RNA干扰PEPC对三角褐指藻油脂含量的影响

The PEPC RNAi regulates lipid content in Phaeodactylum tricornutum

本文应用生物信息学和RT-PCR方法分别获得了三角褐指藻两个PEPC全长cDNA,分别称为：PtPEPC1和PtPEPC2,前者完整开放阅读框3030bp,编码1009个氨基酸,后者完整开放阅读框2634bp,编码877个氨基酸;基于克隆所得PtPEPC的ORF构建了反向互补RNA干扰载体pPha-PtPEPC1RNAi和pPha-PtPEPC2RNAi,并用轰击法（PDS-1000/He）转化野生型三角褐指藻,筛选鉴定到了所需的阳性转基因藻;实时定量RT-PCR结果揭示：含PtPEPC RNA干扰结构的转基因藻的PEPCase基因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高于野生型三角褐指藻,油脂增幅达2%～12%.

Two full-length PEPC cDNAs were isolated from Phaeodactylum tricornutum using bioinfor- matics and RT-PCR methods. They include 3030 bp （PtPEPC1）, encoding 1009 amino acids and 2634 bp （PtPEPC2）, encoding 877 amino acids, respectively. Two shuttle plasmid, named as pPha-Pt- PEPC1RNAi and pPha-PtPEPC2RNAi, separately, which contained an around 400bp invert-repeat RNA construction based on the ORF of PtPEPC, were constructed according to the need for RNA in- terference. The transformants were obtained by the particle delivery system （Biolistic PDS-1000/He）. Real-time PCR analysis revealed there was a significantly reduced expression of PtPEPC in the transfor- mants. An increased lipid content was examined in the transformed algae. These illustrated the PtPEPC RNAi influenced lipid accumulation of P. tricornutum.