MafA gene and application thereof in cultivating sogatella furcifera-resistant transgenic rice

Abstract

The invention relates to application of MafA in cultivation of sogatella furcifera resistant transgenic rice. The invention provides a dsRNA (double-stranded ribonucleic acid) sequence for reducing MafA gene expression of sogatella furcifera and a primer pair for synthesizing the dsRNA. The dsRNA can obviously inhibit the expression of the MafA gene and inhibit the generation of ova of sogatella furcifera, so that the egg laying amount is obviously reduced, and finally, the population quantity is reduced to achieve the purpose of pest control. The dsRNA is expressed by utilizing the cultivated dsMafA transgenic rice, and the expression quantity of MafA in the sogatella furcifera is reduced after the sogatella furcifera continuously feeds, so that the aim of reducing the egg laying quantity of the sogatella furcifera is fulfilled.

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] White-backed planthopper is one of the most destructive rice pests in Asia. It has strong reproductive capacity, strong migration ability, and strong virus transmission ability, causing serious damage to rice. At present, chemical control is still the main method to control white-backed planthopper, but the excessive use of chemical pesticides will lead to increased pesticide resistance and environmental pollution. At the same time, the strong reproductive capacity of white-backed planthopper and its resistance to pesticides have led to outbreaks in more and more regions.

[0003] Reducing the number of pests by interfering with their reproductive process is widely considered to be an effective control strategy. RNA interference (RNAi)-based technology is beginning to be commercialized as a new method for agricultural pest control. Transgenic plants can produce hairpin-shaped dsRNA in vivo, thereby inhibiting insect genes, reducing insect survival and the number of offspring, which is a very economical, convenient and environmentally friendly strategy. However, the feasibility of plant-mediated RNAi methods 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-laying amount of white-backed plant hopper and its target gene MafA. The silencing of the target gene significantly reduces the egg-laying amount 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 as 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 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] The gene fragment was amplified using three pairs of primers, cloned into a vector in the form of a hairpin, 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 white-backed planthopper-resistant transgenic rice products;

[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 laying amount of white-backed plant hopper.

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

[0039] The present invention also provides a method for preventing and controlling white-backed plant hoppers and / or inhibiting the egg production of white-backed plant hoppers to reduce the egg laying amount 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 method also involves using cultivated dsMafA transgenic rice to express dsRNA, and reducing the expression of MafA in white-backed planthoppers after continuous feeding by white-backed planthoppers, thereby reducing the amount of eggs laid by white-backed planthoppers.

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

[0044] Further, step S1) comprises:

[0045] The dsRNA was synthesized using amplification primers. The amplification system was: 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: pre-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 1.5% agarose gel electrophoresis. The current was set to 120A and the voltage was set to 150V for 20 minutes. The electrophoresis fragments were cut under ultraviolet light, and the target fragments for constructing dsRNA genetic transformation interference vectors were recovered and purified. The recovered products were 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.

[0049] Further, step S2) includes:

[0050] 1) Transformation of Agrobacterium EHA105

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

[0052] 2) Agrobacterium EHA105 infection and transformation

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

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

[0055] Differentiation and rooting: Inoculate the positive callus into differentiation medium, culture at 25-27℃ for 15-20 days, and inoculate into rooting medium after 2-5cm buds are differentiated, culture 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, and the T0 generation harvested after maturity was T1 generation. After sowing, the seedlings were further tested for positive results, and Southern blot and RT-qPCR were used to screen the strains suitable for bioassay.

[0057] 3) Molecular testing of transgenic resistant plants

[0058] a. PCR test

[0059] The roots of the kanamycin-resistant plants obtained by rooting screening and the roots of the untransformed plants were taken to extract genomic DNA, and MafA dsRNA was used as the detection target. According to the synthetic amplification reaction primers at both ends, the amplified fragment was 374 bp long. The sequence of the primer MafA374-F / R is shown in SEQ ID NO. 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: 2×PCR Mix×5μL, 0.4μL of forward and reverse primers, 2μL of cDNA template, 2.2μL of ddH2O. The expression level of MafA in each strain was obtained according to the fluorescence quantitative detection data analysis. The sequences of primers MafA-90-qF / R and UBQ-qF / R are shown in SEQ ID NO.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 plant hoppers. The decreased expression level of MafA significantly reduced the egg-laying amount of white-backed plant hoppers and ultimately reduced the population size.

[0071] 2) The present invention provides a dsRNA sequence for reducing the expression of the MafA gene of white-backed plant hopper and a primer pair for synthesizing the dsRNA. The dsRNA can significantly inhibit the expression of the MafA gene, inhibit the egg production of white-backed plant hopper, thereby significantly reducing the egg laying amount, and ultimately reducing its population to achieve the purpose of 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-laying amount 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 drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without 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 was used to detect 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, and M24 are single copies).

[0077] Figure 4 The effect of feeding on MafA RNAi transgenic rice on the number of eggs laid by white-backed planthoppers on the 5th day; M2, M3, and M12 represent the 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 Effect of feeding transgenic rice on the expression of MafA 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. 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] The above primers were used to perform PCR amplification using 2×Phanta Max Master Mix (Dye Plus) enzyme of Nanjing Novozyme Biotechnology Co., Ltd., referring to the enzyme recommended system and recommended procedure. The amplification system is as follows: 2×Phanta Max 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 1.5% agarose gel at a current of 120A and a voltage of 150V for 20 minutes. The band with a size 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 h; 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 min, followed by heat shock at 42°C for 45 sec; subsequently, 500 μL of LB culture medium was added to the centrifuge tube, and the cells were shaken at 37°C and 200 rpm for 1 h; 200 μL of the turbid bacterial solution was taken and evenly spread on a plate containing ampicillin resistance, IPTG and X-gal (ampicillin: SL3810, IPTG: SL38601 and X-gal: SL3800, all purchased from Beijing Coolable Biotech Co., Ltd.) with a spreader, and cultured at 37°C for 12 h. Finally, a single white colony was picked up with a sterile pipette tip and placed in 500 μL of LB medium containing ampicillin, and the culture was shaken at 37°C and 200 rpm 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 the sequence in the database, indicating that the amplification was successful.

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

[0090] 1) According to the MafA gene sequence obtained in Example 1, a suitable dsRNA segment (SEQ ID NO. 3) was screened by preliminary experiments, and three pairs of amplification primers for expressing dsRNA were designed according to 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 Company) 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) Use the above primers and 2×Pfu PCR MasterMix enzyme from Beijing Solebow Biotechnology Co., Ltd. to perform PCR amplification according to the recommended system and recommended procedure of the enzyme. Three pairs of primers are used to make three 50uL systems, and the amplification system is as follows: 2×Pfu PCR Master Mix: 25.0μL; upstream primer: 2.0μL; downstream primer: 2.0μL; H2O: 20.0μL; cDNA: 1.0μl

[0098] The templates for C1 and C3 were the cDNA of the MafA gene of 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. After amplification, the three PCR products were detected by 1.5% agarose gel electrophoresis, and the current was set to 120A and the voltage was set to 150V 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 Yishen Biological Co., Ltd. The recovered product was connected to 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 plant expression vector construction is as follows. Figure 1 The specific implementation measures are as follows:

[0100] 3) Enzyme digestion and ligation were performed simultaneously, and the system was as follows: 10*Buffer: 2μL; restriction endonuclease BsaI: 1μL; T4 ligase: 1μL; pBWA(V)HS-ccdb-RNAi vector: 4μL; H2O: 8.0μL; target fragment gel recovery product: 4μL. The reaction procedure was as follows: 37℃, 20min; enzyme digestion: 37℃, 10min; ligation: 20℃, 10min; incubation: 37℃, 20min; denaturation: 80℃, 5min; storage: 4℃, ∞; 5 cycles were performed from the two steps of enzyme digestion and ligation. After the end, the ligation product was transformed into DH5α competent cells, and then spread on a plate containing kanamycin resistance, IPTG and X-gal. The white single bacteria were picked for expansion and culture and sent to the company for sanger sequencing. The sequencing results showed that the target fragment of tgtF (374bp) MafA (200bp) tgtR (374bp) (tgtF and tgtR are SEQ ID NO: 3 and their reverse complementary sequences, and 200bp is a loop structure) had been connected 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] Take 1 μL of recombinant plasmid and add it to 50 μL EHA105 Agrobacterium competent cells, mix thoroughly and then transfer to the electroporation cup, add 1 mL of LB liquid medium after electroporation, mix thoroughly and then transfer to a 1.5 mL centrifuge tube, shake and culture on a shaker at 30°C and 180 rpm for 30 min, take 50 μL of the activated Agrobacterium liquid and inoculate it on LB solid medium, and culture it in the dark at 30°C for 48 h. After a single colony grows on the culture medium, perform colony PCR identification, add the remaining bacterial samples of the colony with positive identification results to 20 mL of LB resistance culture medium, shake at 28°C (about 16 h) and use it to preserve and infect rice callus.

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

[0105] Induce callus tissue: select rice grains without mildew spots and normal buds, disinfect them with 75% alcohol for 1 min, wash them with sterile water for 1 min each time; disinfect them with 15% sodium hypochlorite for 20 min, wash them 3 times with sterile water for 1 min 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 the bacteria at 28°C to prepare an Agrobacterium resuspension with OD600=0.2; pick the callus into a triangular flask, add the Agrobacterium resuspension, discard the bacterial liquid after infection for 10-15 minutes, inoculate the callus into the co-cultivation medium, and co-cultivate at 20°C for 48-72 hours.

[0107] Callus screening: inoculate the above callus into the screening medium and culture it in the dark at 26°C for 20-30 days; inoculate the positive callus into the secondary screening medium. Be sure to pick the monoclonal callus during the callus picking process and culture it in the dark at 26°C 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 detection: The CTAB method was used to extract rice genomic DNA and perform PCR detection. The detection method was the same as that of Agrobacterium tumefaciens. The positive seedlings were T0 generation, and the T0 generation harvested after maturity was T1 generation. After sowing, the seedlings were further tested for positive results, and Southern blot and RT-qPCR were used to screen strains suitable for bioassay.

[0110] 3) Molecular testing of transgenic resistant plants

[0111] a. PCR test

[0112] The roots of the kanamycin-resistant plants obtained by rooting screening and the roots of the untransformed plants were taken to extract genomic DNA, and MafA dsRNA was used as the detection target. According to the synthetic amplification reaction primers at both ends, the amplified fragment was 374 bp long. The sequence of the primer MafA374-F / R is shown in SEQ ID NO. 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., referring to the enzyme 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 (each sample was repeated 3 times). The amplification system was established according to the instructions of the fluorescence quantitative kit. The amplification system was: 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 dsRNA in each strain was obtained according to 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, MafA dsRNA fragments were highly expressed in transgenic plants compared with 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 restriction endonuclease NdeI (Thermofish) at 37°C for 16 h. The digested DNA was then electrophoresed on a 0.8% agarose gel for 16 h and then transferred to a nylon membrane by capillary blotting. Southern hybridization was performed using a DIG-labeled probe at 48°C 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 h. The results are shown in Figure 2. Figure 3 As shown, 8 of the 12 strains tested in the T1 generation were single copies.

[0125] The probe primer NdeI-CpoI-F / R is 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 against white-backed planthoppers

[0129] In order to detect the resistance of dsMafA transgenic rice to white-backed planthoppers, we used three transgenic rice lines with high dsMafA expression and wild-type rice Zhonghua 11 as research objects. When the rice plants reached the tillering stage, they were transplanted into a cup and inoculated with 20 newly hatched 1st instar nymphs. Each line was set up with 12 replicates and continued to feed. The newly eclosing female adults were moved to the corresponding rice lines. After 5 days of feeding, they were collected and the number of eggs carried by single female adults was detected. 30 heads were dissected and counted for each treatment, and 5 heads / tube were collected for RNA extraction, reverse transcription, and RT-qPCR was used to detect the expression level of MafA in white-backed planthoppers (primers are shown in SEQ ID NO. 20 and 21). Another batch of experimental treatments were the same as above, but the white-backed planthoppers were not transferred and allowed to continue feeding to evaluate the resistance of transgenic rice to white-backed planthoppers until part of the rice plants died and photos were taken.

[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 to 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 shown in Figure 5 As shown in Figure 2, in the female adults collected from each transgenic strain, the expression of SfMafA was significantly inhibited, which was significantly different from the wild type. Figure 6 As shown, dsMafA transgenic rice plants retained most of the green leaves, while the 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 in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A MafA gene for regulating the egg laying amount of white-backed planthopper, characterized in that: The amino acid sequence encoded by the MafA gene is shown in SEQ ID NO:

2.

2. The MafA gene according to claim 1, characterized in that The nucleotide sequence of the MafA gene is shown in SEQ ID NO:

1.

3. A dsRNA for reducing the egg laying amount of white-backed planthopper, characterized in that The nucleotide sequence of the dsRNA is shown in SEQ ID NO:

3.

4. An amplification primer set for synthesizing the dsRNA according to claim 3, characterized in that: The primers included are: C1-F:5'-cagtGGTCTCacaacatccgcacatcattcaacaacaccgtc-3'(SEQ ID NO.4) C1-R:5'-cgatGGTCTCcacaggcacgtctcgaggatacccatgaag-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); The gene fragment was amplified using three pairs of primers, cloned into a vector in the form of a hairpin, and the RNAi vector was generated by recombination reaction.

5. Use of the MafA gene according to any one of claims 1 to 2, the dsRNA according to claim 3, or the primer set according to claim 4, an expression cassette, an expression vector, or a recombinant microorganism containing any one of the above in any of the following: A1) Cultivating transgenic rice resistant to white-backed planthopper; A2) preparing and cultivating white-backed planthopper-resistant transgenic rice products; A3) inhibiting the egg production of white-backed plant hoppers and reducing the egg production of white-backed plant hoppers; A4) preparing a product for inhibiting the egg production of white-backed plant hopper and reducing the egg production of white-backed plant hopper.

6. The use according to claim 5, characterized in that: The cultivated dsMafA transgenic rice expresses dsRNA, and the expression level of MafA in the white-backed planthopper is reduced after the white-backed planthopper continuously feeds on it, thereby reducing the egg laying amount of the white-backed planthopper.

7. A method for controlling white-backed plant hopper and / or inhibiting the egg production of white-backed plant hopper to reduce the egg laying amount of white-backed plant hopper, 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, and the expression of MafA in the white-backed planthopper was reduced after the white-backed planthopper fed on it, thereby reducing the egg-laying amount of the white-backed planthopper; Wherein, the nucleotide sequence of the dsRNA is shown in SEQ ID NO:

3.

8. The method according to claim 7, characterized in that Step S1) comprises: The dsRNA was synthesized using amplification primers. The amplification system was: 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'-cgatGGTCTCcacaggcacgtctcgaggatacccatgaag-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 product was detected by electrophoresis, and the target fragment for constructing the dsRNA genetic transformation interference vector was recovered and purified. 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.

9. The method according to any one of claims 7-8, characterized in that: Step S2) comprises: 1) Transformation of Agrobacterium EHA105: The recombinant RNAi plasmid was transformed into Agrobacterium competent cells, cultured and identified, and the colonies with positive identification results were added to LB resistance medium, and after shaking, they were used to preserve and infect rice callus tissue; 2) Agrobacterium EHA105 infection and transformation: 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, 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 and UBQ-qF / R. MafA-90-qF:ATGCCCTCACAACCTTCCAC (SEQ ID NO.14), MafA-90-qR: GTCACGAACGGTGAGATGGA (SEQ ID NO. 15), <h2 style=";text-align:left;direction:ltr">UBQ-qF:GCGGGAGAAACCACAGGTAA(SEQ ID NO.16), <h2 style=";text-align:left;direction:ltr"> UBQ-qR:GTGATCGTCTTCCCCGTCAG(SEQ ID NO.17)。

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