二酰基甘油转移酶对三角褐指藻油脂积累及相关环境因子响应的研究  

The response of diacylglycerol acyltransferase to lipid accumulation and related environmental factors in Phaeodacylum tricornutum

作　　者：凃丽[1] 于广欣 陈波[1] 曲芳兵[1] 冷鹂[1] 兰利琼[1] 卿人韦[1] 

机构地区：[1]四川大学生命科学学院,成都610064 [2]中海油新能源投资有限责任公司,北京100016

出　　处：《四川大学学报（自然科学版）》2012年第4期901-908,共8页Journal of Sichuan University(Natural Science Edition)

基　　金：国家高技术发展计划(863计划)(2007AA09Z427,2008AA10Z409);国家自然科学基金面上项目(40976092);横向项目(2010H0311);中央高校项目(2010SCU22006)

摘　　要：本文应用RT-PCR和RACE方法扩增出三角褐指藻二酰基甘油转移酶(Ptdgat)全长cDNA,其完整编码框(ORF)为1587 bp,编码528个氨基酸.基于克隆所得Ptdgat的ORF构建了反向互补RNA干扰载体,并用基因枪PDS-1000/He转化野生型三角褐指藻,筛选到了所需的阳性转基因藻.实时荧光定量PCR(qPCR)结果揭示:含Ptdgat RNA干扰结构的转基因藻的二酰基甘油转移酶的表达水平比野生型三角褐指藻该酶的表达水平有显著降低;野生型藻和转基因藻的油脂含量定量分析结果揭示:含RNAi干扰结构的转基因三角褐指藻油脂含量也明显低于野生型三角褐指藻.另外本文也揭示了适当浓度的外源培养因子铁、硅和脱落酸(ABA)提高了三角褐指藻Ptdgat基因的表达水平及其油脂含量.A full-length diacylglycerol acyltransferase cDNA was isolated from Phaeodactylurn tricornu- tum（Ptdgat） using RT-PCR and RACE methods. Sequence analysis indicated that this cDNA has an open reading frame of 1587bp, encoding 528 amino acids. A shuttle plasmid, named as pPha-Ptdgat RNAi, which contained a 460bp invert-repeat RNA construction based on the ORF of Ptdgat, was constructed according to the need for RNA interference. ＂/＇he transformants were obtained by gene gun, bi- olistic PDS-1000/He particle delivery system. Real-time PCR analysis revealed that there was a significantly lower expression level of Ptdgat in the transformants than in the wild diatom. And the analysis of lipid content showed there was a lower lipid content in the transformed algae than in the wild diatom. Furthermore, the Ptdgat expression level and the lipid content were increased when appropriate concen- trations of iron, silicon or ABA was in media for P. tricornutum, respectively.

关 键 词：三角褐指藻 二酰基甘油转移酶 RNAI 油脂积累 生物柴油 

分 类 号：Q786[生物学—分子生物学]