关键词: 球孢白僵菌, 茶卷叶蛾, 几丁质酶基因, 克隆, 序列分析

Abstract: Beauveria bassiana is one of the most important entomopathogenic fungi.It plays a crucial role in biocontrol of the farming and forestry pests.The chitinase gene,Bbchit1,was cloned from JYBb201-11 isolate of Beauveria bassiana,which is highly virulent to Homona coffearia.The expected DNA fragments were amplified by DNA-PCR,and the total RNA-RT-PCR with a pair of primers.The fragments were introduced into DH5α isolate of Escherichia coli by PMD18-T vector,and sequenced subsequently.The results from DNA-PCR and the total RNA-RT-PCR showed that the fragments were both 1047 bp.The gene consisted of an open reading frame with 1047 bp(GenBank accession NO.HQ435871),encoding 348 amino acids,which contains an N-terminal 22 amino acid residue displaying the characteristics of a signal peptide.The mature chitinase had a molecular mass of 36.78 kD and a calculated pI of 5.95.The protein sequence contained two conserved regions including a putative substrate binding site(SIGG)and catalytic domain(DGIDIDIE)of fungal chitinases.The chitinase belonged to the class V of family 18 of glycosyl hydrolases.The analysis showed that the deduced amino acid sequence was 99.43% homologous to that of B.bassiana strain Bb0062(AAN41259)and B.bassiana strain NCIM1216(ACF32998),and 98.28% to that of B.bassiana strain MTCC 2028(ACZ28129).

Key words: Beauveria bassiana, Homona coffearia, chitinase gene, cloning, sequence analysis