains, thereby increasing rice yield. However, whether OsMADS1 is involved in antiviral immunity of rice has not been reported. Summary of the invention

[0006] Based on the defects and shortcomings of the prior art, the present invention relates to a rice flowering-related transcription factor OsMADS1 gene and its encoded protein.

[0007] On the one hand, the nucleotide sequence of the OsMADS1 gene is shown as SEQ ID NO.1, and the amino acid sequence encoded by the OsMADS1 protein is shown as SEQ ID NO.2.

[0008] Nucleotide sequence of OsMADS1:

[0009] SEQ ID NO.1

[0010] ATGGGGAGGGGGAAGGTGGAGCTGAAGCGGATCGAGAACAAGATCAGCCGGCAGGTGACGTTCGCCAAGCGCAGGAACGGCCTGCTCAAGAAGGCCTACGAGCTCTCCCTCCTCTGCGACGCCGAGGTCGCCCTCATCATCTTCTCCGGCCGCGGCCGCCTCTTCGAGTTCTCCAGCTCATCATGCATGTACAAAACCTTGGAGAGGTACCGCAGCTGCAACTACAACTCACAGGATGCAGCAGCTCCAGAAAACGAAATTAATTACCAAGAATACCTGAAGCTGAAAACAAGAGTTGAATTTCTTCAAACCACACAGAGAAATATTCTTGGTGAGGATTTGGGCCCACTAAGCATGAAGGAGCTGGAGCAGCTTGAGAACCAGATAGAAGTATCCCTCAAACAAATCAGGTCAAGAAAGAACCAAGCACTGCTTGATCAGCTGTTTGATCTGAAGAGCAAGGAGCAACAGCTGCAAGATCTCAACAAAGACTTGAGGAAAAAGTTACAGGAAACCAGTGCAGAGAATGTGCTCCATATGTCCTGGCAAGATGGTGGTGGGCACAGCGGTTCTAGCACTGTTCTTGCTGATCAGCCTCATCACCATCAGGGTCTTCTCCACCCTCACCCAGATCAGGGTGACCATTCCCTGCAGATTGGGTATCATCACCCTCATGCTCACCATCACCAGGCCTACATGGACCATCTGAGCAATGAAGCAGCAGACATGGTTGCTCATCACCCCAATGAACACATCCCATCCGGCTGGATATGA

[0011] Amino acid sequence of OsMADS1:

[0012] SEQ ID NO.2

[0013] MGRGKVELKRIENKISRQVTFAKRRNGLLKKAYELSLLCDAEVALIIFSGRGRLFEFSSSSCMYKTLERYRSCNYNSQDAAAPENEINYQEYLKLKTRVEFLQTTQRNILGEDLGPLSMKELEQLENQI EVSLKQIRSRKNQALLDQLFDLKSKEQQLQDLNKDLRKKLQETSAENVLHMSWQDGGGHSGSSTVLADQPHHHQGLLHPPHPDQGDHSLQIGYHHPHAHHHQAYMDHLSNEAADMVAHHPNEHIPSGWI*

[0014] In one aspect, the present invention relates to the use of the rice transcription factor OsMADS1 in the breeding of rice resistant to Tenuivirus and / or Cytoplasmic Rhabdovirus.

[0015] In some embodiments, the Tenuivirus is preferably Ricestripe virus (RSV); the Cytorhabdovirus is preferably Ricestripe mosaic virus (RSMV).

[0016] In some embodiments, the rice includes japonica rice Nipponbare and Zhonghua 11, and indica rice 9311, with Zhonghua 11 being the most preferred.

[0017] In some embodiments, the rice is selected from Zhonghua 11, Nipponbare, and Minghui 63, with Zhonghua 11 being the most preferred.

[0018] In another aspect, the present invention relates to a method for preparing a transgenic rice plant resistant to rice stripe virus and / or rice stripe mosaic virus, the steps of which are as follows:

[0019] (1) Cloning of the rice OsMADS1 gene;

[0020] (2) Construction of OsMADS1 gene interference vector;

[0021] (3) Agrobacterium transformation and callus induction culture;

[0022] (4) Positive identification of transgenic plants.

[0023] In some embodiments, the cloning method of the rice OsMADS1 gene is as follows:

[0024] Primers were designed according to the coding sequence of OsMADS1 SEQ ID NO.1. Total RNA from different rice tissues was extracted by hot phenol method, and cDNA was obtained by reverse transcription as a template. The target gene band of rice OsMADS1 was amplified by nested PCR. The correctness of the amplified fragment of OsMADS1 was confirmed based on the gene size and Sanger sequencing.

[0025] In some embodiments, the method for constructing the interference vector of the rice OsMADS1 gene is as follows:

[0026] The commercial pCAMBIA2300 vector was used as the backbone. After double digestion with Bgl II and BamH I, the intron pUCC fragment was connected to the pCAMBIA2300 vector. The pCAMBIA2300-pUCC-RNAi vector was obtained by recombination, transformation of E. coli competent cells and positive clone screening. The pUCC sequence is as follows: SED ID NO.3. The intron pUCC amplification primer sequences are as follows: SED ID NO.4 and SED ID NO.5. According to the gene interference vector pCAMBIA2300-pUCC-RNAi sequence, primers with recombination linker sequences and cDNA were used as templates to amplify the forward and reverse OsMADS1 interference fragments of about 300 bp, and the obtained PCR products were recovered. At the same time, the pCAMBIA2300-pUCC-RNAi vector was treated with restriction endonucleases and verified by agarose gel electrophoresis. The vector fragment obtained by single enzyme digestion and the forward target fragment were recombined and connected. The connection product was transformed into Escherichia coli, and single clones were selected for PCR detection and Sanger sequencing verification. The recombinant plasmid was extracted from the correct single clone. Subsequently, the recombinant plasmid connected to the forward fragment was digested again, and the reverse target fragment was connected to the interference vector using the same method, and finally the OsMADS1 interference vector containing the forward and reverse target fragments of about 300 bp was obtained.

[0027] Nucleotide sequence of pUCC:

[0028] SEQ ID NO.3

[0029] GGTACGGACCGTACTACTCTATTCGTTTCAATATATTTATTTGTTTCAGCTGACTGCAAGATTCAAAAATTTCTTTATTATTTTAAATTTTGTGTCACTCAAAACCAGATAAACAATTTGATATAGAGGCACTATATATATACATATTCTCGATTATATATGTTAAATGAGTTAACCTTTTTTTCCACTTAAATTATATAGG

[0030] Table 1: Primer sequences for pUCC fragment amplification

[0031] pUCC-F GGTACGGACCGTACTACTCTATTC(SEQ ID NO.4); pUCC-R CCTATATAATTTAAGTGGAAA (SEQ ID NO.5);

[0032] In some embodiments, the Agrobacterium-mediated rice genetic transformation and callus induction culture steps include:

[0033] Transformation and culture of Agrobacterium: Add an appropriate amount of vector containing the recombinant plasmid to Agrobacterium EHA105 competent cells, and use the electric shock method to transfer the OsMADS1 interference vector into Agrobacterium. After the transformation, the bacteria are spread on a solid LB medium containing kanamycin and rifampicin, and cultured inverted at 28°C for 2 days. Single clones are selected for PCR positive screening, and the positive clones obtained are used for subsequent experiments.

[0034] Callus induction: Select mature rice seeds for shelling, sterilize the seeds in 75% ethanol for 3-5 minutes, rinse with an appropriate amount of sterilized water, and then sterilize with 30% sodium hypochlorite for 20-30 minutes, and then rinse thoroughly with an appropriate amount of sterile water for 6-8 times. After the sterilized seeds are dried on the clean bench, they are transferred to the induction medium and cultured in a constant temperature and light incubator at 28°C for 20 days to induce the growth of callus. Subsequently, the formed rice callus is transferred to the subculture medium with sterile tweezers and cultured under the same conditions for 7 days.

[0035] Agrobacterium infection and callus culture: Select the positive clones of EHA105 and inoculate them into LB liquid medium and culture until OD 600 = about 0.5, and the induced callus was placed in a triangular flask containing Agrobacterium suspension culture solution for infection for 25 minutes, and shaken once every 5 minutes to ensure uniform contact. After the infection is completed, the callus is placed on sterilized filter paper to dry the bacterial solution, and then transferred to a solid co-culture medium and cultured at 26°C in the dark for 2-3 days. Subsequently, the callus was placed on a culture medium containing the corresponding antibiotics, and grown at 28°C in the dark for 30 days to screen the successfully transformed cells. Then, the selected callus was placed in a rooting culture medium for light culture. When the seedlings grow obvious roots and green stems and leaves, they were cultured at 28°C in the light for 20 days to obtain transgenic rice seedlings.

[0036] In some embodiments, the positive identification step of the transgenic rice seedlings comprises:

[0037] Total RNA from transgenic rice seedlings was extracted and reverse transcribed into cDNA. OsUBQ5 was used as the internal reference gene to identify positive transgenic rice by RT-qPCR. Compared with the control group, the gene expression level in the positive transgenic rice with OsMADS1 interference was lower than that in Zhonghua 11;

[0038] In some embodiments, the culture medium composition is as follows: the induction culture medium composition is: N6 culture medium 24.1 g / L, 2,4-D working concentration is 2 mg / L, and culture medium pH = 5.8; the subculture culture medium composition is: N6 culture medium 24.1 g / L, 2,4-D working concentration is 2 mg / L, hygromycin working concentration is 50 mg / L, cephalosporin working concentration is 300 mg / mL, and culture medium pH = 5.8; the co-culture medium composition is: N6 culture medium 24.1 g / L, 2,4-D working concentration is 2 mg / L, acetosyringone working concentration is 200 μmol / L, and culture medium pH = 5.2; the rooting culture medium contains: 1 / 2MS 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, and PH = 5.8.

[0039] In another aspect, the present invention also relates to a method for detecting resistance to rice stripe virus and / or resistance to rice stripe mosaic virus.

[0040] In some embodiments, the method comprises: calculating the incidence of rice using a statistical method;

[0041] In some embodiments, the method is: using RT-qPCR to detect the expression of viral genes, and judging the resistance to the virus according to the expression of the relevant viral genes;

[0042] In some embodiments, the method comprises: using Western blot to detect the expression of viral proteins to determine the resistance of rice plants to related viruses;

[0043] In some embodiments, the method of calculating the incidence of rice using statistical methods further includes: counting the rice that naturally becomes diseased in the field 30 days after virus infection, calculating the number of diseased and non-diseased rice in the control group and transgenic rice, respectively, calculating the incidence of the virus, and analyzing the resistance of transgenic rice to the virus by comparing it with the control group.

[0044] In some embodiments, the use of RT-qPCR to detect the expression of viral genes further includes: using the TRIzol method to extract total rice RNA, reverse transcribing the RNA into cDNA, using real-time quantitative fluorescence PCR to detect the expression level of viral genes, using Zhonghua 11 rice as a control, analyzing the relative accumulation of viruses in transgenic rice, and analyzing its resistance to viruses.

[0045] In some embodiments, the use of Western blot to detect the expression of viral proteins further includes: extracting total rice protein with lysis protein lysis solution, using viral protein antibodies prepared and preserved in the laboratory, performing Western blot protein quantitative detection on Zhonghua 11 and transgenic rice, and analyzing the accumulation of viral proteins in the control group and transgenic rice. If the accumulation of viral proteins in Zhonghua 11 is higher than that in transgenic rice, the transgenic rice is a disease-resistant material.

[0046] The present invention mainly adopts the following scheme to achieve the above invention objectives:

[0047] The present invention takes the rice gene OsMADS1 as the research object, clones the OsMADS1 gene and constructs it into an interference expression vector, uses Agrobacterium-mediated genetic transformation technology to introduce the recombinant plasmid into the callus induced by Zhonghua 11 rice, obtains transgenic rice seedlings after growth on subculture, co-culture and rooting medium, and then identifies positive transgenic plants by different methods. Transgenic rice is detected from the RNA level by qRT-PCR, positive seedlings are screened, and then T3 generation homozygous transgenic rice with stable expression is obtained by multiplication and re-identification. RSV and RSMV are artificially inoculated on the homozygous transgenic rice, and the resistance of OsMADS1 transgenic rice is evaluated. Compared with the control group, the transgenic rice interfering with OsMADS1 significantly enhances the resistance to RSV and RSMV.

[0048] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0049] (1) The present invention clones the OsMADS1 gene and constructs its interference vector, obtains stably inherited OsMADS1 transgenic rice through Agrobacterium-mediated genetic transformation technology, and conducts antiviral function analysis on it, clarifies the resistance of transgenic rice to different types of rice viruses, explores gene resources with broad-spectrum resistance, and creates rice antiviral materials.

[0050] (2) The present invention explores the interaction between OsMADS1 and different types of rice viruses, analyzes the important functions of OsMADS1 in virus infection and rice defense, and lays a theoretical foundation for in-depth research on the disease resistance mechanism mediated by OsMADS1. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 :Identification results of transgenic rice interfering with OsMADS1.

[0052] Figure 2 :Symptoms of OsMADS1 transgenic rice and the control group after RSV infection.

[0053] Figure 3: Virus accumulation in OsMADS1 transgenic rice and the control group after RSV infection.

[0054] Figure 4 :Pathogenic symptoms of OsMADS1 transgenic rice and the control group after RSMV infection.

[0055] Figure 5 : Virus accumulation in OsMADS1 transgenic rice and the control group after RSMV infection. DETAILED DESCRIPTION

[0056] The rice varieties used in this series of experiments are: Zhonghua 11

[0057] The relevant culture medium components are as follows:

[0058] Induction medium: N6 medium 24.1 g / L, purchased from Haibo Biotechnology Co., Ltd., product number HBZ0601, 2.5 mg / L 2,4-D, PH=5.8.

[0059] Subculture medium: N6 medium 24.1 g / L, purchased from Haibo Biotechnology Co., Ltd., product number HBZ0601, 2,4-D, 50 mg / L hygromycin, 300 mg / mL cephalosporin, PH = 5.8.

[0060] Co-culture medium: N6 medium 24.1 g / L, purchased from Haibo Biotechnology Co., Ltd., product number HBZ0601, 2.5 mg / L 2,4-D, 200 μmol / L acetosyringone, pH = 5.8.

[0061] Rooting medium: Plant gel medium MS working concentration is 39.45g / L, purchased from Haibo Biotechnology Co., Ltd., product number HB8469-6,

[0062] The working concentration of NAA is 0.5 mg / L, the working concentration of hygromycin is 50 mg / L, and the pH is 5.8.

[0063] Example 1: Construction of rice OsMADS1 interference vector

[0064] (1) Cloning of the rice OsMADS1 gene

[0065] The forward and reverse primers OsMADS1-F / R were designed based on the coding region sequence (CDS) of OsMADS1. The sequences are as follows:

[0066] Table 2: OsMADS1 gene cloning primer sequences

[0067] Using cDNA or plasmid as template, add the above primers for PCR amplification, the system is: 2×PCR Buffer for

[0068] OsMADS1-F ATGGGGAGGGGGAAGGTGGAGCTGAA (SEQ ID NO.6); OsMADS1-R TCATATCCAGCCGGATGGGATGTG(SEQ ID NO.7);

[0069] KOD FX Neo 12.5μL, F and R short primers (10μM) 0.75μL each, dNTP Mix (2.5mM) 2.5μL, cDNA template 1μL, KOD FX Neo 1μL, add ddH2O to a total volume of 25μL.

[0070] The PCR amplification program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 35 s, annealing at 60°C for 35 s, extension at 72°C for 1 min, 32 cycles; final extension at 72°C for 5 min.

[0071] The PCR product was subjected to agarose gel electrophoresis, and the correct target band (774 bp) was purified and recovered. The recovered product was connected to the pMD18-T cloning vector and transformed into DH5a Escherichia coli competent cells. After inverted culture at 37°C for 24 hours, a single clone was picked out and sent to a biological company for Sanger sequencing after PCR positive screening. The sequencing results were compared and clones with completely correct sequences were selected for subsequent experiments.

[0072] (2) Construction of OsMADS1 interference vector

[0073] 1. Using the commercial pCAMBIA2300 vector (purchased from Changsha Aibiwei Biotechnology Co., Ltd.) as the backbone, the intron pUCC fragment was connected to the pCAMBIA2300 vector after double digestion with Bgl II and BamH I, and the pCAMBIA2300-pUCC-RNAi vector was obtained by recombination, transformation of E. coli competent cells and positive clone screening. The pUCC sequence is as follows SED ID NO.3, and the intron pUCC amplification primer sequences are as follows SED ID NO.4 and SED ID NO.5;

[0074] 2. The 298 bp OsMADS1 sequence was selected by bioinformatics prediction to construct the interference vector, and the selected sequence is as follows SEQ ID NO. 8. The pMD18-OsMADS1 plasmid connected to the cloning vector and sequenced correctly was used as a template, and the adapter primers with restriction sites were used for PCR amplification, and the forward fragment and reverse fragment with restriction sites were amplified respectively. The interference vector pCAMBIA2300-pUCC-RNAi was double-digested with Pst I and Sal I restriction endonucleases, and the digestion products were subjected to agarose gel electrophoresis. The large vector fragment was purified and recovered, and the forward fragment of OsMADS1 and the double digestion products of the interference vector were connected by recombination reaction. The connection products were transformed into DH5a Escherichia coli competent cells, and the target fragment was confirmed to be correct by PCR positive screening and Sanger sequencing. The forward fragment vector that was correctly connected was double-digested again, the purified and recovered product was recombined with the reverse fragment, and the connection product was transformed into E. coli competent cells again. The positive clones screened by PCR were sent to a biological company for Sanger sequencing. The clone with completely correct sequencing was the interference vector pCAMBIA2300-pUCC-RNAi-OsMADS1, and the plasmid was extracted for subsequent experiments. The primer sequences, PCR amplification system and procedures used are as follows:

[0075] SEQ ID NO.8:

[0076] ATGGGGAGGGGGAAGGTGGAGCTGAAGCGGATCGAGAACAAGATCAGCCGGCAGGTGACGTTCGCCAAGCGCAGGAACGGCCTGCTCAAGAAGGCCTACGAGCTCTCCCTCCTCTGCGACGCCGAGGTCGCCCTCATCATCTTCTCCGG CCGCGGCCGCCTCTTCGAGTTCTCCAGCTCATCATGCATGTACAAAACCTTGGAGAGGTACCGCAGCTGCGACTACAACTCACAGGATGCAGCAGCTCCAGAAAACGAAATTAATTACCAAGAATACCTGAAGCTGAAAACAAGAGTTG

[0077] Table 3: Primer sequences involved in the construction of interference vectors

[0078]

[0079]

[0080] 1. The PCR amplification system is as follows: 2×PCR KOD FX Neo Buffer 12.5μL, 2mM dNTPs 2μL, KODFX Neo 1μL, F-terminal primer 0.75μL, R-terminal primer 0.75μL, cDNA 1μL, add ddH2O to a total volume of 25μL.

[0081] 2. PCR program: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 33 cycles; final extension at 72°C for 5 min.

[0082] Example 2: Rice genetic transformation

[0083] (1) Callus induction: Select mature rice seeds for shelling, sterilize the seeds in 75% ethanol for 3-5 minutes, rinse with an appropriate amount of sterilized water, sterilize with 30% sodium hypochlorite for 20-30 minutes, and then rinse thoroughly with an appropriate amount of sterile water for 6-8 times. After the sterilized seeds are dried on a clean bench, they are transferred to an induction medium and cultured in a constant temperature and light incubator at 28°C for 20 days to induce the growth of callus. Subsequently, the formed rice callus is transferred to a subculture medium with sterile tweezers and cultured under the same conditions for 7 days.

[0084] (2) Transformation and culture of Agrobacterium: 1 μL of the plasmid containing the expression vector was added to 50 μL of EHA105 competent Agrobacterium, mixed and added to an electrode cup pre-cooled on ice, transformed with 220V of electric shock, added 500 μL of LB without resistance, cultured on a shaker at 28°C and 200rpm for 3h, centrifuged at 5000rpm for 1min, discarded part of the supernatant, and the remaining bacteria were spread on a solid LB medium containing kanamycin and rifampicin, and cultured in an incubator at 28°C for 2 days. Single clones were selected for PCR positive screening, and the positive clones obtained were used for subsequent experiments.

[0085] (3) Agrobacterium infection and callus culture: The EHA105 monoclone containing the recombinant plasmid was inoculated into LB liquid culture medium, and the bacterial solution was cultured at 28°C and 200rpm until OD600 = 0.5. The induced callus was placed in a triangular flask containing Agrobacterium suspension culture solution and infected for 25 minutes, and shaken once every 5 minutes to ensure uniform contact. After the infection was completed, the callus was placed on sterilized filter paper to dry the bacterial solution, and then transferred to a solid co-culture medium and cultured at 26°C in the dark for 2-3 days. Subsequently, the callus was placed on a culture medium containing the corresponding antibiotics and grown at 28°C in the dark for 30 days to select the cells that were successfully transformed. Then, the selected callus was placed in a rooting culture medium and cultured under light. When the seedlings grew obvious roots and green stems and leaves, they were cultured at 28°C in the light for 20 days to obtain transgenic rice seedlings of OsMADS1.

[0086] Example 3: Identification of positive transgenic rice

[0087] (1) Total RNA of OsMADS1-interfered transgenic rice was extracted and reverse transcribed into first-chain cDNA. OsUBQ5 was used as the internal reference gene to identify positive transgenic rice by RT-qPCR. Compared with the control group, the gene expression level in the positive transgenic rice with OsMADS1 interference was lower than that of Zhonghua 11;

[0088] Table 4: Primers used to identify positive transgenic rice

[0089] qRT-OsMADS1-F AGGAGCAACAGCTGCAAGAT(SEQ ID NO.13); qRT-OsMADS1-R GGAGAAGACCCTGATGGTGA (SEQ ID NO.14); OsUBQ5-F ACCACTTCGACCGCCACTACT(SEQ ID NO.15); OsUBQ5-R ACGCCTAAGCCTGCTGGTT(SEQ ID NO.16);

[0090] Example 4: Artificial inoculation of RSV

[0091] (1) The control group Zhonghua 11 and transgenic rice seeds with OsMADS1 interference were soaked in water and placed in a 37°C incubator for 3 days. The water was changed twice a day. After the seeds germinated, they were sown in 500 mL plastic cups, with 25 seeds planted in each cup. Three biological replicates were planted for each transgenic genome material and Zhonghua 11, and cultured in an artificial climate chamber at 26°C (16 h light / 8 h dark). When the seedlings grew to the same level as the cup, they were ready to be inoculated with rice stripe virus.

[0092] (2) An appropriate number of 1-2-year-old non-virus-causing gray planthoppers were selected and inoculated on RSV-infected rice seedlings for 3 days. All insects were then transferred to healthy rice seedlings for circulation. After a 12-day circulation period, a small number of insects were randomly selected to detect their virus-carrying rate.

[0093] (3) The number of inoculated insects per seedling was estimated based on the virus rate, and each seedling was inoculated with infected gray leafhoppers at a ratio of 1:1. All insects were swept out 3 days after inoculation, and the seedlings were placed in a 30°C greenhouse to recover and grow for 2-3 days. All seedlings were then planted in the field. 30 days after inoculation, the symptoms of the plants were observed, the incidence of rice was counted, and the virus content in different rice was detected. The results are shown in the attached figure. The two transgenic lines (#9 and #15) that interfered with OsMADS1 showed obvious disease resistance to RSV, as shown in Figure 1. Figure 2 RT-qPCR and Western blot were used to detect the accumulation of viral RNA and capsid protein CP. The results showed that the accumulation of viral RNA and protein in the transgenic strains with interference of OsMADS1 was significantly lower than that in Zhonghua 11. Figure 3 shown.

[0094] Primer sequences used for RT-qPCR detection of viral RNA:

[0095] RSV-CP-qF: AGGCAATCAATGACATCTCC; SEQ ID NO.17

[0096] RSV-CP-qR: ATCTCTCACAAAGCCAGTGC; SEQ ID NO.18

[0097] Example 5: Artificial inoculation of RSMV

[0098] (1) Referring to the artificial inoculation method of RSV, transgenic rice seeds interfering with OsMADS1 were soaked in a 37°C incubator for 3 days. Seeds with consistent germination levels were selected and sown in 500 mL plastic cups, with 25 seeds planted in each cup, with at least 3 biological replicates, and cultured in an artificial climate chamber at 25°C (16 h light, 8 h dark).

[0099] (2) One-instar leafhopper larvae were selected from artificially reared rice seedlings and infected with RSMV for 3 days. All insects were then transferred to healthy rice seedlings for virus circulation for 14 days. A small number of insects were randomly selected to detect their virus-carrying rate.

[0100] (3) The electric leafhoppers that have passed the cycle period are inoculated on rice seedlings that are about 15 days old. Each seedling is inoculated with the infected electric leafhoppers at a ratio of 1:1 (the number of insects inoculated on each seedling is calculated based on the infection rate). The insects are swept out 3 days after infection, and then all the seedlings are planted in the field to cause high temperature disease. After 45 days, the symptoms of the plants are observed, the incidence of rice is counted, and the virus content in different rice is detected. The results are shown in the attached figure. The two transgenic lines (#9 and #15) that interfere with OsMADS1 show obvious disease resistance to RSMV, such as Figure 4 RT-qPCR and Western blot were used to detect the accumulation of viral RNA and capsid protein CP. The results showed that the accumulation of viral RNA and protein in the transgenic strains with interference of OsMADS1 was significantly lower than that in Zhonghua 11. Figure 5 shown.

[0101] Primer sequences used for RT-qPCR detection of viral RNA:

[0102] RSMV-M4-qF:ACTTCAGTGTCCAGCCTACC; SEQ ID NO.19

[0103] RSMV-M4-qR:CCTCCATCCACCTTGCACTC; SEQ ID NO.20

[0104] The present invention successfully obtains transgenic rice materials resistant to RSV and RSMV infection by transgenic means, achieving unexpected technical effects. The specific examples and schemes described in the present invention are only given as examples for illustrative purposes and are not considered to be limitations of the present invention. Various further modifications or changes within the spirit and scope of the present invention are considered to be included in the disclosure scope of the present invention.

Claims

1. An application of a rice flowering-related transcription factor OsMADS1 in rice breeding for resistance to Cytorhabdovirus and / or Tenuivirus, wherein the nucleotide sequence of the OsMADS1 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, wherein the amino acid sequence of the OsMADS1 gene is shown in SEQ ID NO.

2.

3. The use according to any one of claims 1 to 2, wherein the cytosolic rhabdovirus genus includes Rice stripe mosaic virus (RSMV), Barley yellow streak mosaic virus (BMV), The preferred viruses are rice stripe mosaic virus (RSMV), northern cereal mosaic virus (NCMV), and lettuce necrotic yellows virus (LNYV); preferably, rice stripe mosaic virus.

4. The use according to any one of claims 1 to 3, wherein the Tenuivirus is preferably Rice Stripe Virus.

5. The use according to any one of claims 1 to 4, wherein the rice is selected from Zhonghua 11, Nipponbare, and Minghui 63, and Zhonghua 11 is the most preferred.

6. A method for preparing transgenic rice materials resistant to rice stripe virus and / or rice stripe mosaic virus, the main steps of which are as follows: (1) cloning of rice OsMADS1 gene; (2) construction of OsMADS1 gene interference vector; (3) Agrobacterium transformation and callus induction culture; (4) positive identification of transgenic plants.

7. The preparation method according to claim 6, wherein the nucleotide sequence of the rice OsMADS1 gene is as shown in SEQ ID NO.

1.

8. The preparation method according to any one of claims 6 to 7, wherein the method for cloning the rice OsMADS1 gene comprises: The forward and reverse primers OsMADS1-F / R were designed based on the coding region sequence (CDS) of OsMADS1. The sequences are as follows: OsMADS1-F ATGGGGAGGGGGAAGGTGGAGCTGAA (SEQ ID NO. 6); OsMADS1-R TCATATCCAGCCGGATGGGATGTG (SEQ ID NO.7); Using cDNA as a template, add the above primers for PCR amplification, perform agarose gel electrophoresis on the PCR product, purify and recover the correct target band, connect the recovered product to the pMD18-T cloning vector, transform DH5a Escherichia coli competent cells, and pick out single clones after inverted culture at 37°C for 24 hours. After PCR positive screening, send it to a biological company for Sanger sequencing and then preserve it.

9. The preparation method according to any one of claims 6 to 8, wherein the step of constructing the OsMADS1 gene interference vector further comprises: 1). Using pCAMBIA2300 vector as the backbone, after double digestion with Bgl II and BamH I, the intron pUCC fragment was connected to the pCAMBIA2300 vector, and the pCAMBIA2300-pUCC-RNAi vector was obtained by recombination, transformation of E. coli competent cells and positive clone screening; the pUCC sequence is as follows SED ID NO.3, and the intron pUCC amplification primer sequences are as follows SED ID NO.4 and SED ID NO.5; 2). The OsMADS1 sequence fragment was selected by bioinformatics prediction to construct an interference vector. The selected sequence is as follows: SEQ ID NO.8; pMD18-OsMADS1 plasmid connected to the cloning vector and sequenced correctly was used as a template, and the adapter primers with restriction sites were used for PCR amplification to amplify the forward fragment and the reverse fragment with restriction sites, respectively, and Pst I and Sal I were used to amplify the forward fragment and the reverse fragment with restriction sites. The interference vector pCAMBIA2300-pUCC-RNAi was double-digested with restriction endonuclease I, and the digestion products were subjected to agarose gel electrophoresis. The large vector fragment was purified and recovered, and the forward fragment of OsMADS1 and the double-digestion product of the interference vector were connected by recombination reaction. The connection product was transformed into DH5a Escherichia coli competent cells, and the target fragment was confirmed to be correct by PCR positive screening and Sanger sequencing; the forward fragment vector with correct connection was double-digested again, and the purified and recovered products were recombined and connected with the reverse fragment, and the connection product was transformed into Escherichia coli competent cells again. The positive clones screened by PCR were sent to a biological company for Sanger sequencing. The clone with completely correct sequencing was the interference vector pCAMBIA2300-pUCC-RNAi-OsMADS1, and the amplification primer sequences of the forward fragment and the reverse fragment were: SEQ ID NO.9-10 and SEQ ID NO.11-12, respectively.

10. A method for identifying transgenic rice positive plants resistant to rice stripe virus and / or rice stripe mosaic virus, comprising the following steps: extracting total RNA from transgenic rice, reverse transcribing into cDNA, and detecting the relative expression level of the OsMADS1 gene by real-time fluorescence quantitative PCR, wherein the primer sequences used are shown in SEQ ID NO.13 and SEQ ID NO.14, and the primer sequences of the internal reference gene UBQ5 are shown in SEQ ID NO.15 and SEQ ID NO.16.

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