Cloning of chitinase a gene (chiA) from serratia marcescens Bn10 and its expression in coleoptera-specific bacillus thuringiensis

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2005

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Okay, Sezer

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Chitinases have been shown to be potential agents for biological control of the plant diseases caused by various phytopathogenic fungi and insect pests, because fungal cell walls and insect exoskeletons contain chitin as a major structural component. Chitinase has also been found to increase the efficacy and potency of Bacillus thuringiensis crystal (Cry) proteins toxic to larvae of insect pests. The reason of this synergy is the presence of chitin in the structure of the outer membrane of larval midgut. In this study, the gene encoding chitinase A (chiA) from Serratia marcescens Bn10, a local isolate of Trabzon province was amplified by PCR and cloned into the E.coli/Bacillus shuttle vectors, pNW33N and pHT315. For the expression in B. thuringiensis, the promoter region of cry3Aa11 gene of B. thuringiensis Mm2 was placed at the upstream of chiA. The vectors carrying both chiA and promoter site of cry3Aa11 was first introduced into E. coli and then into Bacillus subtilis 168 which were used as intermediate hosts in this study. pHT315PC carying chiA was then introduced into Coleoptera-specific B. thuringiensis cells (strain 3023) and the specific chitinase activity of the recombinant B. thuringiensis was measured as 5056 U/min/mg which was 6.3 fold higher than that of the parental strain. The specific activity corresponded to about one third of that produced by S. marcescens Bn10. The chiA gene was next sequenced and characterized. The sequence was submitted to GeneBank (Accession No. DQ165083). Chitinase A of S. marcescens Bn10 was found to be a 563 residue protein with a calculated molecular mass of 60.9 kDa. The mean G+C content of the gene is 58.75%. The deduced amino acid sequence was 99.3ا91.5% identical to those of known chitinases from S. marcescens, Burkholderia cepacia and Enterobacter sp. It was found that the chitinase of S. marcescens Bn10 has six amino

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Microbiology.

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http://etd.lib.metu.edu.tr/upload/12606538/index.pdf
https://hdl.handle.net/11511/15322

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Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats

S. Okay, “Cloning of chitinase a gene (chiA) from serratia marcescens Bn10 and its expression in coleoptera-specific bacillus thuringiensis,” M.S. - Master of Science, Middle East Technical University, 2005.