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CN-121992012-A - Application of miniature inverted repeat transposon MITE230 and dsRNA thereof in frankliniella occidentalis prevention and control

CN121992012ACN 121992012 ACN121992012 ACN 121992012ACN-121992012-A

Abstract

The invention relates to the field of biotechnology, and discloses an application of a miniature inverted repeat transposon MITE230 and a dsRNA thereof in frankliniella occidentalis prevention and control, which comprises the following steps of extracting genome DNA of frankliniella occidentalis; mixing dsRNA primer and extracted frankliniella occidentalis genome DNA for PCR amplification, synthesizing dsRNA from the PCR recovered product with T7 RiboMAX Express RNAi kit, connecting MITE230 plasmid to PTCK carrier to constitute recombinant carrier, and agrobacterium transformation to obtain transgenic tobacco. After the transgenic tobacco seedlings obtained by the invention are eaten by frankliniella occidentalis, the sensitivity of the transgenic tobacco seedlings to spinosad is improved, the pests are specifically killed, and the use of chemical pesticides is reduced.

Inventors

  • WU QINGJUN
  • QIAN KANGHUA
  • WAN YANRAN
  • YANG HAOLAN
  • CHEN SIRUI
  • WU YULEI

Assignees

  • 中国农业科学院蔬菜花卉研究所

Dates

Publication Date
20260508
Application Date
20251224

Claims (8)

  1. 1. The application of the miniature inverted repeat transposon MITE230 and the dsRNA thereof in frankliniella occidentalis prevention and control is characterized by comprising the following steps: 1) Collecting adults of a frankliniella occidentalis spinosad sensitive population and a frankliniella occidentalis spinosad resistant population, and extracting genome DNA of the frankliniella occidentalis; 2) Mixing MITE230 primers with extracted frankliniella occidentalis genome DNA for PCR amplification, cloning MITE230, and extracting bacterial liquid with correct sequencing to obtain MITE230 plasmid; 3) Mixing dsRNA primers with the extracted frankliniella occidentalis genome DNA for PCR amplification, and then carrying out gel electrophoresis on a PCR product to recover the product; 4) Performing dsRNA synthesis on the PCR recovered product by using a T7 RiboMAX Express RNAi kit; 5) Feeding the primary emerged frankliniella occidentalis female insects with the synthesized dsRNA; 6) Performing spinosad bioassay on frankliniella occidentalis females fed with dsRNA; 7) Ligating the MITE230 plasmid into PTCK303,303 vector to constitute recombinant vector; 8) Transforming the agrobacterium with the recombinant vector obtained in the step 7) to obtain monoclonal agrobacterium; 9) Inoculating the monoclonal agrobacterium into a culture medium containing antibiotics for culturing to obtain engineering bacterial liquid; 10 Preparing the aseptic tobacco seedlings into explants, then mixing the explants with engineering bacteria liquid for infection, culturing the infected explants in the dark, and then screening and culturing to obtain independent and complete transgenic tobacco strains; 11 Hardening seedlings of the cultured transgenic tobacco lines to obtain transgenic tobacco seedlings capable of improving the sensitivity of frankliniella occidentalis to spinosad; the dsRNA primer comprises ds230-F with a nucleotide sequence shown as SEQ ID NO.1, ds230-R with a nucleotide sequence shown as SEQ ID NO.2, dsEGFP-F with a nucleotide sequence shown as SEQ ID NO.3 and dsEGFP-R with a nucleotide sequence shown as SEQ ID NO. 4; The nucleotide sequence of the miniature inverted repeat transposon MITE230 is shown in SEQ ID No. 5; the MITE230 primer comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer is shown as SEQ ID NO.6, and the sequence of the downstream primer is shown as SEQ ID NO. 7.
  2. 2. The use of mini inverted repeat transposon MITE230 and dsRNA thereof according to claim 1, wherein the PCR amplification reaction system in step 3) comprises the following reagents: 2×ES Taq mix:12.5μL 10. Mu. Mol/L upstream primer 0.5. Mu.L 10. Mu. Mol/L downstream primer 0.5. Mu.L DNA template 1. Mu.L 10.5 Mu L of sterile water; the PCR amplification reaction program is as follows: Pre-denaturation at 95 ℃ for 5min Denaturation at 95℃for 30s Annealing at 58 ℃ for 45s Extension at 72℃for 1min/kb Repeated denaturation-annealing-extension for 35 cycles The final extension was at 72℃for 10min.
  3. 3. The use of mini-inverted repeat transposon MITE230 and dsRNA thereof in frankliniella occidentalis control according to claim 2, wherein the reaction system for dsRNA synthesis in step 4) comprises the following reagents: Template DNA 1. Mu.g Transcription reaction buffer 2. Mu.L 2.5 Mu L of ribonuclease inhibitor T7 RNA polymerase 2. Mu.L ATP adenosine triphosphate at 100mmol/L concentration 1.8. Mu.L CTP cytidine triphosphate at 100mmol/L concentration 1.8. Mu.L GTP guanosine triphosphate at a concentration of 100 mmol/L1.8. Mu.L UTP uridine triphosphate at 100mmol/L concentration 1.8. Mu.L 100Mmol/L concentration of DTP deoxynucleoside triphosphate: 2. Mu.L Nuclease-free water until the total volume of the system is 20. Mu.L.
  4. 4. The use of a mini inverted repeat transposon MITE230 and dsRNA thereof in frankliniella occidentalis prevention and control according to claim 3, wherein the construction method of the recombinant vector in the step 7) is as follows: a. mixing buffer solution, dNTPs, a DNA template of MITE230, DMSO, an upstream primer, a downstream primer and DNA polymerase for PCR amplification; b. performing agarose gel electrophoresis on the PCR amplification product, and cutting and recovering a target DNA band to obtain a DNA product; c. mixing a DNA product, an enzyme digestion buffer solution, a restriction enzyme and water for enzyme digestion to obtain a DNA fragment; d. carrying out agarose gel electrophoresis on the DNA fragments, and cutting and recovering target DNA strips to obtain the DNA fragments; e. D, the DNA fragment and PTCK-303 empty vector are recombined after being digested by SacI/SpeI, bamHI/KpnI enzyme and PTCK-303 empty vector are digested, and finally EasyGenoDNA recombination system is added for connection, thus obtaining recombinant vector PTCK-MITE 230.
  5. 5. The use of mini inverted repeat transposon MITE230 and dsRNA thereof in frankliniella occidentalis prevention and control according to claim 4, wherein the transformation method of the monoclonal agrobacterium in the step 8) is as follows: mixing the agrobacterium competent cells with a recombinant vector, sequentially carrying out ice bath, liquid nitrogen, 37 ℃ water bath and liquid culture medium after ice bath, and shake culturing to obtain monoclonal agrobacterium; The agrobacterium is GV3101.
  6. 6. The application of the mini inverted repeat transposon MITE230 and the dsRNA thereof in frankliniella occidentalis prevention and control according to claim 5, wherein the culture method of the engineering bacterial liquid in the step 9) is as follows: Inoculating a monoclonal agrobacterium colony into a YEB liquid culture medium containing antibiotics, performing shaking culture at 180rpm under 28 ℃ and dark conditions, transferring into a centrifuge tube when the concentration OD600nm of the bacterial liquid is 0.6-0.8, centrifuging at 4000rpm and 4 ℃ for 10min to collect bacterial cells, and then re-suspending by using a heavy suspension YB to obtain engineering bacterial liquid with the concentration OD600nm of 0.5.
  7. 7. The application of the mini inverted repeat transposon MITE230 and the dsRNA thereof in frankliniella occidentalis prevention and control according to claim 6, wherein the infection temperature in the step 10) is room temperature and the time is 10-15 min; The time of culturing in the dark is 2d; the culture medium adopted in the culture process is co-culture medium YC.
  8. 8. The application of the mini inverted repeat transposon MITE230 and the dsRNA thereof in frankliniella occidentalis prevention and control according to claim 7, wherein the temperature of the screening culture in the step 10) is 25 ℃, the illumination intensity is 3000-5000 lux, and the photoperiod is 16h/day; The explants were transferred to new medium once every 2 weeks during the screening culture, and necrotic explants were discarded during the transfer.

Description

Application of miniature inverted repeat transposon MITE230 and dsRNA thereof in frankliniella occidentalis prevention and control Technical Field The invention relates to the technical field of biology, in particular to a miniature inverted repeat transposon MITE230 and application of dsRNA thereof in frankliniella occidentalis prevention and control. Background Frankliniella occidentalis (FRANKLINIELLA OCCIDENTALIS) is a major agricultural pest distributed worldwide, and has a wide host range, and can harm up to 500 plants including vegetables, flowers and various commercial crops. The insect body is tiny, the behavior is hidden, the generation period is short, the reproductive capacity is extremely strong, and the nymphs and adults directly harm through the sap of the epidermal cells of the plant tissues by the file, so that the leaves have the symptoms of white spots, strip spots, curls, wilting of new tips, necrosis and the like. More seriously, frankliniella occidentalis is a high-efficiency transmission medium of various plant viruses, especially tomato spotted wilt virus and impatiens necrotic spot virus, and forms a serious threat to crop yield and quality. At present, the control of frankliniella occidentalis mainly depends on chemical pesticides. However, frankliniella occidentalis has developed a high level of resistance to a variety of commonly used agents including spinosad, pyrethroids due to the short life span, rapid breeding, and long-term high intensity selection pressure of the chemical agents. Studies have shown that specific resistant populations, such as spinosad resistant near isogenic line populations, exhibit very strong tolerance to conventional doses of pesticides, resulting in significant decline or even complete failure of chemical control. The excessive use of chemical pesticides not only faces the dilemma of resistance exacerbation, but also brings a series of serious problems such as environmental pollution, exceeding pesticide residues, biodiversity destruction (such as damage to powdery insects and natural enemies) and the like. In recent years, ribonucleic acid interference technology is regarded as the core direction of new generation pest green prevention and control technology because of its high specificity, environmental friendliness and uneasy occurrence of traditional resistance. RNAi technology can effectively and accurately silence key genes related to growth and development or drug resistance by delivering sequence-specific double-stranded RNA into a pest body, so as to achieve a control effect. Despite the great potential, RNAi technology still faces multiple technical bottlenecks in practical prevention and control applications of frankliniella occidentalis, limiting the large-scale application of the RNAi technology from laboratories to fields: Firstly, naked dsRNA is extremely easy to be degraded by nuclease in natural environment (such as leaf surface) and in internal environment of pests, so that the duration of the dsRNA is shortened, and the prevention and control cost is high. Secondly, how dsrnas penetrate the insect body wall with high efficiency or enter the midgut by feeding and are efficiently absorbed by cells is a key link limiting RNAi efficiency. The frankliniella occidentalis serves as a rasping type oral pest, and the unique feeding mode of the frankliniella occidentalis further increases the difficulty of effectively delivering dsRNA. Again, not all genes are effectively silenced by RNAi and result in a lethal phenotype. At present, a high-efficiency and universal target gene screening system is lacking, and specific effective targets aiming at different physiological stages (such as a case of insects and adults) or different geographical populations (such as sensitive and resistant populations) of frankliniella occidentalis are particularly rare. Finally, the large-scale, low-cost production of dsRNA and its stable formulation technology in the field are not mature, and are important factors limiting commercial application. Therefore, the development of an RNAi technical system capable of overcoming the bottleneck, particularly having an efficient prevention and control effect on a high-resistance frankliniella occidentalis population, has become an urgent need in the current green prevention and control field of agricultural pests. Disclosure of Invention In view of the above, the invention provides a miniature inverted repeat transposon MITE230 and application of dsRNA thereof in frankliniella occidentalis prevention and control, and aims to solve the problems of poor stability, low delivery efficiency and insufficient screening accuracy of target genes of the dsRNA technology in the actual prevention and control application of frankliniella occidentalis. The invention provides an application of a miniature inverted repeat transposon MITE230 and a dsRNA thereof in frankliniella occidentalis prevention and control, which comprises the foll