The Journal of General and Applied Microbiology
Online ISSN : 1349-8037
Print ISSN : 0022-1260
ISSN-L : 0022-1260
Cloning of a chitinase gene into Bacillus thuringiensis subsp. aizawai for enhanced insecticidal activity
Srisurang TantimavanichSomsak PantuwatanaAmaret BhumiratanaWatanalai Panbangred
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1997 Volume 43 Issue 6 Pages 341-347

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Abstract

Chitinase from a high producing strain (TP-1) of Bacillus licheniformis was used with B. thuringiensis subsp. aizawai (B.t.a.) in a combined larvicidal assay against the pest, Spodoptera exigua. With 10mU of this chitinase, the LD50 of B.t.a. was reduced by 7.6, 13.8 and 15 times on days 3, 5 and 7, respectively when compared to use of B.t.a. alone. In addition, a combination of chitinase (10mU) and B.t.a. at a sub-lethal dose retarded growth and development of S. exigua. In preparation for transformation of B.t.a., the TP-1 chitinase gene was cloned in E. coli DH5α and sequenced to reveal a single open reading frame of 1, 815bp. This open reading frame encoded for a protein of 604 amino acids and a characteristic signal peptide sequence of 35 amino acids. The gene was subsequently introduced into B.t.a. where it was expressed constitutively. The transformed strain showed slightly improved activity against S. exigua when compared to the non-transformed strain. This was probably due to the low chitinase activity (15mU/ml) of the transformant, which might be improved by further gene manipulation to overexpress enzyme production.

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