Preparation and use method of RNAi (ribonucleic acid interference) interference fragment of bactrocera dorsalis

A technology of Bactrocera dorsalis and fragments, which is applied in the field of RNAi interference fragment preparation, can solve the problems of high cost, low efficiency, and unfriendly ecological environment, and achieve the effects of clear bands, broad application prospects, and remarkable effects

Pending Publication Date: 2020-06-26
YUNNAN ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

At present, the control methods of this pest mainly include artificial control, chemical control and biological control of sexual attractants, etc. These methods have certain control effects, but generally have problems such as low efficiency, high cost, and unfriendly to the ecological environment. Therefore, RNA Interference, a highly targeted and environmentally friendly technology, shows great application potential in the field of pest control

Method used

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  • Preparation and use method of RNAi (ribonucleic acid interference) interference fragment of bactrocera dorsalis
  • Preparation and use method of RNAi (ribonucleic acid interference) interference fragment of bactrocera dorsalis
  • Preparation and use method of RNAi (ribonucleic acid interference) interference fragment of bactrocera dorsalis

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Embodiment 1

[0029] Preparation of dsRNA interference fragment of Bacteralis dorsalis Flightin gene by RNAi interference

[0030] 1. Interfering primer design

[0031] According to the cloned Bacteralis dorsalis Flightin gene fragment sequence (GenBank accession number: MN557386), use Primer5.0 and Oligo7 to design interference primers, and then add the T7 promoter sequence to the 5' end of the interference primers; similarly, design See Table 1.1 for the interference primers of the control gene EGFP.

[0032]

[0033] 2. DNA template preparation and dsRNA synthesis

[0034] Using the correctly constructed pTOPO-T-Flightin and pTOPO-T-EGFP recombinant vectors as templates, use the primers in Table 1.1 for PCR amplification to obtain the DNA fragments of Flightin and EGFP, which are used as DNA templates for the synthesis of dsRNA after gel recovery.

[0035] The dsRNA was synthesized using an in vitro transcription kit (RiboTM RNAmax-T7).

[0036] (1) Thaw the reagents on ice, centri...

Embodiment 2

[0051] The prepared RNAi fragment product was microinjected into Bacteralis dorsalis.

[0052] 1. Injection of newly emerged adults

[0053] Use a microinjector to inject dsRNA, select adults about 3 hours after eclosion, and use the internode of the prothorax and ventral forefoot as the injection point, and control the depth of the needle to avoid damage to other tissues in the body. The injection volume per head was 1000ng, and 100 female worms and 100 male worms were injected into each group. At the same time, dsEGFP was injected as a negative control, and three biological replicates were set up. The specific method is as follows:

[0054] (1) Thaw dsRNA on ice and adjust the concentration.

[0055] (2) Place a foam board on the stereo microscope, lightly anesthetize the worms with ether, place them on the foam board, adjust their posture gently with tweezers, and then use the pre-made small foam board with grooves suitable for adult worms , gently press the worm into t...

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Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to preparation and a use method of an RNAi (ribonucleic acid interference) interference fragment for interfering a bactrocera dorsalis Flightin gene. The preparation method comprises the following steps of: selecting partial sequences of the bactrocera dorsalis Flightin gene, designing an interference primer, carrying out template DNA amplification, synthesizing dsRNA by taking an amplification product as a template, and purifying to obtain an RNAi interference product with excellent concentration and purity, wherein the RNAi interference product with the substance amount concentration of 1000ng / microliters is selected, and intake is completed through microinjection in the later stage of bactrocera dorsalis pupae or in the just eclosion stage. The technical scheme provided by the embodiment of the invention has a remarkable effect of interfering the bactrocera dorsalis Flightin gene, and a new prevention and control method is provided for safe and efficient biological prevention and control.

Description

technical field [0001] The invention belongs to the technical field of agricultural biology, and in particular relates to the preparation and application method of an RNAi interference fragment for interfering with Bactrocera dorsalis Flightin gene. Background technique [0002] RNA interference (RNA interference, RNAi) is a highly conserved, highly conserved, dsRNA-induced, highly efficient and specific degradation of homologous mRNA widely present in eukaryotes, so that the corresponding gene cannot be expressed, thereby triggering gene silencing at the post-transcriptional level. After the dsRNA is introduced into the organism, it is decomposed into 21~23bp small interfering RNA (siRNA) by the RNase III called Dicer in the cell, and the siRNA binds to the target mRNA under the action of the silencing complex (RISC), sequence-specifically Degrade the target mRNA, prevent the synthesis of the corresponding protein product, resulting in the loss of the function of the target...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A01N57/16A01P7/04
CPCC12N15/113A01N57/16C12N2310/14C12N2320/30
Inventor 袁瑞玲陈鹏郑传伟王艺璇冯丹杜春花
Owner YUNNAN ACAD OF FORESTRY
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