三角褐指藻△5-脂肪酸延长酶基因克隆、过表达载体构建及生物信息学分析

Cloning, Over-expression Vector Construction and Bioinformatics Analysis of △5-fatty Acid Elongase Gene of Phaeodactylum Tricornutum Bohlin

通过RT-PCR的方法克隆三角褐指藻的△5-脂肪酸延长酶基因的cDNA序列,利用双酶切技术构建重组表达载体,并对该基因进行相关的生物信息学分析。结果表明:三角褐指藻△5-脂肪酸延长酶基因的cDNA全长为834 bp,编码278个氨基酸,预测的等电点为9-13,理论分子量32348-91。△5-脂肪酸延长酶基因中存在1个内含子,该酶属于ELO系列延长酶,可能定位于内质网中,包含多个跨膜区域和保守区域。亲缘关系分析表明,三角褐指藻△5-脂肪酸延长酶与假微型海链藻的亲缘关系最近。试验还成功构建了三角褐指藻△5-脂肪酸延长酶基因过表达重组质粒pPha-T1-5e,为下一步三角褐指藻中相关油脂代谢基因的表达和功能验证奠定基础。

Through the method of RT-PCR,cDNA sequence of △5-fatty acid elongase gene of Phaeodactylum tricornutum Bohlin was cloned,and the double enzyme digestion technique was used to construct the recombinant expression vector,and then the related bioinformatics analysis of the gene was carried out.The results showed that the full-length of cDNA of △5-fatty acid elongase gene in Phaeodactylum tricornutum Bohlin was 834 bp,278 amino acids were encoded,the predicted isoelectric point was 9.13,and the theoretical molecular weight was 32348.91.There was an intron existed in △5-fatty acid elongase gene,and the enzyme belonged to ELO series elongase,which could be located in the endoplasmic reticulum and contained multiple transmembrance domains and conserved domains.The genetic relationship analysis showed that △5-fatty acid elongase of Phaeodactylum tricornutum Bohlin had the closest relationship with Thalassiosira pseudonana Hasle et Heimdal.The over-expression recombinant plasmid pPha-T1.5e of △5-fatty acid elongase gene in Phaeodactylum tricornutum Bohlin was also successfully constructed in the experiment,which laid a foundation for the expression and functional verification of related lipid metabolism genes in Phaeodactylum tricornutum Bohlin.