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

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 047bp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36.78kD,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氨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%。

Cloning and Sequence Analysis on A Chitinase Gene of Beauveria bassiana Isolated from Homona cof fearia

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).