Method for cultivating plant resistant to cotton bollworm by utilizing RNAi

A technology of cotton bollworm and RNA interference, applied in botany equipment and methods, DNA/RNA fragments, plant regeneration, etc., to achieve the effect of wide application prospects

Inactive Publication Date: 2012-09-05
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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Problems solved by technology

All the published literature shows that the molting regulation transcription factor HHR3 gene of the cotton bollworm has been cloned, or the expression of the P450 monooxygenase gene CYP6AE14 of the cotton bollworm has been inhibited by RNAi technology, but there is no plant-mediated RNAi technology Inhibition of Molt Regulatory Transcription Factor HaHR3 Expression and Its Application in Cotton Bollworm

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  • Method for cultivating plant resistant to cotton bollworm by utilizing RNAi
  • Method for cultivating plant resistant to cotton bollworm by utilizing RNAi
  • Method for cultivating plant resistant to cotton bollworm by utilizing RNAi

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Embodiment Construction

[0020] The present invention is further set forth below by embodiment.

[0021] Step 1. Cloning of the coding gene HaHR3 of the cotton bollworm molting regulatory transcription factor

[0022] The total RNA of the 5th instar larvae of cotton bollworm was extracted, and its quality was determined by electrophoresis and ultraviolet detection, and then stored for future use. According to the HHR3 gene sequence (AF337637) registered in GenBank, a pair of specific primers were designed for PCR amplification of the target gene, among them, P1: 5'-ATGAACAACAACCAGTTCCACG-3', P2: 5'- TTAACCGTGGGTGTAGTCCAGG-3'. Using the pair of primers and the total RNA of the cotton bollworm as a template to carry out RT-PCR, amplified to obtain a DNA fragment of about 1600bp. The RT-PCR product was purified and cloned into the pMD19-T vector, and the positive clone was picked for PCR identification, and a fragment with the expected size was obtained. Six positive clones with correct PCR identifica...

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Abstract

A molting regulation transcription factor HaHR3 gene of a cotton bollworm is an important gene related to the growth and development of the cotton bollworm. According to the invention, by cloning the HaHR3 gene of the cotton bollworm and selecting partial fragments of the HaHR3 gene, an RNAi intermediate vector pUCCRNAi-HaHR3sa and plant expression vector pCAM-RNAi-HaHR3 are constructed to cultivate a transgenic plant expressing HaHR3 gene dsRNA. After the cotton bollworm is fed by the transgenic plant, the weight, molting, pupating, eclosion and other various aspects of the cotton bollworm can be affected so as to successfully prevent and control the cotton bollworm. The method is a new strategy for biological prevention and control of the cotton bollworm, and has extensive application prospect in agricultural production.

Description

technical field [0001] The invention relates to a method for cultivating cotton bollworm-resistant plants, in particular to a method for cultivating cotton bollworm-resistant transgenic plants using RNAi. Background technique [0002] Molt-regulating transcription factors are important functional proteins that widely exist in a variety of insects and regulate the expression of related genes during the larval molt process, and play a very important role in the regulation of insect molt. The molting regulatory transcription factor proteins belong to the nuclear receptor superfamily members. The proteins that have been cloned and identified mainly include DmHR3 of Drosophila Drosophila, BmHR3 of Bombyx mori, MHR3 of Manduca sexta, GmHR3 of Mellonella mellonella and HHR3 of cotton bollworm ( Zhao et al., Insect Mol Biol, 13(4):407-412.2004) etc. During the growth and development of insects, if the expression of molting-regulating transcription factors is inhibited, the insects ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N5/10A01H4/00A01H5/00
Inventor 曾洪梅邱德文杨秀芬郭立华袁京京徐大伟尹富仕张雨良熊叶辉
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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