链霉菌MY0504纤溶酶YG4基因的克隆及生物信息学分析

Cloning and Bioinformatics Analysis of Fibrinolytic Enzyme YG4 Gene from Streptomyces sp.MY0504

本研究获得了海洋链霉菌MY0504纤溶酶YG4基因,并对其进行生物信息学分析。利用依据同源序列设计的兼并引物,对提取的链霉菌MY0504的基因组DNA进行PCR扩增,将得到的DNA片段连接到pMD18-T载体后转化感受态Trans10细胞,然后对阳性克隆进行测序,并对序列进行生物信息学分析。最终克隆了1083bp的海洋链霉菌MY0504纤溶酶YG4基因。将获得的YG4基因输入Gen Bank网站,进行检索比对,结果显示与丝氨酸蛋白酶基因碱基序列同源性100%。对16s rDNA序列分析结果表明,该菌株与达格斯地链霉菌、氢化链霉菌、嫩白黄链霉菌、浅紫链霉菌的亲缘关系较近;通过对YG4基因编码蛋白的一级结构、二级结构、亚细胞定位和三级结构的预测和分析,结果表明:该基因编码360个氨基酸,其编码产物为稳定的亲水蛋白;二级结构以α螺旋和β折叠为主,无信号肽和跨膜结构域,有40个磷酸化位点;高级结构以无规则卷曲为主。本研究结果为研究海洋链霉菌MY0504纤溶酶YG4基因的表达机制及目的蛋白表达水平的提高提供了重要信息。

In this study,the fibrinolytic enzyme YG4 gene from marine Streptomyces sp.MY0504 was obtained and analyzed by bioinformatics methods.The genomic DNA of the extracted Streptomyces MY0504 was amplified by PCR using the degenerate primers designed based on the homologous sequences.The obtained DNA fragments were ligated to the pMD18-T vectors and transformed into competent Trans10 cells.The positive clones were then sequenced,and the obtained sequences were analyzed bioinformatically.Finally,the fibrinolytic enzyme YG4 gene from marine Streptomyces strain MY0504 sized 1083 bp was cloned.The obtained YG4 gene was introduced into the GenBank website,and then an alignment search and comparison was performed.The result showed that the YG4 gene exhibited 100%homology with the base sequence of the serine protease gene.The results of the 16s rDNA sequence analysis showed that the strain was closely related to Streptomyces daghestanicus,Streptomyces hydrogenans,Streptomyces albidoflavus and Streptomyces violascens.The primary structure,secondary structure,subcellular localization and tertiary structure of the protein encoded by YG4 gene were predicted and analyzed.The analysis results showed that the gene encoded 360 amino acids,and the encoded product was a stable hydrophilic protein.The secondary structure was mainly composed of alpha helix and beta sheet without signal peptides and transmembrane domains but with 40 phosphorylation sites.The high-order structure was dominated by random coils.The results of this study provided important information for investigating the expression mechanism of the fibrinolytic enzyme YG4 gene from marine Streptomyces MY0504 and for improving the expression level of a target protein.