蛹虫草 CmKexin 基因克隆和菌丝体中表达检测

摘要

为了探讨蛹虫草类枯草杆菌蛋白酶（Subtilisin-like protease）的表达特性，以真菌蛹虫草菌丝体为研究对象，通过 RT-PCR 获得蛹虫草 CmKexin 基因的 ORF 序列，并进行序列分析。应用 Northern 杂交和 Real-time PCR 方法，检测蓝光照射后蛹虫草菌丝体中 CmKexin 基因的转录情况。结果获得蛹虫草 CmKexin 基因的 ORF 序列。对翻译蛋白质产物 CmKexin 进行生物信息学分析，表明该蛋白质含1个属蛋白转化酶的肽酶 S8家族功能域（143～429）和1个前体蛋白转化酶 P 功能域（514～600），符合真菌类枯草杆菌蛋白酶的特征。蛹虫草菌丝体在黑暗预培养4 d 后再蓝光照射，Northern Blotting 和 Real-time PCR 检测均可得到相同的结果，即在持续的蓝光照射48～50 h 时，出现 CmKexin 基因的瞬时大量转录。而其他时间内均未检测到该基因的大量转录。试验结果将为蛹虫草类枯草杆菌蛋白酶的利用提供理论依据。%In order to study the characteristics of subtilisin-like protease expression in the mycelia of fungus Cordyceps militaris,CmKexin gene ORF sequences of C.militaris was got by RT-PCR,then CmKexin sequence was analysed.The Real-time PCR and Northern Blotting were used to the CmKexin gene expression analysis in the C.militaris mycelia in blue light irradiation of different time.Results showed the CmKexin gene ORF sequence was obtained from C.militaris mycelia.The bioinformatics analysis for the translation products of CmKexin gene showed that there were 1 peptidase S8 family domain of protein convertases (143 -429)and 1 proprotein convertase P-do-main(5 14 -600)in the protein.The two domains existed in CmKexin demonstrate the characteristics of subtilisin-like protease.Under the dark pre-culture in 96 h,a large amounts of transcript of CmKexin gene in the mycelia could be detected by Northern Blotting and Real-time PCR in the continuous blue light irradiation to 48 -50 hours. But the transcription of CmKexin gene wasn′t detected in the other time of C.militaris mycelia culture.These results would provide the basis for using the subtilisin-like protease in Cordyceps militaris.