三角褐指藻LPAAT基因在酵母中表达对脂肪酸的影响

Influence of expression of Phaeodactylum tricornutum LPAAT gene in yeast on oil content and fatty acid composition of TAG

从三角褐指藻(Phaeodactylum tricornutum)中克隆到1 632bp的溶血磷脂酸酰基转移酶(LPAAT)全长cDNA,其中包含的开放阅读框被命名为PtLPAAT。在毕赤酵母中异源表达PtLPAAT基因,诱导培养45h时随机选取的3个酵母转化子总脂肪酸含量分别提高了2.68%、11.14%、31.28%;诱导培养72h时同样的转化子总脂肪酸含量分别提高11.59%、18.72%、46.63%。用薄层层析法分离诱导培养48h后的酵母菌体的甘油三酯,并用气相色谱法测定其脂肪酸,结果显示:与非转基因对照菌株相比,其油酸提高了0.9倍、亚油酸提高了5倍。用荧光定量PCR检测发现PtLPAAT基因的表达量随三角褐指藻油脂不断积累逐渐升高。表明在酵母中表达PtLPAAT基因不仅能够明显地提高其脂肪酸含量,而且还能选择性结合不饱和脂肪酸到甘油三酯中。

A full-length cDNA encoding a putative lysophosphatidicacid acyltransferase was cloned from Phaeodactylum tricornutum.The 1 632bp open reading frame of the cDNA was designated PtLPAAT.Heterologous expression of PtLPAAT in yeast（Pichia pastoris） increased total fatty acid as 2.68%,11.14% and 31.28%,respectively from three randomly selected yeast transformants based on dry cell weight when inducted for 45h;and 11.59%,18.72% and 46.63% respectively,when inducted for 72h.Triacylglycerols（TAGs） were separated by thin layer chromatography（TLC） from total lipids in transgenic yeasts that were induced for 48h.The analysis of fatty acid composition of TAGs revealed that expression of PtLPAAT in yeast led to a 0.9-fold increase in oleic acid and 5-fold increase in linoleic acid when compared to control strains.Quantitative real-time PCR analysis of gene expression pattern revealed that PtLPAAT was gradually up-regulated during the course of TAG accumulation in P.tricornutum.Expression of PtLPAAT in yeast shows the potential to enhance oil content and selectively incorporate unsaturated fatty acids into TAGs.