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Bacillus thuringiensis vegetative insecticidal protein Vip3AfAa and coding gene thereof, and their applications

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

Inactive 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 Vip3AfAa and coding gene thereof, and their applications
  • Bacillus thuringiensis vegetative insecticidal protein Vip3AfAa and coding gene thereof, and their applications
  • Bacillus thuringiensis vegetative insecticidal protein Vip3AfAa and coding gene thereof, and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Design and synthesis of chimeric genes

[0037] Based on the Vip3Af2 gene and the Vip3Aa1 gene, the chimeric gene Vip3AfAa was designed. The specific steps are as follows:

[0038] (1) Obtain the 1200-base sequence encoding the N-terminal 400 amino acids of the Vip3Af2 protein in the Vip3Af2 gene (GenBank Accession No: DD319822);

[0039] (2) Obtain the 1167 base sequence encoding the 389 amino acids at the C-terminal of the Vip3Aa1 protein in the Vip3Aa1 gene (GenBank Accession No: L48811);

[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 31.05 %;

[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 w...

Embodiment 2

[0043] Example 2 Expression of chimeric genes

[0044] Utilize the Vip3AfAa chimeric gene expression that embodiment 1 obtains to express Bacillus pluginus vegetative phase insecticidal protein Vip3AfAa, specifically include:

[0045] Construct the Vip3AfAa 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 to dilute the culture to OD 600 0.4-0.6, then add IPTG with a final concentration of 1mM to induce expression, and the induction time is 4-6 hours; collect the bacteria by centrifugation, add 20mL sterile water to resuspend, freeze and thaw repeatedly 6 times in liquid nitrogen, and centrifuge to remove the bacteria , to obtain the supernatant.

[0046] The amino acid sequence of Vip3AfAa protein is shown in SEQ ID No.1. The amino acid sequence of the Vip3AfAa protein was compared wit...

Embodiment 3

[0047] Example 3 The insecticidal activity of Vip3AaBb protein

[0048] The supernatant obtained in Example 2 was fed to Lepidoptera insect Spodoptera litura, and the fermentation supernatant of Escherichia coli transformed with pET28a (+) empty vector was used as contrast to detect the killing effect of Vip3AfAa protein on Spodoptera litura. Insect activity, the results are shown in Table 1.

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

[0050]

[0051] It can be seen from Table 1 that the Vip3AfAa 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 vegetative insecticidal protein Vip3AfAa for bacillus thuringiensis, coding genes of the vegetative insecticidal protein Vip3AfAa and application. An amino acid sequence of the vegetative insecticidal protein for the bacillus thuringiensis is shown as SEQ ID (sequence identifier) No.1, and a base sequence of the coding genes is shown as SEQ ID No.2. The vegetative insecticidal protein for the bacillus thuringiensis is applied to preparing insecticides, and the coding genes are applied to cultivating insect-resistant transgenic plants. Compared with the prior art, the vegetative insecticidal proteinVip3AfAa, the coding genes and the application have the advantages that chimeric genes Vip3AfAa are designed and synthesized on the basis of genes Vip3Af2 and genes Vip3Aa1, and the protein Vip3AfAa obtained by means of expressing the chimeric genes Vip3AfAa is high in lepidopterous insecticidal activity; the chimeric genes Vip3AfAa can be efficiently expressed in cells of plants such as cotton, sweet corn, oilseed rape and soybean and can be used for cultivating the insect-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 Vip3AfAa 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): C07K14/325C12N15/32C12N15/82A01H5/00A01N47/44A01P7/04
CPCA01N47/44C07K14/325C12N15/8286
Inventor 潘刚赵晨晨杨茜程方民
Owner ZHEJIANG UNIV
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