Bt cryaAc22 gene with insecticidal activity for Lepidoptera insect and application thereof

A cry1ac22, gene technology, applied in the field of biological control, can solve problems such as the increase in the frequency of pest resistance, and achieve the effects of strong poisonous activity, reduced use, and reduced environmental protection

Inactive Publication Date: 2009-09-16
海南海德热带农业资源研究所有限公司
View PDF1 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The large-scale promotion and application of commercialized engineering bacteria and transgenic plants has greatly reduced the use of chemical pesticides, reduced environmental pollution and brought considerable economic and social benefits, but it has also brought new challenges. The resistance frequency of insect genes has shown an obvious upward trend, and is facing a huge risk of resistance

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Bt cryaAc22 gene with insecticidal activity for Lepidoptera insect and application thereof
  • Bt cryaAc22 gene with insecticidal activity for Lepidoptera insect and application thereof
  • Bt cryaAc22 gene with insecticidal activity for Lepidoptera insect and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Cry1Ac genotype analysis of strain WO15-1

[0066] 1) Total DNA extraction:

[0067] A single Bt colony was inoculated into 7mL LB liquid medium, cultivated overnight at 30℃, 220rpm, 1% transferred to a 250mL Erlenmeyer flask containing 50mL of LB medium, cultured at 30℃, 220rpm for 4 hours to OD600 =1.0~2.0; collect 10mL bacteria by centrifugation, wash the precipitate with 2mL JBuffer (0.1M Tris HCl (pH8.0), 0.1M EDTA (pH8.0), 0.15M Nacl); gently suspend the precipitate in 2mL JBuffer and add 80uL Freshly prepared lysozyme (50mg / mL), mix well and incubate at 37°C for 45min; add 15uLRNase (10mg / mL) and act at 50°C for 15min; then add 200μL SDS (20%) and treat at 70°C for 20min; cool slowly To 37℃, use equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) and chloroform: isoamyl alcohol (24:1) to extract once, then add an equal volume of isoamyl alcohol and mix well and set at -20℃ The supernatant was precipitated for 20 minutes and centrifuged at 12,000 rpm for 10 ...

Embodiment 2

[0072] cry1Ac22 gene cloning

[0073] 1) WO15 plasmid DNA extraction:

[0074] Centrifuge to collect 10 mL of bacteria. Add 150uL SI (10% sucrose, 50mM Tris·Cl (pH 8.0), 20mM EDTA (pH 8.0) Lysozyme 20mg / ml, ready when used) for every 10mL of bacterial liquid, mix well, and bathe in 37℃ water for 3h; add 1350uL SII (10mM Tris·Cl, 1mM EDTA, 0.085MNaOH, 1.1% SDS when used), mix gently, let stand for 10 minutes, then add 750uL SIII (5M KAc, adjust to pH 4.8 with glacial acetic acid), invert the centrifuge tube upside down Mix gently, ice bath for more than 4 hours; centrifuge at 12,000 rpm for 10 minutes, take the supernatant, add 30RNase (10 mg / mL), let it act at 45°C for 30 minutes, add 1 / 10 volume of 3M NaAC and 2 times volume of absolute ethanol, -20°C Shen 30min. Centrifuge at 12,000 rpm for 10 min, discard the supernatant, and rinse the pellet with 70% ethanol. The precipitate was dried in a vacuum dryer and dissolved in 100uL ultrapure water. Take 5uL plasmid DNA sample on 0.8%...

Embodiment 3

[0080] Construction of engineering bacteria and induced expression, isolation and purification of its expression products

[0081] 1) Construction and identification of pQE30-Cry1Ac22 prokaryotic expression vector

[0082] According to the sequence of Bacillus thuringiensis Cry1Ac22, the primers for amplification of Cry1Ac22 were designed and synthesized. The amplified products were subjected to agarose gel electrophoresis, and the gel recovered products were ligated with pMD18-T vector. After transformation, the recombinant plasmid was extracted and digested with BamH I and Sal I. Identification, the digested fragment is 3500bp (such as image 3 Shown), consistent with the expected size, named HIC22 (pMD18-T-Cry1Ac22). The positive clones were sequenced and compared in GenBank after sequencing. The sequence was exactly the same as the Cry1Ac genotype.

[0083] The pMD18-T-Cry1Ac22 plasmid obtained above was digested with BamHI and SalI, and the target gene was recovered with a ge...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention relates to an insecticidal crystalline gene crylAc22 which is separated and cloned from bacillus thuringiensis (Bt) wild strains W015-1 and has high efficient insecticidal activity for lepidoptera insect, and application thereof. The invention adopts a reverse PCR clone method for cloning CrylAc full length gene from Bt strains w015-1, an open reading frame (ORF) of the gene is 3534bp, protein consisting of 1178 amino acids is coded, the whole protein contains three endotoxin protein conserved domains, and is named CrylAc22 by international Bt insecticidal crystalline gene name committee, and GenBank registration sequence number is EU282379. By using engineering bacteria or purified expression protein, the invention has high-active poisoning effect for lepidoptera insect Plutella xylostella.

Description

Technical field [0001] The present invention belongs to the technical field of microbial genetic engineering, and also belongs to the technical field of biological control, and specifically relates to the cloning of insecticidal crystal protein gene from Bacillus thuringiensis, the construction of recombinant engineering bacteria, the induction of expression in host cells (engineered bacteria), and the goal Protein separation and purification and its application in the field of biological control. Background technique [0002] As we all know, Bacillus thuringiensis (hereinafter referred to as Bt) is an insect pathogenic microorganism, and its main insecticidal active substance is called insecticidal crystal protein (ICP), which can be divided into crystal protein (crystal protein, Cry protein) and extracellular soluble protein (cytolytic protein, Cyt protein) are two major categories, which are coded by cry gene family and cyt gene family, respectively. Since Schnepf et al. succe...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/32C12N15/63C12N1/21C07K14/325C12P19/34C12N15/70A01N63/02A01P7/04C12R1/19C12R1/07
Inventor 方宣钧张文飞谢柳刘卓明
Owner 海南海德热带农业资源研究所有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap