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Synthetic Bt bivalent fusion protein with high virulence against lepidoptera, and coding gene thereof

A technology of artificial synthesis and protein, applied in the direction of hybrid peptides, microbial-based methods, biocides, etc., can solve problems such as unprecedented

Inactive Publication Date: 2013-05-01
HUAZHONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Two toxic proteins, Vip3A and ICP, have different action mechanisms in the midgut of insects, and they do not produce cross-resistance to insects. Therefore, feeding the two insecticidal proteins to pests at the same time can slow down the rate of resistance. Currently, Vip3A and Cry9Ca are used as insecticides. Fusion expression unprecedented

Method used

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  • Synthetic Bt bivalent fusion protein with high virulence against lepidoptera, and coding gene thereof
  • Synthetic Bt bivalent fusion protein with high virulence against lepidoptera, and coding gene thereof
  • Synthetic Bt bivalent fusion protein with high virulence against lepidoptera, and coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Vip83* of upland cotton codon bias; Cry9Ca* gene optimization

[0030] Optimization steps: use the codon preference query to use the online database Codon Usage Database (http: / / www.kazusa.or.jp / codon), enter Gossypium hirsutum in the QUERY Box for search with Latin name of organism dialog box, click submit, Select Gossypium hirsutum in the pop-up web page. Obtain the upland cotton preferred amino acid codon sequence list (see figure 2 ). from figure 2 From the analysis, it can be concluded that the GC content of the gene used by upland cotton is 45.83%, and the second and third codons prefer A or T.

[0031] Find the sequences of Vip83 (Accession No. AY044227) and Cry9Ca (Accession No. AX100534) from Genebank, according to the obtained from Codon Usage Database figure 2 The preferred codons are to partially replace the codons corresponding to the original sequences of Vip83 and Cry9Ca by using high-frequency codons used by plant genes, partially remove...

Embodiment 2

[0034] Example 2 Construction of prokaryotic expression plasmids pGEVC9C, pGEVip, pGEC9C.

[0035] Vip83 ＊ Amplification of the gene: according to the Vip83* sequence (see Figure 3A ) See, design amplification primers, add BamH I enzyme cutting site at primer 5 ' end, remove stop codon at 3 ' end, add enzyme cutting site EcoR I, this primer design sequence is as follows:

[0036] Forward primer (5'-Vip83-BamH I): 5'-ACC GGATCC The underlined part of ATGAACAAAACAAT-3' is the enzyme cutting site.

[0037] Reverse primer (3'-Vip83-EcoR I): 5'-AAG GAATTC The underlined part of CTTATCATCATC-3' is the enzyme cleavage site.

[0038] Using the pUC57-V83 plasmid as a template, Vip83* was amplified by PCR.

[0039] The PCR reaction system is as follows (total reaction volume 50 μl):

[0040]

[0041] PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min and 20 s; 30 cycles; extensio...

Embodiment 3

[0063] Example 3 Prokaryotic expression and bioassay of Vip83Cry9Ca* fusion gene in Escherichia coli.

[0064] The pGEVC9C plasmid, pGEVip plasmid, and pGEC9C plasmid were respectively transformed into E. coli expression host BL21. Verify the positive clones, activate the identified clones overnight, transfer to 1L LB liquid medium containing 100μg / ml Amp with a volume ratio of 1%, culture at 37°C for 2-3h, when the A600 reaches about 0.6 , adding the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5mM, and culturing at 22°C and 180rpm for 25h. Collect the thalline by centrifugation, then wash the thalline once with 1% Tris buffer (1% Tris, 50mM NaCl), and then wash it with 50ml 50mM NaNO 3 , add 2% mercaptoethanol solution to resuspend the cells. Then use a high-pressure disruptor (purchased from GEA Niro Soari company model: NSI0012K) to disrupt the cells, and collect the cell disruption solution.

[0065] The expression of fusion protein ...

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Abstract

The invention belongs to the technical field of genetic engineering, and specifically relates to a synthetic bacillus thuringiensis insecticidal gene with virulence against lepidoptera. The synthetic gene comprises a nucleotide sequence of a Vip83Cry9Ca<*> gene and an amino acid sequence coded by the gene. Escherichia coli DH5alpha / Bt. / 6p / pGEVC9C comprising the gene plasmid is collected at China Center for Type CultureCollection (CCTCC), and has a collection number of CCTCC NO: M2011363. As a result of biological experiments, the insecticidal fusion gene has virulence against lepidoptera, and can specifically kill agricultural pests such as diamondback moth and the like. The invention also discloses an application method of the fusion protein in the respect of transgenic insecticidal microbes.

Description

technical field [0001] The present invention relates to the field of microbial genetic engineering, in particular to an artificially synthesized Bt bivalent fusion protein with high toxicity to Lepidoptera and its coding gene, which includes Vip83* and Cry9Ca* genes and their composition and coding sequence. technical background [0002] Bacillus thuringiensis (Bacillus thuringiensis) is the most commonly used commercial insecticidal microorganism in front of the mouth. It expresses insecticidal crystal proteins and vegetative insecticidal proteins. Pests have specific poisoning effect (Yu Ziniu et al., 1996). [0003] At present, insecticidal crystal proteins (ICPs) are used the most in production and application. ICPs are mainly divided into two categories, Cry-like proteins and Cyt-like proteins. As of June 6, 2009, the number of ICPs genes cloned has increased to There are 449 Cry genes in 58 groups, and 27 Cyt genes in 2 groups (Crickmore et al., 1998). Its crystal s...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N1/21C07K19/00A01N63/02A01P7/04C12R1/19
Inventor 刘子铎董方林拥军史瑞平
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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