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ABC transportprotein gene ABCH1 and application of specific dsRNA of ABCH1 in prevention and control of diamond back moth and Bt resistance treatment

A technology of transport protein and diamondback moth, which is applied in the direction of DNA/RNA fragments, applications, genetic engineering, etc., can solve the problems of endangering human health, small promotion area, environmental pollution, etc., and achieve obvious commercial value and application prospects, and obvious technology advantage effect

Inactive Publication Date: 2015-05-13
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The control of agricultural pests has long relied on chemical pesticides. However, the long-term use of chemical pesticides affects the survival of non-target organisms and leads to a series of problems such as pest resistance and environmental pollution, which seriously endanger human health.
Although biological insecticides such as Bacillus thuringiensis (Bacillus thuringiensis, referred to as Bt) have been used in pest control, their action time is usually long and the insecticidal effect is slow, and the promotion area is small

Method used

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  • ABC transportprotein gene ABCH1 and application of specific dsRNA of ABCH1 in prevention and control of diamond back moth and Bt resistance treatment
  • ABC transportprotein gene ABCH1 and application of specific dsRNA of ABCH1 in prevention and control of diamond back moth and Bt resistance treatment
  • ABC transportprotein gene ABCH1 and application of specific dsRNA of ABCH1 in prevention and control of diamond back moth and Bt resistance treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Cloning of Plutella xylostella ABC transporter gene ABCH1 and comparison of Bt Cry1Ac resistance difference

[0023] 1. Sequence cloning of Plutella xylostella ABCH1 gene

[0024] (1) Search of the sequence of ABCH1 gene of Plutella xylostella in the midgut transcriptome database of Plutella xylostella

[0025] Based on the Plutella xylostella midgut transcriptome database in our laboratory, the sequence of the ABCH1 gene of Plutella xylostella was searched using bioinformatics methods. After sequence analysis and comparison, a sequence fragment of the ABCH1 gene of Plutella xylostella was obtained. The cDNA sequence It is the middle sequence fragment of ABCH1 gene.

[0026] (2) Acquisition of the full-length cDNA sequence of the ABCH1 gene of Plutella xylostella

[0027] Based on the obtained ABCH1 gene fragment of Plutella xylostella, 3′, 5′-RACE primers were designed using Primer Premier 5.0 software. All primers were synthesized by Sangon Bioengineer...

Embodiment 2

[0036] Embodiment 2: dsRNA preparation of Plutella xylostella ABCH1 gene

[0037] (1) Design of dsRNA primers for Plutella xylostella ABCH1 gene

[0038] Based on the cloned full-length sequence of Plutella xylostella ABCH1 gene SEQ ID NO.1, using Primer Premier 5.0 software, the unique transmembrane binding domain TMD ( figure 1 ) designed a specific forward primer SEQ ID NO.4 and a reverse primer SEQ ID NO.5 containing a 23-bp T7 promoter. All primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0039] (2) Synthesis of dsRNA of Plutella xylostella ABCH1 gene and preparation before injection

[0040] Use the above primers SEQ ID NO.4 and SEQ ID NO.5 to amplify a single PCR product by PCR, and use T7 Ribomax after further purification TM The Express RNAi System (Promega, Madison, WI, USA) kit was used to synthesize the specific dsRNA of the Plutella xylostella ABCH1 gene by in vitro reverse transcription according to its instructions, using injection...

Embodiment 3

[0041] Example 3: Plutella xylostella ABCH1 gene dsRNA causes lethal phenotypes in the larval and pupal stages of Plutella xylostella

[0042] 1. Microinjection of Plutella xylostella ABCH1 gene

[0043] The third-instar early Plutella xylostella larvae of the Bt insecticidal protein Cry1Ac-sensitive population DBM1Ac-S and the Plutella xylostella Bt insecticidal protein Cry1Ac-resistant near-isogenic line population NIL-R were starved for 6 hours and frozen for 30 minutes, and then 300 μg ( The prepared dsRNA solution (70 μl) was injected into the body cavity from the abdominal intersegmental membrane of the larvae with a Nanoliter 2000 microinjector, and a total of 90 animals were injected into each treatment group, with 3 biological repetitions (30 animals per repetition). The same volume concentration of Buffer and dsEGFP (mixed with the same volume of Metafectene PRO transfection reagent in the treatment group) was injected into the two control groups, and then the inje...

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Abstract

The invention relates to an ABC transportprotein gene ABCH1 and an application of the specific dsRNA of ABCH1 in the prevention and control of diamond back moth and Bt resistance treatment. The invention discloses an insect ABC transportprotein gene and an application of an encoding gene ABCH1 and the double-stranded RNA (dsRNA) of the insect ABC transportprotein gene, and the insect ABC transportprotein gene can specifically silence ABCH1 gene so that Bt Cry1Ac insecticidal protein-sensitive and resistant diamond back moth dies. The gene full-length nucleotide sequence is shown in SEQ ID NO. 1 and is used for encoding the protein as shown in the SEQ ID NO. 2. The invention also discloses ABCH1 gene specific fragments which are used for synthesis of dsRNA; after dsRNA is injected into the Bt Cry1Ac insecticidal protein-sensitive and resistant early 3-year-old diamond back moth larvae body cavity, the diamond back moth slowly develops during the growing process, which results in the death of the diamond back moth in larva and pupa stages. The invention provides a novel target for the prevention and control of field pests and Bt resistance treatment based on RNA interference and thus has good application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of the diamondback moth ABC transporter gene and its specific dsRNA in pest control and Bt resistance management. Background technique [0002] Agricultural pests seriously damage crops and cause huge economic losses worldwide every year. The control of agricultural pests has long relied on chemical pesticides. However, the long-term use of chemical pesticides affects the survival of non-target organisms and leads to a series of problems such as pest resistance and environmental pollution, which seriously endanger human health. Although biopesticides such as Bacillus thuringiensis ( Bacillus thuringiensis , referred to as Bt) etc. are used in pest control, but its action time is usually longer and the insecticidal effect is slow, and the promotion area is small. At present, Bt transgenic insect-resistant crops (Bt crops) are considered to be the preferre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/113A01N57/16A01P7/04
CPCA01N57/16C07K14/43563C12N15/113
Inventor 张友军郭兆将康师朱勋夏吉星吴青君王少丽谢文
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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