硬枝树花中非核糖体多肽合成酶NRPS基因的克隆与鉴定

Isolation and Identification of Nonribosomal Peptides Synthetases(NRPS) from Ramalina conduplicans

以硬枝树花(Ramalina conduplicans)地衣型真菌为研究材料,采用简并引物扩增获得腺苷酰化结构域(A结构域),并以其为模板,通过Gene walking的方法获得非核糖体多肽合成酶(NRPSs)基因的全长,并对Rc NRPS基因进行生物信息学分析、分子系统进化分析及RT-PCR分析。Rc NRPS基因的开放阅读框总长3 150 bp,编码1 049个氨基酸残基。结构域分析显示:硬枝树花的NRPS基因含有腺苷酰化结构域(A结构域)、巯基化结构域(T结构域)、缩合结构域(C结构域)。将硬枝树花的Rc NRPS蛋白序列与曲霉属34条NRPS蛋白构建系统进化树,结果显示其为铁载体。生物信息学分析表明:NRPSs为非分泌蛋白,定位于细胞质基质中。RT-PCR分析表明:酵母粉是Rc NRPS基因诱导表达所必需的,蔗糖可有效促进该基因的诱导表达。

Using the lichen forming-fungi ofRamalina conduplicans as the material, we have obtained the DNA sequences of the adenylation domain （A domain）, eventually gained the full length of the non-ribosomal peptides synthetases （NRPSs） by Gene walking method and used the A domain sequence as template. Then, we checked the NRPS gene in Ramalina eonduplicans through the bioinformatics analysis, phylogenetic tree and RT-PCR analysis. The opening flame reader has the length of 3 150 bp, and codes 1 049 amino acids. The conserved domain showed it included A domain, thiolation domain （T domain） and condensation domain （C domain）; phylogenetic tree decl- ared it was a kind of siderophores with its protein sequence and the other 34 NRPS phylogenetic tree of the Aspergillus; the bioinformatics stated the NRPSs is an unsecreted protein, located in the cytoplasmic matrix. RT-PCR showed that the malt yeast extract plays an important and necessary role in the inducible expression of ReNRPS gene, in the condition existing of the malt yeast extract, the sucrose could promote the induction of RcNRPS.