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Pest-resistant fusion gene, encoded protein, carrier, and applications

A technology of fusion gene and encoded protein, applied in insect resistance fusion gene and its application field, can solve problems such as limited insecticidal activity, narrow insecticidal spectrum, and pest resistance, and achieve wide insecticidal spectrum, improved insecticidal activity, Slowing down the effects of pest resistance

Inactive Publication Date: 2018-02-23
HANGZHOU RUIFENG BIOTECH LIMITED
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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-term use of a single insecticidal protein in large quantities may also cause the emergence and development of pest resistance

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1, the construction of CAL / CCL fusion gene

[0020] The nucleotide sequence of the anti-insect gene Cry1Ab artificially synthesized by Shanghai Sangong is SEQ ID NO:1, the nucleotide sequence of Vip3A is SEQ ID NO:2, and the nucleotide sequence of Vip3C is SEQ ID NO:5. The insect-resistant genes Cry1Ab and Vip3A / Vip3C 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 obtained vectors were named pET-1Ab and pET respectively. -v3A, pET-v3C.

[0021] CAL construction process: The Cry1Ab fragment was obtained by digesting pET-1Ab with BamHI and XmaI, and the Vip3A fragment was obtained by digesting pET-v3A with XmaI and SacI. After the vector pET28a was digested with BamHI and SacI, it was ligated with Cry1Ab and Vip3A to obtain the vector pET-CAL (ie, pET-1Ab-v3A). This vector contains a fusion gene, CAL (shown in SEQ ID NO:3), which encode...

Embodiment 2

[0023] Embodiment 2, the preparation of insecticidal protein

[0024] The vectors pET-1Ab, pET-v3A, pET-v3C, pET-1Ab-v3A, and pET-1Ab-v3C prepared by the method of Example 1 containing insect-resistant genes were introduced into BL21 (DE3) cell line (Escherichia coli) respectively. Culture overnight at 37°C on LB solid medium containing 50 mg / L kanamycin, and select a single clone. 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 ml of 20mM Tris-HCl buffer solution to the precipitate, and ultrasonically pulverize to obtain a crushed mixed solution in which the recombinant protein accounts for more than 60% of the total protein, which is used...

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 and Cry1Ab-Vip3C

[0028] anti-insect protein

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PUM

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Abstract

The invention discloses a pest-resistant fusion gene, an encoded protein, a carrier, and applications. The pest-resistant fusion gene is composed via connection of a nucleotide sequence used for coding BT insecticidal crystal protein Cry1Ab with a nucleotide sequence used for coding BT vegetative insecticidal protein Vip3A or Vip3C. The encoded protein of the pest-resistant fusion gene is high ininsecticidal activity and wider in insecticidal spectrum, is capable of killing the main lepidoptera pests of rice and corn such as armyworm, prodenia litura, corn borer, cotton bollworm, and beet armyworm, and in addition, the encoded protein is capable of delaying generation of 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): C12N15/62C12N5/10C07K19/00A01N47/44A01P7/04
CPCA01N47/44C07K14/325C07K2319/00
Inventor 许超沈志成
Owner HANGZHOU RUIFENG BIOTECH LIMITED
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