Bacillus thuringiensis vegetative insecticidal protein Vip3AaBb as well as encoding gene and applications thereof

A technology of Bacillus chrysogenum and insecticidal protein, applied in application, insecticide, genetic engineering, etc., to achieve the effect of high insecticidal activity

Active Publication Date: 2014-11-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since most commercially used transgenic crops are insecticidal crystal proteins, with the expansion of the planting area of ​​these transgenic crops, the resistance of pests to a single insecticidal protein has become a serious problem

Method used

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  • Bacillus thuringiensis vegetative insecticidal protein Vip3AaBb as well as encoding gene and applications thereof
  • Bacillus thuringiensis vegetative insecticidal protein Vip3AaBb as well as encoding gene and applications thereof
  • Bacillus thuringiensis vegetative insecticidal protein Vip3AaBb as well as encoding gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Design and synthesis of chimeric genes

[0037] Based on the Vip3Bb1 gene and Vip3Aa1 gene, design the chimeric gene Vip3AaBb, the specific steps are as follows:

[0038] (1) Obtain the 1353 base sequence encoding the N-terminal 451 amino acids of the Vip3Aa1 protein in the Vip3Aa1 gene (GenBank Accession No: L48811);

[0039] (2) Obtain the 1050 base sequence encoding the C-terminal 350 amino acids of the Vip3Bb1 protein in the Vip3Bb1 gene (GenBank Accession No: DD319826);

[0040] (3) The two base sequences obtained in the chimeric steps (1) and (2) are obtained to obtain a preliminary modified original base sequence, as shown in SEQ ID No.3, the G+C content of the sequence is only 30.80 %;

[0041] (4) Under the premise of not changing the amino acid sequence, the typical AT-rich sequence and commonly used restriction endonuclease sites existing in the original base sequence that cause instability of plant gene transcripts are replaced with monads The l...

Embodiment 2

[0043] Example 2 Expression of chimeric genes

[0044] Utilize the Vip3AaBb chimeric gene expression that embodiment 1 obtains to express Bacillus pluginus vegetative phase insecticidal protein Vip3AaBb, specifically include:

[0045] Construct the Vip3AaBb chimeric gene into the Escherichia coli plasmid expression vector pET28a(+), and transform the Escherichia coli expression host BL21(DE3); inoculate a single colony in 5 ml of LB medium, culture at 37°C overnight, and then press 1:100 Ratio was diluted and cultivated until OD600 was 0.4-0.6, and then IPTG with a final concentration of 1mM was added to induce expression, and the induction time was 4-6 hours; the bacteria were collected by centrifugation, resuspended by adding 20mL sterile water, and frozen and thawed repeatedly in liquid nitrogen for 6 hours. times, centrifuge to remove the bacteria, and obtain the supernatant.

[0046] The amino acid sequence of the Vip3AaBb protein is shown in SEQ ID No.1. The amino acid...

Embodiment 3

[0047] Example 3 The insecticidal activity of Vip3AaBb protein

[0048] The supernatant obtained in Example 2 was fed to the Lepidoptera insect Spodoptera litura, and the fermentation supernatant of Escherichia coli transformed with pET28a (+) empty vector was used as a contrast to detect the killing effect of the Vip3AaBb protein on Spodoptera litura. Insect activity, the results are shown in Table 1.

[0049] Table 1 Comparison of insecticidal activity of three kinds of samples to Spodoptera litura

[0050]

[0051] It can be seen from Table 1 that the Vip3AaBb protein has significant insecticidal activity against Spodoptera litura. The average mortality of Spodoptera litura after feeding for 24 hours was 86.7%, and the average mortality of Spodoptera litura after feeding for 48 hours was 100%. Spodoptera litura fed the other two samples had lower mortality rates.

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Abstract

The invention discloses a bacillus thuringiensis vegetative insecticidal protein Vip3AaBb and a coding gene thereof and application of the protein and the coding gene. The amino acid sequence of the bacillus thuringiensis vegetative insecticidal protein is represented by SEQ ID No. 1, and the base sequence of the coding gene is represented by SEQ ID No. 2. The application comprises application of the bacillus thuringiensis vegetative insecticidal protein in preparation of a pesticide and application of the coding gene in cultivation of a pest-resistant transgenic plant. Compared with the prior art, the invention has the following advantages: a Vip3Bb1 gene and a Vip3Aa1 gene are used as models for designing and synthesis of a Vip3AaBb chimeric gene, and the Vip3AaBb protein obtained through expression of the Vip3AaBb chimeric gene has high insecticidal activity for lepidopteron; and the Vip3AaBb chimeric gene can be highly expressed in cells of plants like cotton, corn, rape and soybean and is applicable to cultivation of pest-resistant transgenic plants.

Description

technical field [0001] The invention relates to the technical field of biological control, in particular to the vegetative stage insecticidal protein Vip3AaBb of Bacillus thuringiensis and its coding gene and application. Background technique [0002] Crop diseases and insect pests are one of the main agricultural disasters in our country. It has the characteristics of various types, great impact, and frequent outbreaks. The scope and severity of its occurrence and severity often cause heavy losses to our national economy, especially agricultural production. According to statistics, my country loses nearly 50 billion catties of grain and 18 million tons of various economic crops every year due to pests and diseases. In the past, chemical pesticides were widely used to control various pests of crops. However, on the one hand, due to the long-term and large-scale use of pesticides, some insects have developed certain drug resistance or resistance, resulting in an increase in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/325C12N15/32C12N15/82A01H5/00A01N63/02A01P7/04
CPCA01N47/44C07K14/325C12N15/8286Y02A40/146
Inventor 潘刚程方民唐贵香
Owner ZHEJIANG UNIV
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