[1]王彩凤,刘静雯.海洋球石藻病毒甾醇去饱和酶基因的克隆与表达[J].集美大学学报(自然版),2019,24(5):335-342.
 WANG Caifeng,LIU Jingwen.Cloning and Expression of Sterol Desaturase(SD)Gene from Emiliania huxleyi Virus[J].Journal of Jimei University,2019,24(5):335-342.
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海洋球石藻病毒甾醇去饱和酶基因的克隆与表达()
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《集美大学学报(自然版)》[ISSN:1007-7405/CN:35-1186/N]

卷:
第24卷
期数:
2019年第5期
页码:
335-342
栏目:
水产、食品与生物技术
出版日期:
2019-09-28

文章信息/Info

Title:
Cloning and Expression of Sterol Desaturase(SD)Gene from Emiliania huxleyi Virus
作者:
王彩凤12刘静雯12
(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 Caifeng12LIU Jingwen 12
(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)
关键词:
海洋球石藻病毒甾醇去饱和酶基因酿酒酵母克隆表达脂类薄层色谱
Keywords:
Emiliania huxleyi virus sterol desaturase gene Saccharomyces cerevisiaecloningexpressiontotal lipidTLC
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
更新日期/Last Update: 2019-11-04