CLONING OF A CHITINASE GENE INTO BACILLUS-THURINGIENSIS SUBSP AIZAWAIFOR ENHANCED INSECTICIDAL ACTIVITY

Chitinase from a high producing strain (TP-1) of Bacillus licheniformi s was used with B, thuringiensis subsp, aizawai (B.t.a.) in a combined larvicidal assay against the pest, Spodoptera exigua, With 10 mU of t his chitinase, the LD50 of B.t.a. was reduced by 7.6, 13.8 and 15 time s on days 3, 5 and 7, respectively when compared to use of B.t.a. alon e. In addition, a combination of chitinase (10 mU) and B.t.a. at a sub -lethal dose retarded growth and development of S. exigua. In preparat ion for transformation of B.t.a., the TP-1 chitinase gene was cloned i n E. coli DH5 alpha and sequenced to reveal a single open reading fram e of 1,815 bp, This open reading frame encoded for a protein of 604 am ino acids and a characteristic signal peptide sequence of 35 amino aci ds. The gene was subsequently introduced into B.t.a. where it was expr essed 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 (15 mU/ml) of th e transformant, which might be improved by further gene manipulation t o overexpress enzyme production.