MafA gene and its application in breeding white-backed planthopper-resistant transgenic rice

Abstract

The present invention relates to the use of MafA in cultivating transgenic rice resistant to white-backed planthoppers. The present invention provides a dsRNA sequence for reducing the expression of the MafA gene in white-backed planthoppers and a primer pair for synthesizing the dsRNA. The dsRNA can significantly inhibit the expression of the MafA gene, inhibiting the production of eggs in white-backed planthoppers, thereby significantly reducing egg production and ultimately reducing their population size to achieve the purpose of pest control. By using the cultivated dsMafA transgenic rice to express the dsRNA, the expression of MafA in the white-backed planthoppers is reduced after the white-backed planthoppers continuously feed on it, thereby achieving the purpose of reducing the white-backed planthoppers' egg production.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular breeding, and particularly relates to a MafA gene and an application thereof in cultivating white-backed planthopper-resistant transgenic rice. Background Art

[0002] The white-backed planthopper is one of the most destructive rice pests in Asia. Its high reproductive capacity, migratory prowess, and ability to transmit viruses severely damage rice crops. Currently, chemical control remains the primary method for controlling white-backed planthoppers, but excessive use of chemical pesticides has led to increased pesticide resistance and environmental pollution. Furthermore, the planthopper's high reproductive capacity and insecticide resistance have led to outbreaks in a growing number of regions.

[0003] Reducing pest populations by interfering with their reproductive processes is widely considered an effective control strategy. RNA interference (RNAi) technology is beginning to be commercialized as a novel agricultural pest control method. Transgenic plants can produce hairpin-like dsRNA in vivo, thereby inhibiting insect genes and reducing insect viability and offspring number, making it a very economical, convenient, and environmentally friendly strategy. However, the feasibility of plant-mediated RNAi approaches in achieving high RNAi efficiency and effective control of white-backed planthoppers remains uncertain. Summary of the Invention

[0004] The present invention aims to provide a dsRNA for reducing the egg production of white-backed plant hopper and its target gene MafA. Silencing of the target gene significantly reduces the egg production of the white-backed plant hopper.

[0005] The nucleotide sequence of the MafA gene of the white-backed planthopper provided by the present invention is shown in SEQ ID NO.1, and the specific sequence is as follows:

[0006] ATGATGGCTAGCATTGAATCGGAGGACCCGAACCAGTTGGCCGACGAGTATGTCAAGGAGTTTGTGCTCGACCATTTGGACGTTTCGGTGAAACGCGAGCTGCAGCAGCAACAGCAGCAGCAGCCTCCACCACGTCAGCCGCCTCCGATGTCGTCACCGCCCCCACCCCATCATCTACTCACCCCGCCCTCACAGCCTCCACAACCCGACGACTACCAGGAGGTCACCACCGCTGCGGTCACTCACAACCTGACCGCCATGACGGTCACCAACCACCACGATCATCACAACGGCGTGCTGGTCGTCAAACAGAACATGATGTACCCGGGAACACCCCCGGACACGCCGCCCCATGCGGGGGCCTCTCCTCACCACCACTACTTCCAAATGGACGCTCCGCCACTACCGCCTCCGCCGCCGC ATCCGCACATCATTCAACAACACCGTCCCGATC ACGAGATCATGTGGTTGCAGCAGAACGTTCGCATGGACCAGCAGCCGTTGGATTTGAGGCCAAACTCAGATCACGAC AACTGGATGCCTCCTCATCATACCTCTGTCATAGCCGGGGGAAAACGACTACACCATGACTACCAAGATTTGGGTGC ATTGAGTCCACTCAACATCCAGCGTCCCCTCAGCGTTGCTTCAGCGTCCATGTCCCCTGTGATGCCCT CACAACCTTCCACACGCTCCCGACACCACAACAACGTAGACGCTATCAGCGACAAGGAGCTGGTCCATCTCACCGTTCGTGACCTCAACAAACGTCTTCATGGGTATCCTCGAGACGTGGTTGTGCGTTTGAAGCAGAAGCGACGCACACTGAAGAACCGCGGCTACGCGCAGAACTGTCGCACGAAGCGAGTTAACCAGCGAGCGCAGCTGGAACTCACCAACCGCGCGCTGCAAGCCGAGATGCAGAGGATCAAAACTGAGCTGGGACGTGCACTACAGGAACGCGATTATTACCGGCAAAGGTGCGATTCGATGAGACATGCCGCTGCAGCTGCTAACAACTGCGAAGGTTCCGATGGCATCCATTCGAACCCCAGCTCACCCGAGTACATGCTGTGA

[0007] The amino acid sequence of the protein encoded by the Sogatella furcifera MafA gene provided by the present invention is shown in SEQ ID NO.2, and the specific sequence is as follows:

[0008] MMASIESEDPNQLADEYVKEFVLDHLDVSVKRELQQQQQQQPPPRQPPP

[0009] MSSPPPPHHLLTPPSQPPQPDDYQEVTTAAVTHNLTAMTVTNHHDHHNGVL

[0010] VVKQNMMYPGTPPDTPPHAGASPHHHYFQMDAPPLPPPPPHPHIIQQHRPDH

[0011] EIMWLQQNVRMDQQPLDLRPNSDHDNWMPPHHTSVIAGGKRLHHDYQDLG

[0012] ALSPLNIQRPLSVASASMSPVMPSQPSTRSRHHNNVDAISDKELVHLTVRDLN

[0013] KRLHGYPRDVVVRLKQKRRTLKNRGYAQNCRTKRVNQRAQLELTNRALQA

[0014] EMQRIKTELGRALQERDYYRQRCDSMRHAAAAANNCEGSDGIHSNPSSPEY

[0015] ML

[0016] The nucleotide sequence of the Sogatella furcifera MafA dsRNA provided by the present invention is shown in SEQ ID NO.3, and the specific sequence is as follows:

[0017] ATCCGCACATCATTCAACAACACCGTCCCGATCACGAGATCATGTGG

[0018] TTGCAGCAGAACGTTCGCATGGACCAGCAGCCGTTGGATTTGAGGCCAAA

[0019] CTCAGATCACGACAACTGGATGCCTCCTCATCATACCTCTGTCATAGCCG

[0020] GGGGAAAACGACTACACCATGACTACCAAGATTTGGGTGCATTGAGTCCA

[0021] CTCAACATCCAGCGTCCCCTCAGCGTTGCTTCAGCGTCCATGTCCCCTGTG

[0022] ATGCCCTCACAACCTTCCACACGCTCCCGACACCACAACAACGTAGACGC

[0023] TATCAGCGACAAGGAGCTGGTCCATCTCACCGTTCGTGACCTCAACAAAC

[0024] GTCTTCATGGGTATCCTCGAGACGTG

[0025] The present invention also provides an amplification primer set for synthesizing the above-mentioned dsRNA, comprising the following primers:

[0026] C1-F:5'-cagtGGTCTCacaacatccgcacatcattcaacaacaccgtc-3'(SEQ ID NO.4)

[0027] C1-R:5'-cgatGGTCTCcacaggcacgtctcgaggatacccatgaag-3'(SEQ ID NO.5)

[0028] C2-F:5'-cgatGGTCTCacctgcaggtctagtttttctccttcattttc-3'(SEQ ID NO.6)

[0029] C2-R:5'-cgatGGTCTCagcccgggctctgtaactatcatc-3'(SEQ ID NO.7)

[0030] C3-F:5'-cagtGGTCTCagggccacgtctcgaggatacccatgaag-3'(SEQ ID NO.8)

[0031] C3-R:5'-cagtGGTCTCatacaatccgcacatcattcaacaacaccgtc-3' (SEQ ID NO.9);

[0032] Three pairs of primers were used to amplify the gene fragment, cloned into the vector in a hairpin format, and the RNAi vector was generated by recombination reaction.

[0033] The present invention also provides the use of the above-mentioned MafA gene, dsRNA, primer set, or expression cassette, expression vector or recombinant microorganism containing any of the above-mentioned in any of the following:

[0034] A1) Cultivating transgenic rice resistant to white-backed planthopper;

[0035] A2) preparing and cultivating a transgenic rice product resistant to white-backed planthopper;

[0036] A3) inhibiting the egg production of white-backed plant hoppers and reducing the egg production of white-backed plant hoppers;

[0037] A4) preparing a product for inhibiting the egg production of white-backed plant hopper and reducing the egg production of white-backed plant hopper.

[0038] Furthermore, the dsMafA transgenic rice was cultivated to express dsRNA, and the expression level of MafA in the white-backed planthopper was reduced after the white-backed planthopper continuously fed on it, thereby reducing the egg production of the white-backed planthopper.

[0039] The present invention also provides a method for controlling white-backed plant hoppers and / or inhibiting the egg production of white-backed plant hoppers to reduce the egg production of white-backed plant hoppers, comprising the following steps:

[0040] S1) Construction of MafA-RNAi vector,

[0041] S2) Genetic transformation to obtain dsMafA transgenic rice,

[0042] The dsMafA transgenic rice was cultivated to express dsRNA, and the expression of MafA in the white-backed planthopper was reduced after the white-backed planthopper continuously fed on it, thereby reducing the amount of eggs laid by the white-backed planthopper.

[0043] The nucleotide sequence of the dsRNA is shown in SEQ ID NO: 3.

[0044] Furthermore, step S1) includes:

[0045] dsRNA was synthesized using amplification primers. The amplification system was as follows: 2×Pfu PCR Master Mix: 25.0 μL; upstream primer: 2.0 μL; downstream primer: 2.0 μL; H2O: 20.0 μL; cDNA: 1.0 μl;

[0046] The amplification primers include: primers as shown in SEQ ID NO. 4-9, wherein the C1 and C3 templates are white-backed planthopper cDNA, and the C2 template is plasmid pBWA (V) HS-ccdb-RNAi.

[0047] The reaction procedure was as follows: initial denaturation: 94°C, 5 min; denaturation: 94°C, 30 sec; annealing: 50°C, 30 sec; extension: 72°C, 19 sec; final extension: 72°C, 10 min; storage: 16°C, ∞; 30 cycles of denaturation-extension were performed;

[0048] After amplification, the three PCR products were detected by electrophoresis on 1.5% agarose gel, with the current set to 120A and the voltage set to 150V for 20 minutes. The electrophoresed fragments were cut under ultraviolet light, and the target fragments for constructing the dsRNA genetic transformation interference vector were recovered and purified. The recovered products were connected with the pBWA(V)HS-ccdb-RNAi vector and transformed into DH5α competent cells to extract the positive plasmid pBWA(V)HS-MafA-RNAi.

[0049] Furthermore, step S2) includes:

[0050] 1) Transformation of Agrobacterium EHA105

[0051] Take 1 μL of recombinant RNAi plasmid and add it to 50 μL of EHA105 Agrobacterium competent cells, mix thoroughly and then transfer it to an electroporation cup. After electroporation, add 1 mL of LB liquid medium, mix thoroughly and then transfer it to a 1.5 mL centrifuge tube. Incubate it in a shaker at 30°C and 180 rpm for 30 minutes. Pipette 50 μL of the activated Agrobacterium liquid and inoculate it on LB solid medium. Incubate it in the dark at 30°C for 48 hours. After a single colony grows on the medium, perform colony PCR identification. Add the remaining bacterial samples of the positive colonies to 20 mL of LB resistance medium. After shaking at 28°C, use it to preserve and infect rice callus tissue.

[0052] 2) Agrobacterium EHA105 infection and transformation

[0053] Induction of callus: Select rice grains without mold spots and normal buds, disinfect with 75% alcohol for 1 minute, wash with sterile water for 1 minute each time; disinfect with 15% sodium hypochlorite for 20 minutes, and wash with sterile water 3 times for 1 minute each time; inoculate the disinfected rice grains into induction medium and culture under light at 26°C for 20 days, add 100 μL of Agrobacterium EHA105 transformed with the target gene to 20 mL of liquid LB containing Kana+Rif+acetosyringone, shake the culture at 28°C to prepare an Agrobacterium resuspension with an OD600 of 0.2; pick the callus into a triangular flask, add the Agrobacterium resuspension, infect for 10-15 minutes, and then discard the bacterial solution. Inoculate the callus into co-cultivation medium and co-cultivate at 20°C for 48-72 hours.

[0054] Callus screening: inoculate the above callus into screening medium and culture in the dark at 26℃ for 20-30 days; inoculate the positive callus into secondary screening medium and culture in the dark at 26℃ for 7-10 days.

[0055] Differentiation and rooting: Inoculate the positive callus into differentiation medium and culture it at 25-27℃ for 15-20 days. After the buds of 2-5 cm are differentiated, inoculate it into rooting medium and culture it at 30℃ for 7-10 days.

[0056] Positive seedling detection: The CTAB method was used to extract rice genomic DNA and perform PCR detection. The positive seedlings were T0 generation. The T0 generation was harvested after maturity and planted as T1 generation. After sowing, the seedlings were further tested for positive results. Southern blot and RT-qPCR were used to screen the lines suitable for bioassay.

[0057] 3) Molecular testing of transgenic resistant plants

[0058] a.PCR test

[0059] Roots of kanamycin-resistant plants obtained through rooting screening and roots of untransformed plants were collected to extract genomic DNA. MafA dsRNA was used as the detection target. Based on the synthetic amplification reaction primers at both ends, the amplified fragment was 374 bp long. The sequences of primers MafA374-F / R are shown in SEQ ID NOs. 12 and 13.

[0060] MafA374-F:ATCCGCACATCATTCAACAACACC(SEQ ID NO.12)

[0061] MafA374-R:CACGTCTCGAGGATACCCCATGAAG(SEQ ID NO.13)

[0062] b. Fluorescence quantitative RT-qPCR detection

[0063] Total RNA was extracted from leaves of kanamycin-resistant plants and wild-type plants, and reverse transcribed into first-strand cDNA. The expression of MafA gene was detected by fluorescence quantitative RT-qPCR. The amplification system was as follows: 2×PCR Mix×5μL, 0.4μL of forward and reverse primers, 2μL of cDNA template, and 2.2μL of ddH2O. The expression level of MafA in each strain was obtained based on the fluorescence quantitative detection data. The sequences of primers MafA-90-qF / R and UBQ-qF / R are shown in SEQ ID NOs. 14, 15, 16, and 17.

[0064] MafA-90-qF: ATGCCCTCACAACCTTCCAC (SEQ ID NO.14),

[0065] MafA-90-qR: GTCACGAACGGTGAGATGGA (SEQ ID NO. 15),

[0066] UBQ-qF:GCGGGAGAAACCACAGGTAA(SEQ ID NO.16),

[0067] UBQ-qR: GTGATCGTCTTCCCGTCAG (SEQ ID NO. 17).

[0068] According to the results of fluorescence quantitative RT-qPCR detection, the MafAdsRNA fragment was highly expressed in the transgenic plants compared with the wild-type plants.

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] 1) The present invention discovered for the first time that the transcription factor MafA gene is involved in regulating the oogenesis of white-backed planthoppers. The decreased expression of MafA significantly reduces the egg production of white-backed planthoppers and ultimately reduces the population size.

[0071] 2) The present invention provides a dsRNA sequence for reducing the expression of the MafA gene in white-backed planthoppers and a primer pair for synthesizing the dsRNA. This dsRNA can significantly inhibit the expression of the MafA gene, inhibiting egg production in white-backed planthoppers and significantly reducing egg production, ultimately reducing their population size and achieving pest control.

[0072] 3) Using the cultivated dsMafA transgenic rice to express dsRNA, the expression level of MafA in the white-backed planthopper is reduced after the white-backed planthopper continuously feeds on it, thereby achieving the purpose of reducing the egg production of the white-backed planthopper. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0074] Figure 1 This is a map of the pBWA(v)HS-MafA-RNAi plant vector.

[0075] Figure 2 Relative expression levels of MafA dsRNA in single-copy transgenic rice lines and wild-type plants; WT: wild-type plants, M2-M24: MafA RNAi-transgenic lines (among which M2, M3, and M12 have significantly increased expression levels).

[0076] Figure 3 Southern blot detection of the copy number of MafA dsRNA gene transgenic rice, ZH11: wild-type plant, P: positive control, M1-M24: MafA transgenic lines (M2, M3, M8, M11, M12, M13, M15, M24 are single copies).

[0077] Figure 4 Figure 3. Effects of feeding on MafA RNAi transgenic rice on the egg-laying capacity of white-backed planthoppers on the 5th day; M2, M3, and M12 represent treatment groups feeding on different strains of MafA RNAi transgenic rice; ZH11 represents the control group feeding on the wild-type rice variety Zhonghua 11; “Different lowercase letters” indicate p<0.05.

[0078] Figure 5 Effects of feeding transgenic rice on MafA expression in white-backed planthoppers.

[0079] Figure 6 This shows the growth status of wild-type and transgenic rice 20 days after being attacked by white-backed planthoppers. DETAILED DESCRIPTION

[0080] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention with this. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0081] Example 1 Cloning of the MafA gene of the white-backed planthopper

[0082] 1) Obtain the nucleotide sequence of the MafA gene from the open access genome database (http: / / v2.insect-genome.com / ). Use TA cloning to obtain the correct nucleotide sequence. Use NCBI Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to design cloning primers:

[0083] Upstream primer sequence clo-MafA-F: 5'-ATGATGGCTAGCATTGAATCGG-3' (SEQ ID NO. 10)

[0084] Downstream primer sequence clo-MafA-R: 5'-TCACAGCATGTACTCGGGTGAG-3' (SEQ ID NO.11)

[0085] PCR amplification was performed using the above primers and 2× PhantaMax MasterMix (Dye Plus) enzyme from Nanjing Novozymes Biotechnology Co., Ltd., following the enzyme's recommended system and protocol. The amplification system was as follows: 2× PhantaMax Master Mix: 5.0 μL; upstream primer: 0.4 μL; downstream primer: 0.4 μL; H2O: 3.2 μL; cDNA: 1.0 μL.

[0086] The reaction procedure is as follows:

[0087] Pre-denaturation: 95°C, 5 min; denaturation: 95°C, 15 sec; annealing: 60°C, 15 sec; extension: 72°C, 1 min; final extension: 72°C, 5 min; storage: 16°C, ∞; 32 cycles of denaturation-extension were performed.

[0088] 2) The amplified product was electrophoresed on a 1.5% agarose gel at a current of 120 A and a voltage of 150 V for 20 min. The band of 1077 bp was identified as the correct band and recovered from the gel. Subsequently, 4.0 μL of the gel recovery product, 5 μL of Solution I, and 1 μL of PMD-18T cloning vector (Takara Biotech Dalian Co., Ltd.) were mixed in a PCR tube and connected in a 16°C water bath for 12 hours. After the connection, all the connection products were added to a centrifuge tube containing 100 μL DH5α competent cells (Takara Biotech Dalian Co., Ltd.), gently mixed, and ice-bathed for 30 minutes. After the ice bath, heat shock was performed at 42°C for 45 seconds. Subsequently, 500 μL LB medium was added to the centrifuge tube, and the cells were shaken at 37°C and 200 rpm for 1 hour. 200 μL of the turbid bacterial solution was evenly spread on a plate containing ampicillin resistance, IPTG, and X-gal (ampicillin: SL3810, IPTG: SL38601, and X-gal: SL3800, all purchased from Beijing Coollab Co., Ltd.) using a spreader and cultured at 37°C for 12 hours. Finally, a single white colony was picked with a sterile pipette tip and placed in 500 μL of LB medium containing ampicillin. The culture was shaken at 200 rpm at 37°C for 4 hours before being sent to Changsha Qingke Biotechnology Co., Ltd. for Sanger sequencing. The sequencing results showed that the cloned sequence was consistent with that in the database, indicating successful amplification.

[0089] Example 2 Construction of pBWA(V)HS-MafA-RNAi vector for genetic transformation

[0090] 1) Based on the MafA gene sequence obtained in Example 1, a suitable dsRNA segment (SEQ ID NO. 3) was screened through preliminary experiments. Three pairs of amplification primers for expressing dsRNA were designed based on the SEQ ID NO. 3 sequence combined with the hairpin and loop structure of the backbone plasmid pBWA(V)HS-ccdb-RNAi vector (Wuhan Boyuan Biological Co., Ltd.) used for subsequent genetic transformation:

[0091] C1-F:5'-cagtGGTCTCacaacatccgcacatcattcaacaacaccgtc-3'(SEQ ID NO.4)

[0092] C1-R:5'-cgatGGTCTCcacaggcacgtctcgaggatacccatgaag-3'(SEQ ID NO.5)

[0093] C2-F:5'-cgatGGTCTCacctgcaggtctagtttttctccttcattttc-3'(SEQ ID NO.6)

[0094] C2-R:5'-cgatGGTCTCagcccgggctctgtaactatcatc-3'(SEQ ID NO.7)

[0095] C3-F:5'-cagtGGTCTCagggccacgtctcgaggatacccatgaag-3'(SEQ ID NO.8)

[0096] C3-R:5'-cagtGGTCTCatacaatccgcacatcattcaacaacaccgtc-3'(SEQ ID NO.9)

[0097] 2) Perform PCR amplification using the above primers and 2×Pfu PCR MasterMix enzyme from Beijing Solaibao Biotechnology Co., Ltd., following the recommended system and protocol. Three 50 μL amplification systems were prepared for each of the three primer pairs. The amplification system was as follows: 2×Pfu PCR Master Mix: 25.0 μL; Upstream Primer: 2.0 μL; Downstream Primer: 2.0 μL; H2O: 20.0 μL; cDNA: 1.0 μL.

[0098] The templates for C1 and C3 were the cDNA of the MafA gene of the white-backed planthopper, and the template for C2 was the plasmid pBWA(V)HS-ccdb-RNAi.

[0099] The reaction procedure is as follows: pre-denaturation: 94°C, 5min; denaturation: 94°C, 30sec; annealing: 50°C, 30sec; extension: 72°C, 19sec; final extension: 72°C, 10min; storage: 16°C, ∞; 30 cycles of denaturation-extension were performed. After the amplification, the three PCR products were detected by electrophoresis on 1.5% agarose gel, and the current was set to 120A and the voltage was set to 150V for electrophoresis for 20min. The three electrophoresis fragments of C1, C2, and C3 were cut under ultraviolet light, placed in a system for gel recovery, and recovered and purified using the gel recovery kit of Shanghai Yisheng Biological Co., Ltd. The recovered product was connected with the pBWA(V)HS-ccdb-RNAi vector, transformed into DH5α competent cells, and the positive plasmid pBWA(V)HS-MafA-RNAi was extracted; the process of constructing the plant expression vector is as follows. Figure 1 The specific implementation measures are as follows:

[0100] 3) Enzyme digestion and ligation were performed simultaneously using the following system: 10*Buffer: 2 μL; restriction endonuclease BsaI: 1 μL; T4 ligase: 1 μL; pBWA(V)HS-ccdb-RNAi vector: 4 μL; H2O: 8.0 μL; target fragment gel recovery product: 4 μL. The reaction procedure was as follows: 37°C, 20 min; enzyme digestion: 37°C, 10 min; ligation: 20°C, 10 min; incubation: 37°C, 20 min; denaturation: 80°C, 5 min; storage: 4°C, ∞; 5 cycles of enzyme digestion and ligation were performed. After completion, the ligation product was transformed into DH5α competent cells and plated on plates containing kanamycin resistance, IPTG, and X-gal. A single white bacterium was cultured and sent to the company for Sanger sequencing. The sequencing results showed that the target fragment tgtF (374 bp) MafA (200 bp) tgtR (374 bp) (tgtF and tgtR are SEQ ID NO: 3 and their reverse complementary sequences, and 200 bp is a loop structure) had been ligated to the pBWA (V) HS-ccdb-RNAi vector. The complete pBWA (V) HS-MafA-RNAi vector sequence is shown in SEQ ID NO. 18.

[0101] Example 3 Cultivation of dsMafA transgenic rice

[0102] 1) Transformation of Agrobacterium EHA105

[0103] Add 1 μL of the recombinant plasmid to 50 μL of EHA105 competent Agrobacterium cells, mix thoroughly, and transfer to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix thoroughly, and transfer to a 1.5 mL centrifuge tube. Incubate on a shaker at 30°C, 180 rpm, for 30 minutes. Inoculate 50 μL of the activated Agrobacterium culture onto LB solid medium and incubate in the dark at 30°C for 48 hours. Once a single colony has grown on the medium, perform colony PCR analysis. Add the remaining sample of positive colonies to 20 mL of LB resistance medium and shake at 28°C for approximately 16 hours before use for preservation and inoculation of rice callus.

[0104] 2) Transformation of japonica rice ZH11 by infection with Agrobacterium tumefaciens EHA105

[0105] Induce callus tissue: Select rice grains without mold spots and normal buds, disinfect them with 75% alcohol for 1 minute, wash them with sterile water for 1 minute each time; disinfect them with 15% sodium hypochlorite for 20 minutes, and wash them with sterile water 3 times for 1 minute each time; inoculate the disinfected rice grains into induction culture medium and culture them under light at 26℃ for 20 days.

[0106] Add 100 μL of Agrobacterium EHA105 transformed with the target gene to 20 mL of liquid LB containing Kana + Rif + acetosyringone, shake at 28°C to prepare an Agrobacterium resuspension with an OD600 of 0.2; pick calli into a triangular flask, add the Agrobacterium resuspension, infect for 10-15 minutes, and then discard the bacterial liquid. Inoculate the calli into the co-cultivation medium and co-cultivate at 20°C for 48-72 hours.

[0107] Callus screening: inoculate the above callus into screening medium and culture in the dark at 26℃ for 20-30 days; inoculate the positive callus into secondary screening medium. Be sure to pick single clone callus during callus picking process and culture in the dark at 26℃ for 7-10 days.

[0108] Differentiation and rooting: Inoculate the positive callus into differentiation medium and culture it at 25-27℃ for 15-20 days. After the 2-5cm buds are differentiated, inoculate it into rooting medium and culture it at 30℃ for 7-10 days.

[0109] Positive seedling testing: Rice genomic DNA was extracted using the CTAB method and then tested by PCR, using the same method as Agrobacterium testing. Positive seedlings were designated as the T0 generation. Mature T0 seedlings were harvested and planted as the T1 generation. After sowing, seedlings were further tested for positive results. Southern blot and RT-qPCR were then used to screen for suitable lines for bioassay testing.

[0110] 3) Molecular testing of transgenic resistant plants

[0111] a.PCR test

[0112] Roots of kanamycin-resistant plants obtained through rooting screening and roots of untransformed plants were collected to extract genomic DNA. MafA dsRNA was used as the detection target. Based on the synthetic amplification reaction primers at both ends, the amplified fragment was 374 bp long. The sequences of primers MafA374-F / R are shown in SEQ ID NOs. 12 and 13.

[0113] MafA374-F:ATCCGCACATCATTCAACAACACC(SEQ ID NO.12)

[0114] MafA374-R:CACGTCTCGAGGATACCCCATGAAG(SEQ ID NO.13)

[0115] PCR amplification was performed using the above primers and 2×Phanta Max MasterMix (Dye Plus) enzyme produced by Nanjing Novozymes Biotechnology Co., Ltd., according to the enzyme's recommended system, recommended program, and fragment length.

[0116] b. Fluorescence quantitative RT-qPCR detection

[0117] Total RNA was extracted from leaves of kanamycin-resistant plants and wild-type plants, reverse transcribed into first-strand cDNA, and the expression of MafA dsRNA gene was detected by fluorescence quantitative RT-qPCR (repeated 3 times for each sample). The amplification system was established according to the instructions of the fluorescence quantitative RT-qPCR kit: 2×PCR Mix×5μL, 0.4μL each of forward and reverse primers, 2μL of cDNA template, and 2.2μL of ddH2O. The expression level of MafA dsRNA in each strain was obtained based on the fluorescence quantitative detection data analysis, as shown in Figure 2. Figure 2 The sequences of primers MafA-90-qF / R and UBQ-qF / R are shown in SEQ ID NO. 14, 15, 16, and 17.

[0118] MafA-90-qF: ATGCCCTCACAACCTTCCAC (SEQ ID NO.14),

[0119] MafA-90-qR: GTCACGAACGGTGAGATGGA (SEQ ID NO. 15),

[0120] UBQ-qF:GCGGGAGAAACCACAGGTAA(SEQ ID NO.16),

[0121] UBQ-qR: GTGATCGTCTTCCCGTCAG (SEQ ID NO. 17).

[0122] According to the results of fluorescence quantitative RT-qPCR detection, the MafA dsRNA fragment was highly expressed in the transgenic plants compared with the wild-type ZH11 plants.

[0123] c. Southern blot detection

[0124] To determine the copy number of dsMafA transgenic rice, 10 μg of DNA was extracted from leaves of transgenic and wild-type rice plants at the tillering stage and digested with the restriction endonuclease NdeI (Thermofish) at 37°C for 16 hours. The digested DNA was then electrophoresed on a 0.8% agarose gel for 16 hours and then transferred to a nylon membrane by capillary blotting. Southern hybridization was performed at 48°C using a DIG-labeled probe according to the instructions of the DIG-High Prime DNA Labeling and DIG Nucleic Acid Detection Kit (Roche). The membrane was photographed using X-ray film and exposed in a darkroom at 80°C for 4 hours. The results are shown in Figure 2. Figure 3 As shown, 8 of the 12 T1 generation lines tested were single-copy.

[0125] The probe primers NdeI-CpoI-F / R are shown in SEQ ID NO. 19 and 20.

[0126] NdeI-CpoI-F: GACCTAACAGAACTCGCCGT (SEQ ID NO.19)

[0127] NdeI-CpoI-R: AAAGTCCCCCGTGTTCTCTC (SEQ ID NO. 20)

[0128] Example 4: Effect of dsMafA transgenic rice on resistance to white-backed planthoppers

[0129] To test the resistance of dsMafA transgenic rice to white-backed planthoppers, we used three transgenic rice lines expressing high dsMafA expression and the wild-type rice variety Zhonghua 11. When rice plants reached the tillering stage, they were transplanted into a cup and inoculated with 20 newly hatched first-instar nymphs. Twelve replicates were set up for each line and continuously reared. Newly emerged female adults were transferred to the corresponding rice line and collected after 5 days of rearing. The number of eggs per female adult was determined. Thirty individuals were dissected from each treatment, and RNA was extracted from 5 individuals per tube. RNA was then reverse-transcribed and analyzed by RT-qPCR to determine MafA expression levels in white-backed planthoppers (primers shown in SEQ ID NOs. 20 and 21). Another batch of rice plants was treated as above, but without the transfer of white-backed planthoppers. The plants were allowed to continue feeding to assess the resistance of the transgenic rice plants to white-backed planthoppers. The plants were then partially succumbed and photographed.

[0130] The quantitative primers for detecting the expression level of the MafA gene in white-backed planthoppers after feeding on dsMafA transgenic rice are shown in SEQ ID NOs. 21 and 22:

[0131] MafA-qF:CTCACCGTTCGTGACCTCAA(SEQ ID NO.21)

[0132] MafA-qR: GTTAACTCGCTTCGTGCGAC (SEQ ID NO. 22)

[0133] The results showed that after feeding on transgenic MafA rice plants, a significant decrease in the number of eggs in the ovaries of individual females was observed on the fifth day after treatment, compared with ZH11. The average number of eggs carried by female adults after feeding on transgenic lines M2, M3, and M12 was 22.6, 18.6, and 26.2, respectively. In comparison, the average number of eggs carried by female adults in the control group was 35.2 ( Figure 4 ). In addition, RT-qPCR was used to detect the expression of SfMafA in white-backed planthoppers; the results are as follows Figure 5 As shown in Figure 2, the expression of SfMafA in female adults collected from each transgenic line was significantly suppressed, with significant differences from the wild type. Figure 6 As shown, dsMafA transgenic rice plants retained most green leaves, while wild-type plants had completely withered, proving that it has good insect-resistant and growth-promoting effects and has great industrial value and application prospects.

[0134] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A dsRNA that reduces egg production in white-backed planthoppers, characterized in that: The nucleotide sequence of the dsRNA is shown in SEQ ID NO:

3.

2. An amplification primer set for synthesizing the dsRNA according to claim 1, characterized in that: The following primers are included: C1-F: 5' -cagtGGTCTCacaacatccgcacatcattcaacaacaccgtc- 3' (SEQ ID NO.4) C1-R: 5'-cgatGGTCTCacaggcacgtctcgaggatacccatgaag- 3' (SEQ ID NO.5) C2-F: 5' - cgatGGTCTCacctgcaggtctagtttttctccttcattttc- 3' (SEQ ID NO.6) C2-R: 5'-cgatGGTCTCagcccgggctctgtaactatcatc- 3' (SEQ ID NO.7) C3-F: 5' -cagtGGTCTCagggccacgtctcgaggatacccatgaag- 3' (SEQ ID NO.8) C3-R: 5'-cagtGGTCTCatacaatccgcacatcattcaacaacaccgtc- 3' (SEQ ID NO. 9); Three pairs of primers were used to amplify the gene fragment, cloned into the vector in a hairpin format, and the RNAi vector was generated by recombination reaction.

3. Use of the dsRNA according to claim 1 or the primer set according to claim 2 in any of the following: A1) Developing transgenic rice resistant to white-backed planthopper; A2) Preparation and cultivation of transgenic rice products resistant to white-backed planthoppers; A3) Inhibit the egg production of white-backed planthoppers and reduce the egg production of white-backed planthoppers; A4) Preparation of products for inhibiting the egg production of white-backed plant hoppers and reducing the egg production of white-backed plant hoppers.

4. The use according to claim 3, characterized in that The dsMafA transgenic rice was cultivated to express dsRNA, which reduced the amount of dsMafA in the white-backed planthopper after continuous feeding. MafA , thereby reducing the egg production of white-backed planthoppers.

5. A method for controlling white-backed planthoppers and / or inhibiting the egg production of white-backed planthoppers to reduce the egg production of white-backed planthoppers, characterized in that: The steps include: S1) Construction of MafA-RNAi vector; S2) Genetic transformation to obtain dsMafA transgenic rice; The dsMafA transgenic rice was cultivated to express dsRNA, which reduced the amount of dsMafA in the white-backed planthopper after feeding. MafA , thereby reducing the egg production of white-backed planthoppers; The nucleotide sequence of the dsRNA is shown in SEQ ID NO:

3.

6. The method according to claim 5, characterized in that Step S1) includes: dsRNA was synthesized using amplification primers. The amplification system was as follows: 2×Pfu PCR Master Mix: 25.0 μL; upstream primer: 2.0 μL; downstream primer: 2.0 μL; H2O: 20.0 μL; cDNA: 1.0 μL; The amplification primers include: C1-F: 5' -cagtGGTCTCacaacatccgcacatcattcaacaacaccgtc- 3' (SEQ ID NO.4) C1-R: 5'-cgatGGTCTCacaggcacgtctcgaggatacccatgaag- 3' (SEQ ID NO.5) C2-F: 5' - cgatGGTCTCacctgcaggtctagtttttctccttcattttc- 3' (SEQ ID NO.6) C2-R: 5'-cgatGGTCTCagcccgggctctgtaactatcatc- 3' (SEQ ID NO.7) C3-F: 5' -cagtGGTCTCagggccacgtctcgaggatacccatgaag- 3' (SEQ ID NO.8) C3-R: 5'-cagtGGTCTCatacaatccgcacatcattcaacaacaccgtc- 3' (SEQ ID NO. 9); After amplification, the PCR products were detected by electrophoresis, and the target fragments for constructing dsRNA genetic transformation interference vectors were recovered and purified by gel cutting. The recovered products were connected with the pBWA(V)HS-ccdb-RNAi vector and transformed into DH5α competent cells to extract the positive plasmid pBWA(V)HS-MafA-RNAi.

7. The method according to any one of claims 5-6, characterized in that: Step S2) includes: 1) Transformation of Agrobacterium tumefaciens EHA105: The recombinant RNAi plasmid was transformed into competent Agrobacterium cells, cultured and identified, and colonies with positive results were added to LB resistance medium. After shaking, the cells were used to preserve and infect rice callus tissue. 2) Transformation by Agrobacterium tumefaciens EHA105: callus induction, callus screening, differentiation and rooting, and positive seedling detection; 3) Molecular detection of transgenic resistant plants: a. PCR detection: Take the roots of resistant plants, extract genomic DNA, and use MafA dsRNA as the detection target and amplify it with primers MafA374-F / R, where MafA374-F: ATCCGCACATCATTCAACAACACC (SEQ ID NO. 12) MafA374-R: CACGTCTCGAGGATACCCCATGAAG (SEQ ID NO.13) b. Fluorescence quantitative RT-qPCR detection: Total RNA was extracted from leaves of resistant plants and reverse transcribed into first-strand cDNA. The expression of MafA dsRNA gene was detected by fluorescence quantitative RT-qPCR using primers MafA-90-qF / R; UBQ-qF / R. MafA-90-qF:ATGCCCTCACAACCTTCCAC (SEQ ID NO.14), MafA-90-qR: GTCACGAACGGTGAGATGGA (SEQ ID NO. 15), UBQ-qF:GCGGGAGAAACCACAGGTAA (SEQ ID NO.16), UBQ-qR: GTGATCGTCTTCCCGTCAG (SEQ ID NO. 17).

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