牛樟芝非核糖体多肽合成酶基因(AcNRPS)的克隆与鉴定

Isolation and Identification of Nonribosomal Peptides Synthetase( AcNRPS) from Antrodia camphorata

胶霉毒素属于真菌天然次生代谢产物epipolythiodioxopiperazine(ETP)家族,具有免疫抑制剂、抗真菌等多种生物活性,是由非核糖体多肽合成酶(NRPSs)催化合成。从牛樟芝(Antrodia camphorata)基因组中挖掘出非核糖体多肽合成酶基因(AcNRPS,NCBI登录号为KX430967),克隆获取其全长cDNA,并对其进行生物信息学分析和表达谱分析。结果显示AcNRPS基因cDNA全长6 687 bp;与其DNA序列比对发现AcNRPS基因含有12个内含子;其开放阅读框编码2 229个氨基酸残基,BLAST比对发现其含有2个A-C-T结构域,底物需2个氨基酸;系统发育树结果显示AcNRPS与其他合成产物为胶霉毒素的NRPS基因聚为一类,其可合成胶霉毒素类化合物;表达谱分析显示,以葡萄糖和土豆蛋白胨作为碳、氮源的培养基能够有效促进牛樟芝NRPS基因的表达。

Gliotoxin is a kind of fungal natural secondary metabolites,which belongs to the family of epipolythiodioxopiperazine（ ETP）,it has much bioactivity such as immunosuppressant,antifungal etc. The synthesis of gliotoxin could be catalyzed by NRPSs（ nonribosomal peptides synthetase）. In this paper the NRPS gene（ AcNRPS,NCBI ID：KX430967） of the NRPSs was tapped from the genome of Antrodia camphorata,acquired the full length of AcNRPS,and analyzed AcNRPS gene with bioinformatic methods and expression spectrum analysis. The results showed that the full length of AcNRPS cDNA has 6 687 bp including 12 introns by alignment the cDNA and genomic DNA of Antrodia camphorata,the opening frame reader of AcNRPS gene could encode 2 229 amino acids. NRPSs have two repeats of A-C-T domains by BLAST,and need 2 amino acids as substrates to synthetase the gliotoxin precursor. Phylogenetic analysis clarified that the Ac NRPS and the other NRPS gene that they produced the gliotoxin was clustered in a branch.The expression results revealed that the medium,having the glucose and potato peptone as carbon and nitrogen source,respectively,could exalt the production of NRPS gene,effectively.