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Xenorhabdusbovienii GroEL gene, protein and application thereof

A technology of insecticidal protein and protein, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as rising, increasing pest resistance, and limitation, and achieve broad application prospects, high toxicity, and high biological activity. Effect

Inactive Publication Date: 2013-12-18
LIAONING ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Since the current commercialized transgenic insect-resistant crops contain relatively limited types of insect-resistant genes, planting transgenic crops on a large scale will increase the risk of increased pest resistance

Method used

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  • Xenorhabdusbovienii GroEL gene, protein and application thereof
  • Xenorhabdusbovienii GroEL gene, protein and application thereof
  • Xenorhabdusbovienii GroEL gene, protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, bacterial strain XB1027 insecticidal activity assay

[0032] Inoculate the strain on LB solid medium and cultivate for 72 hours until the OD of the bacterial solution 600The value is 0.6, and the insecticidal activity of the strain XB1027 against Lepidoptera pests was determined by taking cotton bollworm larvae as an example. First, the surface smearing method was used to measure the biological activity of the bacterial strain XB1027. The prepared artificial feed was added to a 96-well culture plate with a continuous pipette, and 200 μl of bacterial solution was added to each well. After the bacterial liquid is air-dried, the worms are inoculated. Each bacterial solution (each treatment) was added to 6 wells (1 row on a 24-well plate), and clear water and Bt strains (100 million spores per milliliter) were set on the same plate as negative and positive controls. Pick up the newly hatched larvae of cotton bollworm, place the larvae directly in a 24-well p...

Embodiment 2

[0034] Example 2, in bacterial strain XB1027 XbGroEL gene cloning

[0035] A set of primers (P1 / P2) for amplifying the full length of the gene was designed, and the primer sequences are:

[0036] P1: 5'-CTGCTCGCCTTCAAAGTTT-3'

[0037] P2: 5'-CGGCAATGGTCGTATCCTT-3'

[0038] 1. XbGroEL Amplification of full-length gene sequences

[0039] Extract the total DNA of bacterial strain XB1027 as the template of amplification, carry out PCR amplification with pfuDNA polymerase, the result (see attached figure 2 ), the result showed that the amplified product was a single band of 1.6kb. The PCR amplification system is as follows:

[0040] PCR reaction system

[0041]

[0042]

[0043] The amplified fragment was purified and recovered and ligated with the vector pMD18-T, transformed into Escherichia coli DH5α competent cells, and ligated and transformed according to the following scheme.

[0044] 2. Connection and conversion

[0045] PCR product ligation reaction system...

Embodiment 3

[0062] Embodiment 3, the determination of gene expression and biological activity

[0063] 1. XbGroEL Construction of Gene Recombinant Expression Vector

[0064] At the 5' end of forward primer P1R and reverse primer P2R, respectively introduce Ned I and xho I recognition site, the underlined sequence in the primer sequence is the endonuclease site.

[0065] Forward primer: P1R 5'-GC CATATG GCAGCTAAAGACGTAAA-3'

[0066] Reverse primer: P2R 5'-GC CTCGAG GCAGCACTACATCATGCCGC-3'

[0067] It was synthesized by Beijing Sanbo Polygala Biotechnology Co., Ltd.

[0068] With P1R (introducing Ned I restriction site) and P2R (import xho I restriction site) primer, pT- XbGroEL The plasmid was used as a template, and PCR amplification obtained two ends containing 5'- Ned I and 3'- xho I endonuclease insertion site XbGroEL Gene. The PCR amplification system and reaction procedures are as follows:

[0069]

[0070] The PCR product was connected with pMD18-T...

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Abstract

The invention relates to Xenorhabdusbovienii GroEL gene, protein and application thereof, belonging to the field of agricultural biotechnology and biological control. The insecticidal protein related to the invention has an amino acid sequence as shown in SEQ NO.2 and a gene encoding the insecticidal protein, and the nucleotide sequence of the gene is shown in SEQ NO.1. The gene has very high toxicity on newly hatched larvae of cotton bollworm, so that the gene is used for being transformed into microorganisms and plants so that the microorganisms and the plants can express toxicity on related pests, and the generation of insecticide resistance of lepidoptera pests on genetically engineered strains and transgenic plants is overcome and delayed.

Description

[0001] technical field [0002] The present invention relates to cotton bollworm ( Helicoverpa armigera ) highly effective pathogenic bacteria nematophila GroEL Genes, proteins and their applications belong to the category of agricultural biological control and biotechnology. Background technique [0003] Agricultural insect pests are one of the important reasons for crop yield reduction, and reducing the loss of insect pests is an important way to increase the yield of food and feed crops. Cotton bollworm ( Helicoverpa armigera ) is a common insect pest, which is very serious and miscellaneous. It not only harms cotton, but also harms various crops such as wheat, corn, pepper, tomato and sunflower. Cotton bollworm mainly harms the cotton area and vegetable area of ​​North China, and also seriously harms areas such as South China and West China. Cotton bollworm is the largest pest of cotton, causing the natural loss rate of cotton to be about 50% all the year round. To...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C07K14/195C12P21/02A01H5/00A01N63/00A01N47/44A01P7/04
Inventor 薛仁峰张庆黄艳孟黎歌
Owner LIAONING ACAD OF AGRI SCI
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