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Bt protein Cry1Hc1 and coding gene and application thereof

A technology that encodes genes and bt proteins, applied in applications, genetic engineering, plant genetic improvement, etc., to achieve the effects of reducing environmental pollution, improving insect resistance, and reducing usage

Active Publication Date: 2016-03-02
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, studies have proved that Bti has not yet found resistance problems in the use of field, but the problem of mosquito resistance to it has been confirmed in the laboratory, and this situation may also appear in field (GeorghiouGP, 1997. Applied and Environmental Microbiology, 63 :1095-1101.)

Method used

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  • Bt protein Cry1Hc1 and coding gene and application thereof
  • Bt protein Cry1Hc1 and coding gene and application thereof
  • Bt protein Cry1Hc1 and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Cry1Hc1 Gene cloning

[0033] The present invention is a Bacillus thuringiensis isolated from the soil in Chengdu, Sichuan Province ( Bacillusthuringiensis ) The new strain BN23-5, which has been in the General Microbiology Center of the China Microbial Culture Collection Management Committee on July 14, 2014 (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip code 100101) preserved, classified as Bacillus thuringiensis ( Bacillusthuringiensis ), the deposit number is CGMCCNo.9448.

[0034] This example was cloned by the following method Cry1Hc1 The full-length sequence of the gene.

[0035] Use genomic DNA purification kit (purchased from Cybersun) to extract the total DNA of strain BN23-5 as amplification Cry1Hc1 Gene template, design primer sequence is as follows:

[0036] P1: 5’-ATGGAGAATAAAAATCAACAC-3;

[0037] P2: 5’-CTATTCCTCCATAAGGAG-3’

[0038] 25μl PCR reaction system:

[0039] 10...

Embodiment 2

[0048] Example 2 Cry1Hc1 Protein acquisition

[0049] according to Cry1Hc1 Design and synthesis of a pair of specific primers 1HTF: 5'-GCC GGATCC ATGGAGAATAAAAATCAACAC-3', 1HTR:5'-CCC CTCGAG CTATTCCTCCATAAGGAG-3', 5'end primers underlined bases are respectively BamHI with XHoI Restriction sites. Use the total DNA of BN23-5 as a template for amplification, and the digested product is ligated with the vector pET-28a(+) after the same double digestion, and transformed E.coli After extracting DH5α competent cells, the plasmids were digested and electrophoresed to verify that the size of the inserted fragment was in line with the expected target fragment ( figure 2 ), then transferred to the recipient bacteria E.coli .BL21(DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd.). The recombinant plasmid was named pET-1H, and the recombinant containing the recombinant plasmid was named E.coli .BL21(2L). The positive transformants were cultured in LB medium at 200r / min...

Embodiment 3

[0051] Example 3 Cry1Hc1 Protein insecticidal activity determination

[0052] Example 2 is obtained Cry1Hc1 The protein was tested for its insecticidal activity against Plutella xylostella and Corn Borer. Bioassay of Plutella xylostella: Will Cry1Hc1 The protein is formulated into 6 different concentration gradients of 200, 100, 50, 25, 12.5, 1.25ug / mL, etc.; select the moderately old and tender cabbage leaves, wash and dry; irradiate under UV light for 15min, and cut into 2×2cm 2 Size, put it in different concentrations of bacteria liquid, soak for 5min; take out the excess liquid, put it in a sterilized petri dish to dry, E.coli. BL21 (DE3) was used as a negative control, and clean water was a blank control. Each petri dish was placed with 4 leaves; 20 healthy 2-3 instar diamondback moths were selected; each treatment was repeated 3 times, placed in a room, and the larvae death was investigated after 3 days Happening. Bioassay of corn borer: will Cry1Hc1 The protein is for...

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Abstract

The invention provides a Bt protein Cry1Hc1 and a coding gene thereof. The protein is of an amino acid sequence shown in the SEQIDNo.2 or of an amino acid sequence obtained by replacing, missing and / or adding one or more amino acids of the amino acid sequence shown in the SEQIDNo.2, wherein the amino acid sequence is the same as the amino acid sequence shown in the SEQIDNO.2 in activity. The protein can be used for preparing a Bt pesticide, the gene for coding the protein can convert cotton, corns, rice, vegetables and other crops, and the corresponding insect resistant activity is achieved, so that the pesticide using amount is lowered, environment pollution is reduced, and the important economic value and application prospects are achieved.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a Bt protein and its coding gene and application. Background technique [0002] In the process of human production, insect pests are an important factor that causes losses in agricultural production and affects human health. According to FAO statistics, the annual economic loss caused by insect pests in agricultural production around the world is as high as 14%, disease loss is 12%, and weed loss is 11%. The loss amounted to US$126 billion, equivalent to half of China's total agricultural output value and more than four times that of the UK. In order to reduce these losses, for many years, crop pests and mosquitoes have been generally controlled by chemical control methods. However, due to the long-term and large-scale use of chemical pesticides, the environment has been polluted. The amount of pesticide residues in agricultural and sideline products has increased, which is vital to huma...

Claims

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Application Information

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IPC IPC(8): C07K14/325C12N15/32C12N15/82A01H5/00A01N47/44A01P7/04
Inventor 郑爱萍余宗兰陈磊王娜李巧李平王玲霞刘怀年李双成朱军邓其明王世全
Owner SICHUAN AGRI UNIV
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