牛樟芝鲨烯合酶基因的克隆与表达分析

The Cloning and Expression Analysis of Squalene Synthetase Gene(AcSQS) in Antrodia camphorata

为了解牛樟芝三萜类生物合成、调控机理,以多孔菌科真菌茯苓(Wolfiporia cocos,AFR13032.1)的SQS基因为模板对牛樟芝基因组数据进行本地Blast,分离获得牛樟芝鲨烯合酶(AcSQS)基因.以该序列为模板设计特异引物Ac SQSF0和Ac SQSR0,通过PCR扩增得到Ac SQS c DNA全长,并对其进行生物信息学分析,检测不同碳氮源添加物的培养基上该基因的表达水平.结果显示:克隆得到的基因为牛樟芝鲨烯合酶(Ac SQS),该基因含4个外显子,3个内含子,外显子拼接总长1 803 bp,编码600个氨基酸;其蛋白序列的1~17位为信号肽位点,具两段跨膜结构,可能定位于内质网;位于该蛋白189~204位的CHYVAGLVGEGLTRL和225~238位的MGLMLQKTNIIRDY的保守基序为鲨烯合酶识别位点,DTIEDD和D(Y/F)RED为FPP绑定位点;蛋白网络分析显示其位于三萜类化合物的代谢网络中;聚类分析显示Ac SQS蛋白和茯苓、硫磺多孔菌、拟蜡菌、灵芝、云芝等的SQS蛋白聚为一支;不同碳氮源添加物培养基上的表达分析显示:果糖诱导该基因表达的能力最强,而不同氮源添加物诱导该基因表达的能力无明显差异.

In order to understand the biosynthesis and regulation mechanism of the triterpene compounds in Antrodia camphorata,the SQS gene of Wolfiporia cocos（ AFR13032. 1） from Fomitopsidaceae fungi was used as template to isolate the Ac SQS gene from A. camphorata genome through the method of local Blast,we obaited the DNA sequence of squalene synthase gene（ Ac SQS） in A.camphorata. Then,we designed the special primers Ac SQSF0 and Ac SQSR0 following the Ac SQS sequence,and got the full length of Ac SQS c DNA by PCR amplification. Nextly, we analyzed Ac SQS gene by bioinformatics, and detected its expression level growing in these medium that adding different carbon or nitrogen source. The results showed that the gene we cloned is squalene synthase gene of A. camphorata（ Ac SQS）,which includes 4 extrons,3 introns and 5＇-untransla-tional region,its opening reading frame has 1 803 bp,encoding 600 amino acids. Its amino acids lying from first to seventeenth place is the signal peptide site,which has 2 transmembrane structure,could locate in the endoplasmic reticulum. The conserved proteinic sequence CHYVAGLVGEGLTRL locating in 189 ~ 204 of the domain and MGLMLQKTNIIRDY locating in 225 ~ 238 is the squelene synthetase characterized sites,DTIEDD and D（ Y/F） RED is the FPP binding sites.The results of Multiple Em for Motif Elicitation showed that this protein could exist in the metabolism network of triterpene. The phylogenetic analysis revealed A. camphorata, Laetiporus sulphureus,Obba rivulosa,Ganoderma lucidum,Trametes versicolor,Trametes pubescens,Phlebia centrifuga,Moniliophthora roreri clustered one clan. The expression analysis in different carbon or nitrogen source clarified the induction ability of fructose is the best,and different nitrogen source has no significant effect on the ability of gene induction expression.