蝉拟青霉类枯草杆菌蛋白酶基因的克隆及其序列和蛋白质分析  

Cloning of sublitisin-like protease gene cDNA from Isaria cicadae and characterization of its sequence and deduced protein

作　　者：陈官菊[1] 柴一秋[1] 厉晓腊[1] 方鸣[1] 刘又高[1] 金轶伟[1] CHEN Guanju;CHAI Yiqiu;LI Xiaola;FANG Ming;LIU Yougao;JIN Yiwei(Zhejiang Institute of Subtropical Crops,Wenzhou 325005,China)

机构地区：[1]浙江省亚热带作物研究所

出　　处：《浙江农业学报》2019年第10期1663-1670,共8页Acta Agriculturae Zhejiangensis

基　　金：浙江省自然科学基金(LY12C14004)

摘　　要：蝉拟青霉是一种常见的昆虫病原真菌,被广泛应用于生物防治。本研究根据GenBank中已登录的淡紫拟青霉、粉拟青霉、球孢白僵菌和金龟子绿僵菌的类枯草杆菌蛋白酶基因序列的同源性比较,以它们高度保守的核苷酸序列设计一对引物,采用RT-PCR和3′/5′-RACE相结合的方法,从蝉拟青霉中克隆出完整的类枯草杆菌蛋白酶(subtilisin-likeprotease)cDNA基因序列。cDNA全序列为2031bp,该序列5′-端和3′-端的非编码序列长度分别为170bp和262bp,开放阅读框为1599bp,编码532个氨基酸,信号肽长度为18个氨基酸,前肽长度为133个氨基酸。该基因编码的蛋白序列与虫草棒束孢(Isariafarinosa)、球孢白僵菌(Beauveriabassiana)、蛹虫草(Cordycepsmilitaris)和哈茨木霉(Trichodermaharzianum)类枯草杆菌蛋白酶有较高的同源性,同源性分别为88%、88%、89%和71%。成熟蛋白具有典型的丝氨酸蛋白酶活性位点,同时有5个半胱氨酸,4个潜在的N-糖基化位点,位于细胞的液泡中与分泌途径相关,二级结构中以α-螺旋和无规则卷曲为主。Isaria cicadae is one of the most common species of entomopathogenic fungi,and has been used as biocontrol agents against pests.In order to obtain a complete cDNA of sublitisin-like protease from I.cicadae,a pair of primers was designed using MegAign of DNAStar analysis software according to the high conserved nucleotide sequences in GenBank ,and RT-PCR,3′/5′-RACE PCR were used.The whole sequence of cloned cDNA was2 031 bp,and the results showed that it had 170 bp and262 bp on the 5′-terminal and3′-terminal respectively,encompassed an open reading frame (ORF) with1 599 bp encoding 532 amino acid (aa) residues,including18-aa signal peptide,133-aa propeptide.Alignments with the deduced amino acid of proteins in the species of Isaria farinose, Beauveria bassiana,Cordyceps militaris and Trichoderma harzianum showed a homology of 88%,88%,89% and 71%, respectively.The sublitisin-like protease of I.cicadae was located in the vacuoles of cells associated with the secretory pathway,with 5 cycteines and 4 putative N-glycosylation sites. The secondary structure of the protein was mainly the α-helix and randon coil.

关 键 词：虫生真菌 蝉拟青霉 类枯草杆菌蛋白酶基因 克隆 

分 类 号：S476.12[农业科学—农业昆虫与害虫防治]