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Bt protein Cyt2-like and gene and application thereof

A bt protein and gene technology is applied in the fields of application, genetic engineering, plant genetic improvement, etc., to achieve the effect of reducing usage, reducing cost, and good insecticidal activity

Active Publication Date: 2019-07-30
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, studies have shown that resistance to Bti has not been found in field use, but mosquito resistance to it has been continuously confirmed in the laboratory, and this situation may also appear in field

Method used

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  • Bt protein Cyt2-like and gene and application thereof
  • Bt protein Cyt2-like and gene and application thereof
  • Bt protein Cyt2-like and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Cloning of embodiment 1 Cyt2-like gene

[0032] The new bacterial strain CS72-2 of Bacillus thuringiensis isolated from the soil in the plateau area of ​​Guizhou Province in the present invention is classified as Bacillus thuringiensis CS72-2 (the preservation number of the bacterial strain is: CCTCCNO: M 2018930; its preservation time As: December 27, 2018; the preservation address is: China Typical Culture Collection Center of Wuhan University, Bayi Road, Hongshan District, Wuhan City, Hubei Province), through the toxicity test of CS72-2, it was shown that CS72-2 is not harmful to Lepidoptera Pests, etc. have extremely high toxicity.

[0033] Then design a pair of specific primers according to the conserved sequence of the Cyt2-like gene, and amplify its genomic DNA. The results show that there is a Cyt2-like gene in the bacterial strain. Further design its full-length gene primers and clone the Cyt2-like gene. The nucleotide sequence is as follows: As shown in the seq...

Embodiment 2

[0045] Example 2 Obtaining of Cyt2-like protein

[0046] The complete open reading frame of the Cyt2-like gene was obtained by sequencing, and a pair of full-length amplification primers PET-Cyt2F / PET-Cyt2R containing restriction sites were designed according to the sequences at both ends of the open reading frame of the Cyt2-like gene, and the Bt strain CS72 -2 Genomic DNA was used as a template, PCR amplification was performed using FastPfu high-fidelity polymerase, and the digested product was connected with the vector pET-28a after the same double digestion, and transformed into E.coli DH5α competent cells, and its plasmid was digested After electrophoresis verified that the size of the inserted fragment conformed to the expected target fragment ( figure 2 ), and then transferred into the recipient bacteria E.coli.BL21 (DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd.).

[0047] Name the recombinant plasmid pET-Cyt2, culture the positive transformant in LB...

Embodiment 3

[0049] Embodiment 3 protein insecticidal activity assay

[0050] The Cyt2-like protein obtained in Example 2 was tested for its insecticidal activity against corn borer and cotton bollworm.

[0051] Bioassay of corn borer: Cyt2-like protein was formulated into 6 different concentration gradients of 400, 200, 100, 50, 25, 0.1 μg / mL, and the protein was added to the feed for raising corn borer and mixed, E.coli .BL21(DE3) was used as negative control, clear water was used as blank control, and then 20 2-3 instar corn borers were put into each treatment, and each treatment was repeated 3 times, and the results were counted after 7 days.

[0052] Bioassay of cotton bollworm: Cyt2-like protein was formulated into 6 different concentration gradients of 400μg / mL, 200μg / mL, 100μg / mL, 50μg / mL, 25μg / mL and 0.1μg / mL, and the protein was added to feed cotton bollworm Mixed in the feed, E.coli.BL21 (DE3) was used as a negative control, and clear water was used as a blank control, and then...

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Abstract

The invention discloses Bt protein Cyt2-like and a gene and application thereof. The protein has good insecticidal activity. When the protein is used for preparing transgenic plants, pests can be specifically killed, the use amount of pesticide is reduced, the cost is reduced, and environmental pollution is reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Bt protein Cyt2-like and 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 the statistics of FAO, the economic loss caused by insect pests in the world's agricultural production is as high as 14%, the loss of diseases is 12%, and the loss of weeds is 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 order to reduce these losses, for many years, chemical control methods have been widely used to control crop pests and mosquitoes. However, due to the long-term and large-scale use of chemical pesticides, the pollution to the environment has been caused, and the amount of pesticide residue...

Claims

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Application Information

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IPC IPC(8): C07K14/325C12N15/32C12N15/82A01H5/00A01H6/46
CPCC07K14/325C12N15/8286
Inventor 郑爱萍李平孙宏伟马丽邓其明王世全李双成朱军王玲霞刘怀年
Owner SICHUAN AGRI UNIV
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