茶卷叶蛾寄生真菌球孢白僵菌几丁质酶基因的克隆与序列分析

摘要

球孢白僵菌是最重要的虫生真菌之一,在农林害虫生防中发挥着重要的作用.本研究从茶卷叶蛾球孢白僵菌JYBb201-11菌株中克隆了几丁质酶Bbchit1基因(GenBank登录号:HQ435871).根据GenBank上昆虫病原真菌几丁质酶基因序列同源性设计引物,分别进行DNA-PCR和总RNA-RT-PCR反应,对得到的目的片段,回收纯化后通过PMD18-T载体转化到大肠杆菌DH5α中,获得几丁质酶基因序列的重组质粒PMD18-chit,并测序.结果表明,DNA-PCR和总RNA-RT-PCR获得的基因序列完全一样,都是一个完整ORF序列,含1 047 bp核酸序列,编码348个氨基酸,信号肽长度为22个氨基酸,成熟蛋白理论分子量约为36.78 kD,理论等电点为5.95.该蛋白序列中包含2个保守区域,即底物结合区域( SIGG)和几丁质的活性位点(DGIDIDIE),该蛋白可归为几丁质酶18族V类.氨基酸序列同源性分析表明,球孢白僵菌JYBb201-11菌株几丁质酶与球孢白僵菌Bb0062菌株(AAN41259)和NCIM1216菌株(ACF32998)几丁质酶chit1氮基酸序列同源性都达99.43％;与球孢白僵菌MTCC 2028菌株(ACZ28129)几丁质酶chit1氨基酸序列同源性达98.28％.%Beauveria bassiana is one of the most important entomopathogenic fungi. It plays a crucial role in biocontrol of the fanning and forestry pests. The chitinase gene, Bbchitl, was cloned from JYBb201-ll 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).