Screening of Bacillus thuringiensis Isolates Recovered from Diverse Habitats in India for the Presence of cry1A-type Genes and Cloning of a cry1Ac33 Gene Toxic to Helicoverpa armigera (American Bollworm)
Rajendra K. Meena,
Gunda Gouthami Krishna Kumari,
Helicoverpa armigera (American bollworm) is a severe pest of many economically important crops such as cotton, pigeon pea, chickpea and tomato in the Indian subcontinent. Insecticidal cry genes from the bacterium Bacillus thuringiensis (Bt) have been used for developing transgenic crops. Transgenic cotton expressing cry1Ac gene has shown good level of protection from H. armigera. However, there is a threat of eventual development of resistance in insects upon large-scale cultivation of transgenic crops. Therefore, prospecting of Bt strains for new types of cry genes is desirable. This study was undertaken for screening of native Bt isolates for the presence of cry1Aa,b,c-type genes with the objective of cloning, sequence analysis, expression and evaluation of toxicity. Sixty three native Bt isolates recovered from different soil and grain samples from diverse agricultural and non-agricultural locations in India, along with 10 known Bt strains used as reference, were screened for the presence of full length cry1A-type genes by Polymerase Chain Recation (PCR) using a set of primer. The full length gene was obtained in the native Bt isolate SK-711 recovered from Red gram field, Lam, Guntur Andhra Pradesh and in 3 Bt strains. The gene from the native Bt isolate was cloned into an E. coli expression vector. The sequence of the cloned gene (GenBank accession No. GQ866913) was analyzed by comparison with previously known cry1A-type genes and was assigned the name cry1Ac33 by the Bacillus thuringiensis Nomenclature Committee. The gene was expressed in E. coli and evaluated for toxicity towards H. armigera. The cry1Ac33 gene, cloned from a native Bt isolate, has been found to be more toxic towards H. armigera than the holotype cry1Ac1 used as a control, based on LC50 toxicity analysis.
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