Bt (Bacillus thuringiensis) protein Cry70Ba1 as well as encoding gene and application thereof

A technology of bt protein and gene, applied in application, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2013-11-27
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a new Bt protein Cry70Ba1, its coding gene and its application to the problem that insects produce resistance to Bt and its insecticidal crystal protein

Method used

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  • Bt (Bacillus thuringiensis) protein Cry70Ba1 as well as encoding gene and application thereof
  • Bt (Bacillus thuringiensis) protein Cry70Ba1 as well as encoding gene and application thereof
  • Bt (Bacillus thuringiensis) protein Cry70Ba1 as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1c

[0024] Cloning of embodiment 1cry70Ba1 gene

[0025] The present invention is a new bacterial strain of Bacillus thuringiensis (Bacillus thuringiensis) isolated from the soil in the virgin forest area of ​​Muchuan, Sichuan Province. No. 3, Datun Road, Chaoyang District, City, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101) is preserved, and the classification is named Bacillus thuringiensis (Bacillus thuringiensis), and the preservation number is CGMCC No.2719.

[0026] In this example, the full-length sequence of the cry70Ba1 gene was cloned by the following method.

[0027] The total DNA of strain BtMC28 was extracted using a genomic DNA purification kit (purchased from Saibaisheng Company). The primer sequences were designed as follows:

[0028] P1: 5'-ATGACAAAGAAACACAAAAAAATAT-3'

[0029] P2: 5'-TTATTTTTTCGCACCGATAACTTTAAC-3'

[0030] PCR reaction system:

[0031]

[0032]

[0033] Thermal cycle reaction: pre-denaturation at 94°C for 5 min...

Embodiment 2

[0034] Example 2 Obtaining of Cry70Ba1 protein

[0035] According to the sequences at both ends of the open reading frame of the cry70Ba1 gene, a pair of specific primers were designed and synthesized:

[0036] cry70F: 5′ CGG GGTACC (Kpn I)ATGACAAAGAAACACAAAAAAA-3′

[0037] cry70R:5′GCC GGATCC (BamH I)TTATTTTTTCGCACCGATAACT-3'

[0038] Kpn I and BamH I restriction sites were introduced at the 5' and 3' ends, respectively. The total DNA of BtMC28 was used as a template for amplification. The reaction procedure and amplification conditions were the same as in Example 1. The amplified product was double-digested with Kpn I and BamH I, and the digested product was the same as the carrier pET- 32a(+) was connected, transformed into E.coli DH5α competent cells, and its plasmid was extracted and digested by electrophoresis to verify that the size of the inserted fragment was in line with the expected purpose ( figure 2 ), and then transferred into the recipient bacteria E.col...

Embodiment 3

[0040] Example 3 Cry70Ba1 Protein Insecticidal Activity Determination

[0041] The Cry70Ba1 protein obtained in Example 2 was tested for its insecticidal activity against Aedes mosquitoes. First, the Cry70Ba1 protein was formulated into 6 different concentration gradients of 0.1, 4, 8, 16, 32 and 64mg / mL, and then 20 Aedes larvae were put into each treatment, and each treatment was repeated 3 times. E.coli.BL21(DE3 ) as a negative control, clear water as a blank control; 12h after the statistical results, LC 50 Analyzed with SPSS10.0 software.

[0042] The result shows (table 1): expression product has certain insecticidal activity to Aedes mosquito, LC 50 It was 49.61μg / mL, while E.coli.BL21(DE3) and the blank control had no insecticidal activity against Aedes mosquitoes.

[0043] Table 1 Insecticidal activity of Cry70Ba1 against Aedes mosquitoes

[0044]

[0045] Those skilled in the art can operably link the cry70Ba1 gene disclosed in the present invention with an ex...

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Abstract

The invention provides a new Bt (Bacillus thuringiensis) protein Cry70Ba1 and an encoding gene thereof. The protein has the amino acid sequence shown in SEQ ID No.1 or the amino acid sequence which is formed by substituting, deleting and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID No.1 and has the same function; and the encoding gene of the protein has the nucleotide sequence shown in SEQ ID No.2. The Cry70Ba1 protein can be used for preparing Bt insecticide; and the encoding gene of the protein can be used for transforming cotton, corn, rice, vegetable and other crops, so that the crops have corresponding anti-insect activity, thus the pesticide consumption is reduced, the environmental pollution is reduced and the Bt protein Cry70Ba1 and the encoding gene have important economic value and application prospects.

Description

technical field [0001] The invention relates to a new Bt protein, in particular to a Bt protein Cry70Ba1, its coding gene and application. Background technique [0002] In the process of human production, insect pests are an important factor that causes agricultural production losses and affects human health. According to FAO statistics, the annual economic losses caused by insect pests in agricultural production worldwide are as high as 14%, disease losses reach 12%, and weed damage losses reach 11%. %. The loss was as high as 126 billion US dollars, equivalent to half of China's total agricultural output value, and more than four times that of the United Kingdom. In addition, mosquito-borne diseases occupy an important position in preventive medicine, among which mosquito-borne diseases such as dengue fever and yellow fever have strong transmissibility, wide prevalence, high incidence and great harm. According to WHO statistics, as many as 80 million people are infected ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/325C12N15/32C12N15/63C12N1/15C12N1/19C12N1/21A01H5/00A01N63/02A01P7/04
Inventor 郑爱萍李平关鹏朱军邓其明刘怀年
Owner SICHUAN AGRI UNIV
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