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Disinsection gene and purpose thereof

A technology of insecticidal genes and genes, which is applied in the fields of genetic engineering, plant genetic improvement, application, etc.

Inactive Publication Date: 2006-09-27
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But a variety of insects have now been found to develop resistance to crystalline insecticidal proteins

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1. Gene cloning and obtaining:

[0013] The steps of gene cloning are as follows:

[0014] 1. Isolation method of Bacillus thuringiensis:

[0015] Take 0.5 g of the soil sample and place it in about 40 ml of BPA (0.5% beef extract, 1% peptone, 3.4% sodium acetate, pH 7.2-7.4) medium, shake it well; then place it in a shaker at 30°C 4.0 hours, take out and put it in a water bath at 80℃ for 30 minutes, or in an incubator for 1.0 hour; take 0.5 ml and transfer to BP medium (0.3% beef extract, 0.5% peptone, 0.5% sodium chloride, 1.8% agar, penicillin G1000u / ml, polymyxin B3ppm, PH7.2-7.4), spread evenly, and incubate at 28°C for 24 hours.

[0016] 2. Screening of new genes:

[0017] Draw a line on the candidate strain on LB solid medium supplemented with 50 μg / ml ampicillin, pick a single clone, and transfer it to 5 ml LB liquid medium supplemented with 50 μg / ml ampicillin. Cultivation with shaking at 190r / min to the mid-log growth phase; centrifugation at 12000rpm for...

Embodiment 2

[0023] Example 2. Preparation of protein:

[0024] The expression vector pET28a-LG01 containing the above-mentioned genes was transformed into E. coli BL21star using a general standard method and a colony containing the plasmid pET28a-LG01 was obtained. Inoculate a single colony into 100 ml of LB bacterial culture solution, culture it with shaking at 37℃ to OD 600 =0.6, add IPTG to 0.5mM, and continue to incubate for 4 hours under the same conditions. The culture solution was centrifuged at 5000g for 10 minutes to pellet the E. coli cells, and then the supernatant was discarded to collect the pellet. Add 30 ml of 20mM Tris-HCl buffer to the precipitate, which can be used for the determination of insecticidal activity after ultrasonic pulverization.

Embodiment 3

[0025] Example 3. The protein expressed in Escherichia coli has a killing effect on various lepidopteran insects:

[0026] Mythimna separata

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PUM

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Abstract

The invention discloses an insecticidal gene wherein the nucleotide sequence of the gene is SEQ ID NO: 1. The invention also discloses the insecticidal protein coded by the insecticidal gene, the amino acid sequence of the protein is SEQ ID NO: 2. The insecticidal protein has rather high insecticidal activity to the main agricultural pest insects including Ostrinia furnacalis, beet armyworm and Pyralidae.

Description

Technical field [0001] The invention relates to an insecticidal gene and its use. Background technique [0002] Insecticidal toxin is a kind of protein with important application value. It can be used to modify insecticidal microbial pesticides, and it can also be used in genetically modified insect-resistant crops. At present, transgenic insect-resistant technology has been widely used commercially (James C. 2004 ISAAA Briefs No. 32. Ithaca, NY: ISAAA. 43 pp), providing a new low-cost technology for pest management. At present, the plant transgenic technology has matured, and the key to transgenic insect resistance is to obtain insecticidal proteins with excellent performance. [0003] There are many insecticidal proteins. Bt crystal proteins are more commonly used, such as Cry1Ab, Cry1C, Cry2, Cry3, etc. (Crickmore, N., Zeigler, DR, Feitelson, J., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J. & Dean, DH1998 Microbiol. Mol. Biol. Rev. 62, 807-813). The genetically modified in...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/32C12N15/82C07K14/325A01N37/44
Inventor 沈志成李春雨
Owner ZHEJIANG UNIV
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