Insect-fusion-resistant gene, coding protein, carrier and application thereof

A technology of fusion gene and encoded protein, which is applied to the anti-insect fusion gene and its application field, can solve the problems of limited insecticidal activity, narrow insecticidal spectrum, pest resistance, etc., achieves wide insecticidal spectrum, improved insecticidal activity, The effect of slowing down pest resistance

Inactive Publication Date: 2016-06-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A single insecticidal toxin often has a narrow insecticidal spectrum and limited insecticidal activity; at the same time, long

Method used

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  • Insect-fusion-resistant gene, coding protein, carrier and application thereof
  • Insect-fusion-resistant gene, coding protein, carrier and application thereof
  • Insect-fusion-resistant gene, coding protein, carrier and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, construction of Cry1Ab-Vip3A fusion gene

[0021] The nucleotide sequence of the anti-insect gene Cry1Ab artificially synthesized by Shanghai Sangong is SEQ ID NO: 4, the nucleotide sequence of Vip3A is SEQ ID NO: 5 and the nucleotide sequence of connecting peptide CL is SEQ ID NO: 6. The insect-resistant genes Cry1Ab and Vip3 were respectively cloned into the expression vector pET28a (Novagen, 69864-3, the United States) between the sites of the restriction endonucleases BamHI and SacI, and the resulting vectors were named pET-1Ab and pET-v3 respectively .

[0022] Cry1Ab-Vip3A construction process: The Cry1Ab fragment was obtained by digesting pET-1Ab with BamHI and XmaI, and the Vip3A fragment was obtained by digesting pET-v3 with XmaI and SacI. After the vector pET28a was digested with BamHI and SacI, it was connected with Cry1Ab and Vip3A to obtain the vector pET-1Ab-v3. The vector pET-1Ab-v3 was single-digested with XmaI and ligated with CL to obta...

Embodiment 2

[0023] Embodiment 2, the preparation of insecticidal protein

[0024] The vectors pET-1Ab, pET-v3, pET-1Ab-v3, and pET-1Ab-CL-v3 prepared by the method of Example 1 containing insect-resistant genes were introduced into BL21 (DE3) cell line (Escherichiacoli) respectively, and contained 50mg / Culture overnight at 37°C on LB solid medium with L kanamycin, and select single clones. Inoculate a single colony into 100 ml of LB culture medium, shake culture at 37°C to OD 600 =0.6, then IPTG (Isopropylβ-D-1-thiogalactopyranoside) was added to 0.5 mM, and culture was continued for 4 hours under the same conditions. The culture solution was centrifuged at 5000 g for 10 minutes to pellet E. coli cells, and then the supernatant was discarded to collect the pellet. Add 30 milliliters of 20mM Tris-HCl buffer solution to the precipitation, and ultrasonically pulverize to obtain a crushed mixed solution in which the recombinant protein accounts for more than 60% of the total protein conten...

Embodiment 3

[0025] Embodiment 3, the insect-resistant activity determination of the insect-resistant protein expressed in Escherichia coli

[0026] Each 200 microliters of the anti-insect protein obtained in Example 2 (that is, the broken mixture) was used alone or mixed on the surface of 1 square centimeter of insect artificial feed, and fed to newborn first-instar larvae for the determination of anti-insect activity. The preparation method of the negative control was the same as in Example 2, but the plasmid was the pET28a vector itself without any inserted DNA. After raising for 7 days, the insecticidal rate was counted, and the results are shown in Table 1:

[0027] Table 1. Insecticidal rate of Cry1Ab-Vip3A

[0028]

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Abstract

The invention discloses an insect-fusion-resistant gene, coding protein, a carrier and application thereof. The insect-fusion-resistant gene is formed by using a connecting peptide coding gene to connect a nucleotide sequence of coding BT insecticidal crystal protein Cry1Ab with coding BT trophophase insecticidal protein Vip3A, the connecting peptide amino acid sequence is shown as SEQ ID NO:1. Insecticidal activity of the insect-fusion-resistant gene is improved, and insecticidal spectrum is wider. The insect-fusion-resistant gene can kill main lepidopterous insects like armyworm, Spodoptera litura, Ostrinia furnacalis, cotton bollworm and Spodoptera exigua of paddy and maize. In addition, the insect-fusion-resistant gene can effectively slow down insect resistance.

Description

(1) Technical field [0001] The invention relates to an insect-resistant fusion gene and its application. (2) Background technology [0002] Pests cause an annual loss of about 8 billion US dollars to global agricultural production. The control of pests currently mainly relies on the use of chemical pesticides, but the residues of pesticides will have adverse effects on human health and the environment. The use of biotechnology to cultivate transgenic insect-resistant crops containing insect-resistant genes can greatly reduce the use of chemical pesticides and effectively protect crops from pests. At present, transgenic insect-resistant corn and cotton have been widely planted. [0003] The key technology for transgenic crops to resist insects is to obtain insecticidal proteins with excellent performance, and there are many kinds of insecticidal proteins. More commonly used are BT insecticidal crystal proteins, such as Cry1Ab, Cry1C, etc., which have been widely used in tran...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/82A01H5/00
CPCC07K14/325C07K14/32C07K2319/00C12N15/8205C12N15/8286
Inventor 许超沈志成张先文林朝阳
Owner ZHEJIANG UNIV
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