球孢白僵菌降解昆虫体壁蛋白酶基因 CDEP-1的克隆与序列分析

构建了球孢白僵菌不同诱导时间的混合cDNA文库。根据丝氨酸类蛋白酶的保守区域设计引物，以构建cDNA文库同样的mRNA为模板，采用RT-PCR法得到长度为594bp的片段BbP。序列测定表明BbP是球孢白僵菌类枯草杆菌蛋白酶Prl的一部分，以BbP为探针，从上述cDNA文库筛选得到长度为1557bp的克隆CDEP-1。CDEP-1含有一个1134bp的开放阅读框（ORF），编码377个氨基酸，分子量为38616、PI=8.302的蛋白酶前体。CEDP-1的核苷酸序列与蛋白酶K、金龟子绿僵菌Prl、球孢白僵菌Prl的同源性分别为：57.9%、54.7%、83.3%。根据cDNA序列扩增到CDEP-1的基因组序列，分析表明其中含有3个内含子。Southern杂交表明CDEP-1在球孢白僵菌是单拷贝。

Cloning and CharacteriZation of Cuticle Degrading Enzyme CDEP-1 from Beauveria bassiana

Beauveria bassiana extracellular subtilisin like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA library was constructed using mRNA from mycelia of Beauveria bassiana grown on cuticle/chitin cultures. A cDNA clone of the protease, designated CDEP 1, was isolated from cDNA library. CDEP 1 contained an 1?134bp ORF that predicted a protein of 377 amino acids with M r=38?616 and PI=8.302. The amino acid sequence of the gene shows 57.9%,83.3% and 54.7% identity to Metarhizium nisopliae Pr1, Beauveria bassiana Pr1 and proteinase K, respectively. Southern analysis indicated that CDEP 1 was present as singly copy in Beauveria bassiana.