海洋球石藻病毒甾醇去饱和酶基因的克隆与表达-《集美大学学报（自然科学版）》

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Title:: Cloning and Expression of Sterol Desaturase(SD)Gene from Emiliania huxleyi Virus

作者:: 王彩凤1; 2; 刘静雯1; 2; (1.集美大学食品与生物工程学院, 福建厦门 361021 ;2.福建省食品微生物与酶工程重点实验室, 福建厦门 361021)

Author(s):: WANG Caifeng1; 2; LIU Jingwen 1; 2; (1.College of Food and Biological Engineering,Jimei University,Xiamen 361021,China2.Key Laboratory of Food Microorganism and Enzyme Engineering of Fujian Province,Xiamen 361021,China)

Keywords:: Emiliania huxleyi virus ; sterol desaturase gene; Saccharomyces cerevisiae; cloning; expression; total lipid; TLC

摘要:: 以海洋球石藻（Emiliania huxleyi）病毒EhV-99B1基因组为模板，用一对病毒甾醇去饱和酶（sterol desaturase，SD）基因的特异性引物进行PCR扩增，获得EhV-SD基因，将扩增产物克隆至pMD19-T载体后进行测序，并对该基因编码的蛋白质序列进行生物信息学分析。将测序正确的目的基因亚克隆入酿酒酵母表达载体pYES2/CT中，转化酿酒酵母缺陷型菌株YMR015C，并用D-半乳糖诱导表达，提取重组酵母和野生型酵母细胞总脂并进行薄层色谱（thin layer chromatography，TLC）分析。结果显示：EhV-SD基因全长为987 bp，编码328个氨基酸，该蛋白质的理论等电点为8.31，预测的蛋白质分子质量为37.83 ku，为疏水性、不稳定蛋白质，存在6个跨膜结构域，不存在信号肽；其二级结构中,α-螺旋含量最多。氨基酸序列比对结果显示，EhV-SD基因与宿主球石藻及金色藻属（Chrysochromulina sp.CCMP 291 ）亲缘关系较近。TLC结果表明，EhV-SD基因的表达导致酵母细胞极性较强的脂类明显减少，而极性较弱的脂质成分显著增加，表明EhV-SD具有一定的催化活性。

Abstract:: The genomic DNA of Emiliania huxleyi virus EhV-99B1 was used as the template for amplification of sterol desaturase SD gene by using PCR with a pair of specific primers.The amplified product was cloned into pMD19-T vector and then sequenced.The deduced protein sequence of this gene was further analyzed by bioinformatics.The target gene was subcloned into yeast pYES2/CT expression vector.The recombinant plasmid was transformed into SD gene defective Saccharomyces cerevisiae YMR015C and the target gene was induced to express by using D-galactose.The total lipids of recombinant and defective yeast cells were detected by thin layer chromatography (TLC).The results showed that EhV-SD gene was 987 bp,encoding a protein of 328 amino acids with deduced molecular mass of 37.83 ku and a theoretical isoelectric point (pI) of 8.31,respectively.It contained six transmembrane domains and there was no signal peptide.The α-helix is the most abundant secondary structure in protein.Phylogenetic analysis showed that EhV-SD gene was closely related to those of its host E.huxleyi and Chrysochromulina sp.Compared to the defective yeast cells,the content of high-polarity lipids were significantly decreased,while the content of low-polarity lipids were significantly increased in recombinant yeast cells,indicating that the EhV-SD had certain catalytic activity