Method for feeding dsRNA restraint insect gene expression

Abstract

The invention provides a method for suppressing the gene expression of insects through feeding dsRNA, which belongs to the growth development field of adjusting and controlling insects through naturally feeding the dsRNA of the silenced and specific gene. The method selects a gene fragment SeA3 on chitin synthetase enzyme gene SeCHS-A, and studies the impact on the insects after the natural feeding. The method mainly comprises the steps: a proper expression vector L4440 is selected to construct a recombinant expression vector L4440-SeA3 with a purpose gene fragment SeA3; the recombinant vector is induced and expressed in specific recipient cell coliform bacteria HT115 (DE3) under vitro adaptive condition; and the recipient bacterium htLSA3 which expresses dsRNA is added into the feedstuff of the insects, the insect larvae are naturally fed with the feedstuff, the situation of the insects suppressing the gene expression is detected after the feeding, and the change of the phenotype of the insects is recorded. By adopting natural feeding to suppress the expression of the insect genes, the method explores new paths for controlling the insects.

Description

technical field [0001] The present invention relates to the field of growth and development regulation of insects, and it relates to the use of dsRNA for feeding and silencing specific genes in the prevention and control of pests. Specifically, the present invention relates to inhibiting gene expression of lepidopteran insects by feeding dsRNA containing chitin synthase gene interfering with insects, thereby regulating their growth and development. Background technique [0002] RNA interference, or RNA interference or RNAi, is a phenomenon of specific gene expression silencing mediated by dsRNA. It is an ancient and evolutionarily highly conserved gene expression regulation mechanism in the biological world. It was first discovered in the nematode Caenorhabditis elegans in 1998 this phenomenon. The general process of RNAi action is: dsRNA introduced into the body or generated by endogenous transcription is cut into 21~25nt (base) siRNA by Dicer enzyme, siRNA is further comb...

AI Technical Summary

Problems solved by technology

Although the injection method of RNAi has achieved great results in the study of insect gene functions, it also has the following disadvantages: firstly, it has high injection technical requirements for experimental operators, and the labor intensity is high; secondly, the injection of dsRNA or siRNA is basically Kits are required to prepare relatively pure double-stranded RNA, which increases the cost of the experiment; the re-injection method can only be used to study the function of a certain gene in a certain insect, and it cannot explain if a certain insect eats a certain gene. Whether the dsRNA of the gene affects its growth and development; and the injection method needs to be implemented individually, and the RNAi research of the injection method cannot be directly and widely used in the effective control of pests

The following reports have been made in insects: In 2006, it was reported that Araujo et al. fed blood-sucking stinkbug Rhodnius prolixus containing dsRNA targeting anticoagulant gene NP2, which reduced the expression level of NP2 gene mRNA and made the hemagglutination initiation time of blood-sucking stink bug 4 times shorter than the control group (R.N.Araujo et al., InsectBiochemistry and Molecular Biology 36, 683 (2006).); However, this feeding method RNAi research still has the following defects: dsRNA is synthesized and purified using kits, and the cost is high , cannot meet the requirements of large-scale applied research

In 2006, it was reported that Turner et al. fed the dsRNA solution of EposCXE1 and EposPBP1 to the apple penetrating moth Epiphyas postvittana, and the EposCXE1 mRNA transcription level was reduced by half after feeding for 2 days, and the EposPBP1 mRNA transcription level of the adult antennae was reduced (C.T.Turner et al., Insect Molecular Biology 15 , 383(2006).); but this feeding method RNAi research also adopts the synthetic kit of purification, the cost is high, and adopts the microsampler to send the dsRNA solution into the mouthparts of each test insect, and such feeding is just another Injection in this sense, the operation is complicated, the workload is heavy, and it is impossible to carry out large-scale research on gene function and screening of genes of interest.

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