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Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

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[Construction of insecticidal recombinant Bacillus thuringiensis using an integrative vector].

Chao-Yin Yue¹, Ming Sun, Shou-Wen Chen

¹Huazhong Agricultural University, College of Life Science and Technology, Key Laboratory of Agricultural Microbiology of Ministry of Education, Wuhan 430070, China. yz41@public.wh.hb.cn

Yi Chuan Xue Bao = Acta Genetica Sinica

|December 20, 2003

Summary

Researchers integrated the insecticidal cry1C gene into Bacillus thuringiensis chromosomes, enhancing its toxicity against Spodoptera exigua. This genetic modification shows promise for improved biological pest control strategies.

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Area of Science:

Molecular Biology
Entomology
Biotechnology

Background:

Bacillus thuringiensis (Bt) produces insecticidal crystal proteins.
The cry1C gene exhibits high toxicity against Spodoptera exigua.
Integrating insecticidal genes into Bt chromosomes can enhance efficacy.

Purpose of the Study:

To clone and integrate the cry1C gene into a wild-type Bt strain.
To assess the stability and expression of the integrated cry1C gene.
To evaluate the insecticidal activity of the engineered Bt strain.

Main Methods:

Cloning of the cry1C gene into the integrative vector pBMB-F7E.
Transformation of Bacillus thuringiensis strain YBT803-1 with the recombinant plasmid.
Selection and characterization of chromosomal integrants using heat treatment and Southern blotting.
Bioassays to determine insecticidal toxicity against Spodoptera exigua and Plutella xylostella.

Main Results:

Successfully obtained a transformant BMB803-A with the cry1C gene integrated into the chromosome at a frequency of 3.4 x 10(-5).
Southern blotting confirmed integration at different chromosomal sites.
The integrated cry1C gene was effectively expressed.
Recombinant strains BMB803-X and BMB803-Z showed enhanced toxicity against Spodoptera exigua compared to the wild-type strain.
Toxicity against Plutella xylostella remained similar to the wild-type strain.

Conclusions:

Chromosomal integration of the cry1C gene in Bacillus thuringiensis is stable and effective.
Engineered Bt strains exhibit increased toxicity against Spodoptera exigua.
This approach offers potential for developing novel biological control agents against specific insect pests.