Bivalent RNAi expression vector for resisting rice viruses RRSV and SRBSDV as well as construction method and application of bivalent RNAi expression vector

Abstract

The invention belongs to the field of modern molecular biotechnology and plant disease-resistant breeding, and particularly relates to a bivalent RNAi expression vector for resisting rice virus RRSV (rice serrated leaf dwarf virus) and SRBSDV (southern rice black-streaked dwarf virus) as well as a construction method and application of the bivalent RNAi expression vector. According to the method, a hairpin RNA expression construct of targeted virus gene segments (RRSV Pns10 and SRBSDV Pns5) regulated and controlled by a phloem specific promoter RTBV (Ribonucleic Acid Ribonucleic Acid) is introduced into rice, so that efficient and lasting genetic resistance to the two main rice viruses is realized. According to the method, the broad-spectrum resistance of rice to RRSV and SRBSDV is remarkably improved, meanwhile, the common metabolic interference problem of a constitutive RNAi system is avoided, and a safe, efficient and generalizable innovative approach is provided for rice antiviral molecular breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of modern molecular biotechnology and plant disease-resistant breeding, and particularly relates to a bivalent RNAi expression vector for resisting rice viruses RRSV and SRBSDV, and a construction method and application thereof. BACKGROUND

[0002] Rice ( Oryza sativa ) is the staple food crop for nearly half of the world's population, and its stable production is of great significance to food security. However, rice production is increasingly threatened by rice ragged stunt virus (RRSV) and southern rice black-streaked dwarf virus (SRBSDV). Both of these two viruses belong to the Reoviridae family and are transmitted by the brown planthopper (BPH) ( Nilaparvata lugens ) and white-backed planthopper (WBPH) ( Sogatella furcifera ), respectively, frequently causing epidemic outbreaks and complex infections in Asian rice-growing regions, often resulting in significant yield reduction or even complete loss, posing a serious challenge to agricultural sustainable development.

[0003] Traditional disease-resistant breeding strategies rely on natural resistance gene resources, but effective resistance genes against rice ragged stunt virus (RRSV) and southern rice black-streaked dwarf virus (SRBSDV) are very limited, and the rapid variation of virus populations makes it difficult to achieve the goal of stable and durable resistance breeding. Therefore, developing new, broad-spectrum, and less affecting plant normal growth antiviral molecular strategies has become an important direction of current research.

[0004] RNA interference (RNAi) is a highly conserved gene silencing mechanism that can produce small interfering RNA (siRNA) in plant cells by expressing hairpin RNA (hpRNA) derived from viral genes, thereby specifically degrading viral RNA and achieving precise inhibition of viral replication. Previous studies have shown that RNAi technology can confer plants with broad-spectrum resistance to multiple viruses. However, if a constitutive promoter is used to drive the continuous high-level expression of hpRNA, it may trigger metabolic disorders, developmental abnormalities, and other side effects, affecting agronomic traits and biological safety.

[0005] In view of the fact that RRSV and SRBSDV mainly replicate and spread in the phloem system of rice, the targeted expression of hpRNA driven by a phloem-specific promoter can effectively trigger the RNAi response at the site of virus infection, thereby significantly reducing the interference with the overall metabolism and growth and development of the plant while maintaining strong resistance. In particular, the phloem-specific promoter represented by the Rice tungro bacilliform virus (RTBV) promoter has the advantages of high expression strength and precise positioning, and can achieve efficient expression of hpRNA in the phloem. Therefore, the use of the RTBV promoter to drive the expression of hpRNA against the RRSV Pns10 gene fragment and the SRBSDV Pns5 gene fragment is an innovative anti-viral strategy that combines high resistance, tissue specificity and biological safety. SUMMARY

[0006] In view of the above problems, the purpose of the present application is to provide a bivalent RNAi expression vector against rice viruses RRSV and SRBSDV and a construction method thereof, which realizes efficient and durable genetic resistance to two major rice viruses (RRSV and SRBSDV) by introducing a hairpin RNA expression construct targeting the viral gene fragments (RRSV Pns10 and SRBSDV Pns5) regulated by the phloem-specific promoter RTBV in rice.

[0007] In order to achieve the above technical purposes, the technical solutions adopted by the present application are as follows: In a first aspect, the present application provides a construction method of a bivalent RNAi expression vector against rice viruses RRSV and SRBSDV, comprising the following steps: 1) Design specific primers to perform PCR amplification of the virus RRSV Pns10 fragment and the virus SRBSDV Pns5 fragment, wherein an XhoI restriction site is introduced at the 5' end of the RRSV Pns10 fragment, a 12 bp region overlapping with the SRBSDV Pns5 fragment is introduced at the 3' end of the RRSV Pns10 fragment, a BglII restriction site is introduced at the 3' end of the SRBSDV Pns5 fragment, and a 12 bp sequence overlapping with the RRSV Pns10 fragment is introduced at the 5' end of the SRBSDV Pns5 fragment; use primers iRRSV10-XhoI-f and iSRBSDV5-BglII-r to perform overlapping PCR amplification to obtain a viral fusion fragment containing XhoI and BglII restriction sites at both ends; the RRSV Pns10 fragment gene sequence is shown in SEQ ID NO. 1, and the SRBSDV Pns5 fragment gene sequence is shown in SEQ ID NO. 2; 2) The obtained fusion fragment and the intermediate vector pUCC containing intron structure were double digested by XhoI and BglII respectively, and then ligated by T4 DNA ligase to obtain the intermediate vector pUCC-RRSV&SRBSDV-intron with the fusion fragment introduced in one direction; the vector pUCC-RRSV&SRBSDV-intron was double digested by SalI and BamHI, and then the fusion fragment was inserted reversely to form a hairpin structure, thereby obtaining the final expression vector pUCC-RRSV&SRBSDV-intron-SRBSDV&RRSV; 3) The vector pUCC-RRSV&SRBSDV-intron-SRBSDV&RRSV was digested by PstI to obtain the pUCC-RRSV&SRBSDV-intron-SRBSDV&RRSV fragment containing PstI site; the plant expression vector CAMBIA1300 was digested by PstI; p CAMBIA1300 empty vector, and the digested p The CAMBIA1300 vector and the pUCC-RRSV&SRBSDV-intron-SRBSDV&RRSV fragment containing PstI site were ligated to obtain the p1300-RRSV&SRBSDV-intron-SRBSDV&RRSV vector; the plant expression vector p1300-RRSV&SRBSDV-intron-SRBSDV&RRSV was double digested by EcoRI and BamHI, the amplification primer of the fusion enzyme digestion site of the RTBV promoter was designed, and the fragment RTBV was obtained by amplifying the DNA sample of the rice infected with RTBV; pYSL16 was double digested by EcoRI and BamHI; the double digestion products of pYSL16 and p1300-RRSV&SRBSDV-intron-SRBSDV&RRSV were ligated by T4 DNA ligase to prepare the RNA interference expression vector based on the rice phloem specific promoter RTBV (p1300-pRTBV-RRSV&SRBSDV-intron-SRBSDV&RRSV, the nucleotide sequence is shown as SEQ ID NO. 5).

[0008] Further, the RRSV Pns10 fragment PCR primer is iRRSV10-XhoI-f, iRRSV10-SRBSDV5-r; the SRBSDV Pns5 fragment PCR primer is iSRBSDV5-RRSV10-f, iSRBSDV5-BglII-r; the pRTBV promoter fusion enzyme digestion site amplification primer is OL-EcoRI-pRTBV-F, OL-BamHI-pRTBV-R; The sequence of primer iRRSV10-XhoI-f is 5'-CCGCTCGAGATTCGCAAGACAAGGCAAGA-3'; The sequence of primer iRRSV10-SRBSDV5-r is 5'-GCATCGCTAACTCTTACCTGTGGCGTCAACCTCA-3'; The sequence of primer iSRBSDV5-RRSV10-f is 5'-GCCACAGGTAAGAGTTAGCGATGCCTTTGTTGC-3'; The sequence of primer iSRBSDV5-BglII-r is 5'-GCAGATCTATCATAGCGGAAACGTACTCAT-3'; The sequence of primer OL-EcoRI-pRTBV-F is 5'-ATGACCATGATTACGAATTCACGAATCAATAAAGAAGGACCAGAA-3'; The sequence of primer OL-BamHI-pRTBV-R is 5'-CTTGCTCACCATGGATCCTCTTGCTCTCTTAGAAGTTTGAGCTC-3'.

[0009] Further, the double promoter vector comprises a CaMV 35S promoter for driving the Hygromycin resistance gene and a RTBV promoter for driving the expression of the virus fusion hairpin RNA structure; the nucleotide sequence is shown as SEQ ID NO. 5.

[0010] In the second aspect, the present application provides a double-valent RNAi expression vector against rice viruses RRSV and SRBSDV, which is obtained by the construction method of the first aspect.

[0011] Further, the double-valent RNAi expression vector against rice viruses RRSV and SRBSDV contains the nucleotide sequence shown as SEQ ID NO. 3.

[0012] In the third aspect, the present application provides an Agrobacterium, which is Agrobacterium tumefaciens EHA105, which is transfected with the double-valent RNAi expression vector of the second aspect.

[0013] In the fourth aspect, the present application provides the use of the double-valent RNAi expression vector of the second aspect in enhancing the resistance of rice to RRSV and SRBSDV viruses.

[0014] In the fifth aspect, the present application provides the use of the Agrobacterium of the third aspect in enhancing the resistance of rice to RRSV and SRBSDV viruses.

[0015] Furthermore, the transformation of rice using Agrobacterium as described in the third aspect specifically includes the following steps: Rice seeds without mold and with normal germ were selected as transformation materials. The seeds were disinfected with 75% ethanol for 1 min and washed with sterile water for 1 min, and rinsed 3 times. Then they were treated with 15% sodium hypochlorite for 20 min and then rinsed with sterile water 3 times. The disinfected seeds were inoculated into induction medium and cultured at 26 ℃ under light conditions to induce callus tissue. Prepare Agrobacterium resuspension, immerse callus tissue in the bacterial suspension for 10-15 min, then discard the bacterial suspension; inoculate callus tissue into co-culture medium and culture in the dark at 20 ℃ for 48-72 h; subsequently, transfer callus tissue sequentially to primary screening medium and culture for 20-30 days, then transfer to secondary screening medium for single-clonal callus selection and culture in the dark at 26 ℃ for 7-10 days; positive callus tissue is transferred to differentiation medium and cultured under light at 25-27 ℃ for 15-20 days to form shoots, then transferred to rooting medium and cultured under light at 30 ℃ for 7-10 days to obtain complete plants.

[0016] Furthermore, the induction medium was selected as 2 mg / L 2,4-D; the Agrobacterium resuspension OD 600 = 0.2; the primary screening medium contains 50 mg / L Hygromycin.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art: The phloem-specific RTBV promoter-driven bivalent hairpin RNA vector constructed in this invention can be specifically expressed in the rice phloem and exhibits significant resistance to both RRSV and SRBSDV. Field and greenhouse validation results show that this transgenic rice exhibits extremely low disease incidence and virus accumulation levels after virus inoculation, while plant growth, development, and agronomic traits are not significantly affected.

[0018] The strategy of this invention effectively solves the problems of metabolic burden and growth inhibition in traditional constitutive RNAi expression systems. It has the advantages of broad spectrum, high efficiency, safety and wide applicability, and has important prospects for promotion and application in molecular breeding of antiviral rice. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0020] Figure 1Schematic diagram of the bivalent RNA interference (RNAi) hairpin structure composed of Rice ragged stunt virus (RRSV) Pns10 and Southern rice black-streaked dwarf virus (SRBSDV) Pns5. Intron indicates the intron sequence, which is used to connect the reverse repeat viral gene fragments and form a stable hairpin structure, thereby inducing the production of specific small interfering RNA (siRNA) in the plant body.

[0021] Figure 2 Schematic diagram of the virus fusion hairpin RNA expression vector (1300_pRTBV) driven by the phloem-specific promoter pRTBV. In the vector: LB T-DNA repeat indicates the T-DNA left border sequence; RB T-DNA repeat indicates the T-DNA right border sequence; pRTBV is the Rice tungro bacilliform virus (RTBV) promoter, which is used to drive the tissue-specific expression of the fusion viral fragment hairpin structure; CaMV 35S promoter is the Cauliflower mosaic virus (CaMV) 35S promoter, which is used to drive the expression of the resistance screening gene; Hygromycin (R) is the hygromycin resistance gene (hyg) used for eukaryotic cell screening. This vector can efficiently express RNAi structures in the phloem of rice, thereby achieving targeted interference of double viruses.

[0022] Figure 3 Northern blot analysis results of small RNA (siRNA) accumulation in transgenic rice T3 generation plants. U6: internal control gene, used as RNA loading control; ZH11: wild type control rice variety "Zhonghua 11"; RRSV S10Ri: siRNA signal detected with RRSV Pns10 fragment as probe; SRBSDV S5Ri: siRNA signal detected with SRBSDV Pns5 fragment as probe. The results show that specific siRNA accumulation can be detected in transgenic plants, indicating that the hairpin structure is correctly transcribed and effectively processed into siRNA in the plants.

[0023] Figure 4 Disease resistance identification results of transgenic rice materials driven by phloem-specific promoters. ZH11: wild type control material; #22 and #26: transgenic lines carrying the construct pRTBV:RRSV Pns10 + SRBSDV Pns5. Through artificial insect inoculation and virus inoculation experiments, #22 and #26 showed obvious resistance enhancement, with significantly lower incidence than the control.

[0024] Figure 5The statistical analysis results of main agronomic traits of the transgenic rice materials. The indexes such as plant height, thousand grain weight and tiller number were compared respectively. The results show that the RNAi transgenic material driven by the RTBV promoter does not show adverse changes in agronomic traits while maintaining significant resistance. DETAILED DESCRIPTION

[0025] The application will be further described in detail below in combination with the drawings and examples. It is particularly pointed out that the following examples are only used to illustrate the application, but do not limit the scope of the application. Similarly, the following examples are only part of the examples of the application rather than all the examples, and all other examples obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0026] The application provides a phloem-specific promoter-driven rice bivalent RNA interference vector and a construction method thereof, which is used for imparting double resistance of rice to rice sawtoothed leaf stunt virus (RRSV) and southern rice black-streaked dwarf virus (SRBSDV).

[0027] 1. Virus RNAi fragment design and source The RNAi fragment for targeting and silencing rice rough stunt virus (RRSV) is obtained from the Pns10 gene of RRSV (nucleotide sequence as shown in SEQ ID NO. 1), and the RNAi fragment for targeting and silencing southern rice black-streaked dwarf virus (SRBSDV) is obtained from the Pns5 gene of SRBSDV (nucleotide sequence as shown in SEQ ID NO. 2).

[0028] 2. Bivalent hairpin structure design and construction method In order to obtain a bivalent hairpin RNA structure capable of simultaneously targeting RRSV and SRBSDV, the application provides a fusion sequence (nucleotide sequence as shown in SEQ ID NO. 3) and a partial sequence of a constructed vector (nucleotide sequence as shown in SEQ ID NO. 4). The construction method is as follows: Specific primers were designed to amplify the viral gene fragments by PCR, wherein XhoI enzyme cutting site was introduced at the 5' end of RRSV Pns10 fragment, and 12 bp region overlapping with SRBSDV Pns5 fragment was introduced at the 3' end, so as to realize fragment fusion by overlapping PCR; meanwhile, BglII enzyme cutting site was introduced at the 3' end of SRBSDV Pns5 fragment, and 12 bp sequence overlapping with RRSV Pns10 fragment was introduced at the 5' end, so as to realize overlapping connection with the previous fragment; overlapping PCR amplification was carried out by using primers iRRSV10-XhoI-f and iSRBSDV5-BglII-r, and a viral fusion fragment with a length of about 564 bp was obtained, which contains XhoI and BglII enzyme cutting sites at both ends.

[0029] The obtained fusion fragment and the intermediate vector pUCC containing intron structure were subjected to XhoI and BglII double enzyme cutting respectively, and then T4 DNA ligase was used for ligation, so as to obtain the intermediate vector pUCC-RRSV&SRBSDV-intron for single guide introduction of the fusion fragment. Since XhoI and SalI are same tail enzymes, and BglII and BamHI are same tail enzymes, the pUCC-RRSV&SRBSDV-intron vector was further subjected to SalI and BamHI double enzyme cutting, and then the fusion fragment was reversely inserted to form a hairpin structure, so as to obtain the final pUCC-RRSV&SRBSDV-intron-SRBSDV&RRS expression vector.

[0030] Table 1, primer sequence table for cloning RRSV Pns10 and SRBSDV Pns5 RNAi fragments of rice virus

[0031] 3. Expression vector design The application also provides a double promoter vector (nucleotide sequence as shown in SEQ ID NO. 5) for expressing the viral fusion hairpin RNA structure. Figure 2 The pRTBV promoter fusion enzyme cutting site amplification primer is shown in Table 2.

[0032] Table 2, pRTBV promoter fusion enzyme cutting site amplification primer

[0033] The vector comprises two independent promoter modules: (a) The Hygromycin resistance gene driven by the CaMV 35S promoter is used for screening transformed plants; (b) The expression of viral fusion hairpin RNA structures was driven by the promoter of Rice tungro bacilliform virus (RTBV) to achieve RNAi effects targeting the phloem.

[0034] This configuration ensures efficient and specific expression of hpRNA in the phloem, while reducing interference with the overall metabolism and development of the plant.

[0035] 4. Transgenic screening and functional verification system To screen transgenic rice that normally expresses RRSV Pns10 and SRBSDV Pns5 siRNA, this invention establishes a dedicated screening system: Northern blot analysis was performed on small RNA extracted from transgenic plants using DNA molecular probes containing RRSV and SRBSDV fragments (SEQ ID NO.1 and SEQ ID NO.2). The results showed (see...) Figure 3 Transgenic rice can normally transcribe hairpin structures and generate corresponding siRNAs, indicating that the hairpin RNA structure can be effectively processed in the plant and trigger RNA interference responses.

[0036] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments: Example 1: Obtaining RRSV Pns10 and SRBSDV Pns5 RNAi fragments (1) Design and amplification of RNAi fusion fragments Rice samples identified as infected with RRSV and SRBSDV were collected from the field, and total RNA was extracted from each sample. RNA extraction was performed using the ERIK AG RNAex Pro Reagent kit, and reverse transcription was performed using the Novoprotein cDNA synthesis kit.

[0037] According to the sequences published in the NCBI database, the RRSV Pns10 gene is located in the 142-1035 bp region of the 10th double-stranded RNA fragment in the RRSV genome. This protocol selects the 183-444 bp region (262 bp in length, as shown in SEQ ID NO.1) as the RNAi target sequence. The SRBSDV Pns5 gene sequence ranges from 16 to 2835 bp. This protocol selects the 258-542 bp region (285 bp in length, as shown in SEQ ID NO.2) as the RNAi target sequence.

[0038] Specific primers were designed to amplify the above fragments. The 5' end of RRSV Pns10 fragment was introduced with Xhol enzyme site, and the 3' end was added with 12 bp region overlapping with SRBSDV Pns5 fragment to realize overlapping PCR fusion. The 3' end of SRBSDV Pns5 fragment was introduced with Bglll enzyme site, and the 5' end was added with 12 bp region overlapping with the end of RRSV Pns10.

[0039] The overlapping PCR was performed using primers iRRSV10-Xhol-f and iSRBSDV5-Bglll-r to obtain a fusion fragment with a length of about 564 bp, which has Xhol and Bglll enzyme sites at both ends. The fragment was used for subsequent construction of hairpin structure. The sequences of primers used for virus gene cloning are shown in Table 1 above.

[0040] (2) Construction of virus fusion hairpin structure The intermediate vector pUCC containing intron structure and the virus fusion fragment were double digested with Xhol and Bglll respectively, and then ligated using T4 DNA ligase to obtain the intermediate vector pUCC-RRSV&SRBSDV-intron inserted in one direction.

[0041] Since Xhol and Sail are homologous enzymes, and Bglll and BamHI are homologous enzymes, the vector was again digested with Sail and BamHI, and the fusion fragment was inserted in reverse direction to form a reverse repeat hairpin structure. Finally, the complete pUCC-RRSV&SRBSDV-intron-SRBSDV&RRSV construct was obtained (structure shown in Figure 1 ). The hairpin structure can be transcribed into bivalent hpRNA in plants to induce double RNA interference of RRSV and SRBSDV.

[0042] Example 2: Obtaining of p1300-pRTBV-RRSV&SRBSDV-intron-SRBSDV&RRSV The pUCC-RRSV&SRBSDV-intron-SRBSDV&RRSV vector was digested with Pstl to obtain the pUCC-RRSV&SRBSDV-intron-SRBSDV&RRSV fragment containing Pstl site; the plant expression vector p CAMBIA1300 empty vector was digested with Pstl using T4 DNA ligase to ligate the digested pCAMBIA1300 vector and pUCC-RRSV & SRBSDV-intron-SRBSDV & RRSV fragment containing Pst I site were connected to obtain p1300-RRSV & SRBSDV-intron-SRBSDV & RRSV vector; the plant expression vector p1300-RRSV & SRBSDV-intron-SRBSDV & RRSV was double-digested with EcoRI and BamHI, and the RTBV promoter fusion enzyme cutting site amplification primer (see Table 2 above) was designed, and a rice DNA sample infected with RTBV was amplified to obtain the fragment RTBV; pYSL16 was double-digested with EcoRI and BamHI; the double-digested pYSL16 product and the p1300-RRSV & SRBSDV-intron-SRBSDV & RRSV product were connected by T4 DNA ligase to prepare an RNA interference expression vector based on the rice phloem-specific promoter RTBV (p1300-pRTBV-RRSV & SRBSDV-intron-SRBSDV & RRSV, the nucleotide sequence is shown as SEQ ID NO. 5) Example 3: Obtaining of phloem-specific promoter transgenic rice (1) Agrobacterium competence transformation 1 μL of plasmid p1300-pRTBV-RRSV & SRBSDV-intron-SRBSDV & RRSV was added to 50 μL of EHA105 competent cells, mixed, and then placed in an electric transformation cup for electric transformation. After electric shock, 1 mL of LB liquid medium was immediately added, and the culture was incubated at 30 °C, 180 rpm on a shaker for 30 min. Then 50 μL of bacterial solution was inoculated on LB solid medium, and incubated at 30 °C in the dark for 48 h. Single colonies were picked and subjected to colony PCR detection, and positive colonies were used for rice transformation. Agrobacterium tumefaciens

[0043] (2) Rice genetic transformation and regeneration Mildew-free and normal embryo "Shuang Kangmingzhan" rice seeds were selected as the transformation material. The seeds were sterilized with 75% ethanol for 1 min and washed with sterile water for 1 min × 3; then treated with 15% sodium hypochlorite for 20 min, and then washed with sterile water for 3 times. The sterilized seeds were inoculated in induction medium (2,4-D 2 mg / L) and cultured at 26 °C under light conditions for about 20 days to induce callus.

[0044] Preparation of Agrobacterium resuspension liquid (OD 600 ​= 0.2), the callus was immersed in the bacterial solution for 10-15 min and then the bacterial solution was discarded. The callus was inoculated in the co-culture medium and cultured in dark at 20 °C for 48-72 h. Then, the callus was transferred to the primary screening medium (containing 50 mg / L Hygromycin) for 20-30 days, and then to the secondary screening medium for monoclonal callus selection, and cultured in dark at 26 °C for 7-10 days. The positive callus was transferred to the differentiation medium, and buds were formed after cultured in light at 25-27 °C for 15-20 days. Then, the buds were transferred to the rooting medium and cultured in light at 30 °C for 7-10 days to obtain the whole plant. The genomic DNA of the transgenic rice was extracted by the CTAB method, and PCR identification was performed using specific primers to confirm the positive transformed plants.

[0045] Example 4: Functional identification of the hairpin structure of the transgenic rice To verify whether the hairpin structure in the transgenic plants can be correctly transcribed and processed into siRNA, small RNA Northern blot analysis was used. Specific probes against RRSV Pns10 (262 bp) and SRBSDV Pns5 (257 bp) were designed to detect the accumulation of siRNA in the T3 generation of transgenic plants. The detection results are shown in Figure 3 : siRNA signals corresponding to the transgenic rice were detected, while no signals were detected in the control variety ZH11, indicating that the hairpin structure can be normally transcribed in the plant body and effectively trigger the RNA interference pathway.

[0046] Example 5: Antiviral identification of the transgenic rice The T3 generation of transgenic rice obtained was subjected to field resistance identification. The results (see Figure 4 ) showed that the transgenic lines carrying the pRTBV:RRSV Pns10 + SRBSDV Pns5 construct (#22, #26) showed significant resistance to RRSV and SRBSDV under artificial insect infection and natural infection conditions, and the incidence rate was much lower than that of the wild type ZH11. In addition, statistical analysis (see Figure 5 ) of the main agronomic traits (plant height, tiller number, and thousand-grain weight) showed that there was no significant difference between the transgenic lines and the control variety, indicating that the dual RNAi expression system driven by the RTBV promoter did not cause obvious abnormal growth and development while enhancing the antiviral resistance.

[0047] In summary, the application combines the rice East Gru virus (RTBV) phloem-specific promoter with the RRSV and SRGBSDV bivalent RNAi structure, and for the first time realizes the targeted expression of hpRNA in the rice phloem to resist two major rice blast viruses. The method not only significantly improves the broad-spectrum resistance of rice to RRSV and SRGBSDV, but also avoids the metabolic interference problem commonly seen in the constitutive RNAi system, and provides a safe, efficient and popularized innovative approach for rice antiviral molecular breeding.

[0048] The above only describes some embodiments of the application, and does not limit the protection scope of the application, and any equivalent device or equivalent process transformation using the content of the specification and drawings, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the application.

Claims

1. A method for constructing a bivalent RNAi expression vector against rice viruses RRSV and SRBSDV, characterized by, Comprise the following steps: 1) Design specific primers, PCR amplify the virus RRSV Pns10 segment and the virus SRBSDV Pns5 segment, wherein XhoI enzyme cutting site is introduced at the 5' end of the RRSV Pns10 segment, 12 bp region overlapping with the SRBSDV Pns5 segment is introduced at the 3' end, BglII enzyme cutting site is introduced at the 3' end of the SRBSDV Pns5 segment, and 12 bp sequence overlapping with the RRSV Pns10 segment is introduced at the 5' end; use primers iRRSV10-XhoI-f and iSRBSDV5-BglII-r to perform overlapping PCR amplification, and obtain a virus fusion segment containing XhoI and BglII enzyme cutting sites at both ends; the RRSV Pns10 segment gene sequence is shown as SEQ ID NO. 1, and the SRBSDV Pns5 segment gene sequence is shown as SEQ ID NO. 2; 2) After the obtained fusion segment and the intermediate vector pUCC containing an intron structure are double enzyme cut by XhoI and BglII respectively, use T4 DNA ligase for ligation, and obtain the intermediate vector pUCC-RRSV&SRBSDV-intron into which the fusion segment is introduced in a single direction; after the pUCC-RRSV&SRBSDV-intron vector is double enzyme cut by SalI and BamHI, the fusion segment is reversely inserted to form a hairpin structure, and the final pUCC-RRSV&SRBSDV-intron-SRBSDV&RRSV expression vector is obtained; 3) pUCC-RRSV&SRBSDV-intron-SRBSDV&RRSV vector was digested with PstI to obtain pUCC-RRSV&SRBSDV-intron-SRBSDV&RRSV fragment containing PstI site; plant expression vector pYSL16 was digested with PstI p CAMBIA1300 empty vector, the digested pUCC-RRSV&SRBSDV-intron-SRBSDV&RRSV fragment containing PstI site and CAMBIA1300 vector were connected with T4 DNA ligase to obtain p1300-RRSV&SRBSDV-intron-SRBSDV&RRSV vector p CAMBIA1300 empty vector, the digested pUCC-RRSV&SRBSDV-intron-SRBSDV&RRSV fragment containing PstI site and CAMBIA1300 vector were connected with T4 DNA ligase to obtain p1300-RRSV&SRBSDV-intron-SRBSDV&RRSV vector; plant expression vector p1300-RRSV&SRBSDV-intron-SRBSDV&RRSV was double-digested with EcoRI and BamHI, and RTBV promoter fusion enzyme digestion site amplification primer was designed; RTBV infected rice DNA sample was amplified to obtain fragment RTBV; pYSL16 was double-digested with EcoRI and BamHI; double-digested pYSL16 product and p1300-RRSV&SRBSDV-intron-SRBSDV&RRSV product were connected with T4 DNA ligase to prepare RNA interference expression vector based on rice phloem specific promoter RTBV.

2. The method for constructing a bivalent RNAi expression vector against rice viruses RRSV and SRBSDV according to claim 1, wherein, The RRSV Pns10 segment PCR primers are iRRSV10-XhoI-f and iRRSV10-SRBSDV5-r; the SRBSDV Pns5 segment PCR primers are iSRBSDV5-RRSV10-f and iSRBSDV5-BglII-r; and the pRTBV promoter fusion enzyme cutting site amplification primers are OL-EcoRI-pRTBV-F and OL-BamHI-pRTBV-R; The sequence of the primer iRRSV10-XhoI-f is 5'-CCGCTCGAGATTCGCAAGACAAGGCAAGA-3'; The sequence of the primer iRRSV10-SRBSDV5-r is 5'-GCATCGCTAACTCTTACCTGTGGCGTCAACCTCA-3'; The sequence of the primer iSRBSDV5-RRSV10-f is 5'-GCCACAGGTAAGAGTTAGCGATGCCTTTGTTGC-3'; The sequence of the primer iSRBSDV5-BglII-r is 5'-GCAGATCTATCATAGCGGAAACGTACTCAT-3'; The sequence of the primer OL-EcoRI-pRTBV-F is 5'-ATGACCATGATTACGAATTCACGAATCAATAAAGAAGGACCAGAA-3'. The sequence of the primer OL-BamHI-pRTBV-R is 5'-CTTGCTCACCATGGATCCTCTTGCTCTCTTAGAAGTTTGAGCTC-3'.

3. The method for constructing a bivalent RNAi expression vector against rice viruses RRSV and SRBSDV according to claim 1, wherein, The double-promoter vector comprises a CaMV 35S promoter for driving the expression of a Hygromycin resistance gene and an RTBV promoter for driving the expression of a viral fusion hairpin RNA structure; the nucleotide sequence of which is shown as SEQ ID NO.

5.

4. A bivalent RNAi expression vector against rice viruses RRSV and SRBSDV, characterized in that, The construction method of any one of claims 1-3.

5. The expression vector of claim 4, wherein, The bivalent RNAi expression vector against the rice viruses RRSV and SRBSDV comprises the nucleotide sequence shown as SEQ ID NO.

3.

6. An Agrobacterium characterized in that, The Agrobacterium is Agrobacterium tumefaciens EHA105 transfected with a bivalent RNAi expression vector as claimed in claim 4 or 5.

7. Use of the bivalent RNAi expression vector of any one of claims 4-5 in enhancing the resistance of rice to RRSV and SRBSDV viruses.

8. Use of the Agrobacterium of claim 6 in enhancing the resistance of rice to RRSV and SRBSDV viruses.

9. Use according to claim 8, characterized in that, The rice is transformed by the Agrobacterium of claim 6, specifically comprising the following steps: The rice seeds without mildew and with normal germ are selected as the transformation material, the seeds are sterilized by 75% ethanol for 1 min and washed by sterile water for 1 min for 3 times, then treated by 15% sodium hypochlorite for 20 min and washed by sterile water for 3 times, the sterilized seeds are inoculated in the induction medium and cultured at 26°C under light to induce callus; The Agrobacterium resuspension liquid is prepared, the callus is immersed in the bacterial solution for 10-15 min and then the bacterial solution is discarded, the callus is inoculated in the co-culture medium and cultured at 20°C in dark for 48-72 h, then the callus is transferred to the primary screening medium for 20-30 days, then to the secondary screening medium for single callus selection, and cultured at 26°C in dark for 7-10 days, the positive callus is transferred to the differentiation medium, and the bud is formed after cultured at 25-27°C under light for 15-20 days, then transferred to the rooting medium and cultured at 30°C under light for 7-10 days to obtain the complete plant.

10. Use according to claim 9, characterized in that, The induction medium was selected as 2 mg / L 2,4-D; the Agrobacterium resuspension liquid OD 600 = 0.2; and the preliminary screening medium contained 50 mg / L Hygromycin.

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