Cotton bollworm calcium mucoprotein gene fragment hacad1 with insect disinfestation building action and uses thereof

A cadherin gene, cotton bollworm technology, applied in the field of agricultural microbiology, can solve the problems of resistance restricting the application and popularization of Bt insecticides and Bt transgenic plant products, threatening development, etc., so as to delay the problem of drug resistance and improve the Insecticidal activity, the effect of wide application value

Inactive Publication Date: 2009-07-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existence of resistance problems seriously restricts the application and promotion of Bt pesticides and Bt transgenic plant products, threatening the rapid development of the entire industry

Method used

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  • Cotton bollworm calcium mucoprotein gene fragment hacad1 with insect disinfestation building action and uses thereof
  • Cotton bollworm calcium mucoprotein gene fragment hacad1 with insect disinfestation building action and uses thereof
  • Cotton bollworm calcium mucoprotein gene fragment hacad1 with insect disinfestation building action and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Cloning of the cadherin-like cadherin gene fragment hacad1 of the cotton bollworm

[0028] The present invention uses cotton bollworm as the source of the cadherin gene fragment hacad1:

[0029] 1. Extraction and quality detection of total RNA in midgut of cotton bollworm

[0030] A few healthy cotton bollworms raised to the 5th instar were taken, their midguts were dissected, and total RNA was extracted from the midguts of cotton bollworms according to the "guanidine isothiocyanate-phenol-chloroform" (TRIzoL) extraction method (specific method reference: Fu Yuejun et al. 2003. An effective method for extracting total RNA from cotton bollworm. Biotechnology Bulletin 5: 40-42). The specific steps of total RNA extraction by TRIzoL method are as follows:

[0031] (1) Thoroughly clean the mortar with deionized water, add a certain amount of quartz sand, add an appropriate amount of alcohol, and ignite it. After the mortar is naturally cooled, pour liquid nitroge...

Embodiment 2

[0046] Example 2 Overexpression and Purification of Cotton Bollworm Cadherin Fragment HaCad1

[0047] 1. Construction of the prokaryotic overexpression vector of the cadherin fragment HaCad1 in cotton bollworm

[0048] The recombinant plasmid pUC18-hacad1 containing hacad1 obtained in Example 1 was digested with EcoRI and SalI, and the expression vector pGEX-6p-1 (purchased from Novagen) was subjected to the same double digestion. After the above-mentioned vectors and exogenous digestion products were separated by 0.8% agarose gel, the target fragment was excised and purified and recovered by a DNA gel recovery kit (purchased from Omega Biotechnology Company). Afterwards, the two were connected by T4 ligase (purchased from TaKaRa Company), thereby obtaining the recombinant expression plasmid pEMB1104 containing hacad1 (see image 3). The recombinant expression plasmid was transformed into Escherichia coli E.coli DH5α (purchased from Tiangen Company), and the plasmid was extr...

Embodiment 3

[0058] Example 3 Biological Test on the Synergistic Activity of Cotton Bollworm Cadherin Fragment HaCad1 to Bt Toxin Cry1Ac10

[0059] 1. Purification and quantitative analysis of insecticidal crystal protein Cry1Ac10

[0060] The Bacillus thuringiensis recombinant strain BMB871 carrying the insecticidal crystal protein gene cry1Ac10 (see the literature for the source of the strain: Wu Lan, Sun Ming, Yu Ziniu. 2000. The dissociation vector containing the cry1Ac10 gene was constructed by using the Bacillus thuringiensis transposon Tn4430. Acta Microbiology Sinica 40: 264-269.) strains in ICPM medium (ICPM medium formula: peptone 0.6%, glucose 0.5%, CaCO 3 0.1%, MgSO 4 0.05%, KH 2 PO4 0.05%, pH7.0-7.2) After cultivating for 48-52h, take the fermented liquid and stain according to conventional fuchsin (for the fusin staining method, see: edited by Shen Ping, "Microbiology", Higher Education Press, Beijing, 2000 ) to make lenses, observe with an optical microscope, after more...

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Abstract

The invention relates to the technical field of agriculture microorganism genetic engineering, in particular to separation and cloning of a Helicoverpa armigera cadherin gene fragment hacad1and encoded polypeptide fragment HaCad1. By separation, the cadherin gene fragment hacad1 of 735bp is obtained from the midintestine of the Helicoverpa armigera, an encoded product thereof is the polypeptide fragment HaCad1 which can be combined with Bt toxin CrylAc10. Biological experiments indicate the polypeptide prepared in the invention has very strong enhancing insecticidal activity on insecticidal crystal protein CrylAc10 of lepidoptera insects. The invention also includes preparation and an application for recombinant Escherichia coli EMB1104 expressing the Helicoverpa armigera cadherin fragment HaCad1. The recombinant Escherichia coli EMB1104 is preserved in CCTCC, the preservation number is CCTCC NO: M209010.

Description

technical field [0001] The invention belongs to the technical field of agricultural microbiology application. It specifically relates to the isolation and cloning of a cotton bollworm cadherin gene fragment with insecticidal synergistic effect and the application of its coded polypeptide fragment in microbial pesticides. The invention relates to the technical fields of microbial genetic engineering and microbial pesticides. Background technique [0002] Bacillus thuringiensis (Bt) is a Gram-positive rod-shaped bacterium that widely exists in various ecological environments. The biggest feature of this species is that it can form a dormant body of endogenous spores during its growth cycle, and can produce insecticidal crystal proteins (Insecticidal Crystal Proteins, ICPs) that are specifically toxic to many insects during the formation of spores. companion crystals. These insecticidal crystal proteins are effective against more than 500 insects in 10 orders of Insecta such...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N1/21C07K14/435A01H5/00A01N63/02A01P7/04C12R1/19
Inventor 孙明彭东海徐肖会阮丽芳喻子牛
Owner HUAZHONG AGRI UNIV
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