牛樟芝中一个新型还原型聚酮合酶基因的克隆及表达分析

Cloning and expression analysis of a new reducing polyketide synthase gene in Antrodia camphorata

为了研究牛樟芝中PKS基因与化合物之间的关系,该研究通过对牛樟芝基因组分析获得牛樟芝聚酮合酶基因,以此序列为模板设计含有起始密码子和终止密码子的特异引物并以牛樟芝c DNA为模板克隆获得一个高度还原型PKS(HR-PKS)基因全长,命名为AcPKS2;对AcPKS2基因进行生物信息学分析,并比较该基因在不同培养基上的表达量。结果表明:AcPKS2全长7 842 bp,有24个内含子,其外显子共编码2 613个氨基酸,该蛋白的相对分子质量为293.5 kDa,理论等电点pI为5.78。用CDD分析其结构域显示,该基因属于HR-PKS,其结构域组织排列为KS-AT-DH-MT-ER-KR-ACP-TE,8个结构域其活性位点分别为β-酮基合成酶(DTACSSSL)、酰基转移酶(GHSIGETA)、脱水酶(RNDGSTSPL)、甲基转移酶(SFDIITAFDV)、烯酰还原酶(HAGVSSPAA)、酮基还原酶(GSPGQANYTAA)、酰基转移酶(YGLDSLTSVRL)、硫酯酶(KQPNGPY)。系统发育树显示AcPKS2与其他化合物未知的HR-PKS蛋白聚为一支,结构域和系统进化树分析显示该基因可能编码一种新的含TE结构域高度还原型聚酮合酶;表达分析结果显示葡萄糖和果糖能够诱导该基因的表达。

We isolated the polyketide synthase gene with anlyzing the genome of Antrodia camphorata,designed special primers including initial coden and stop coden according to the DNA sequence of this gene.Then,we cloned the full length of a PKS gene using the cDNA of A.camphorata,it is a part of HR-PKS and named as AcPKS2.We used bioinformatic methods to analyze AcPKS2 gene and its proteinic sequence,and compared the expression level of AcPKS2 gene culturing in different media.The results showed that the gene AcPKS2 had 7 842 bp including 24 introns,all the exons encoded 2 613 amino acids;and its relative molecular weight was 293.5 kDa,the theoretical isoelectric point(pI)was 5.78.The result of conserved domain database(CDD)analysis revealed that the organization domain of AcPKS2 was KS-AT-DH-MT-ER-KR-ACP-TE,the active sites of eight domains wereβ-ketosynthase(DTACSSSL),acyltransferase(GHSIGETA),dehydratase(RNDGSTSPL),methyl-transferase(SFDIITAFDV),enoyl reductase(HAGVSSPAA),ketoreductase(GSPGQANYTAA),acyl carier protein(YGLDSLTSVRL),thioesterase(KQPNGPY),respectively.Phylogenetic analysis clarified that AcPKS2 and another HR-PKS that their products had not identified clustered together,the domain and phylogenetic tree analysis could anthenticated that AcPKS2 gene encoded a new HR-PKS that includes TE domain.The gene expression analysis showed glucose and fructose could improve the ability of AcPKS2 gene expression.