过表达亚甲基四氢叶酸脱氢酶对高山被孢霉脂质合成的影响

Effects of MTHFD1 Overexpression on Lipid Synthesis in the Oleaginous Fungus,Mortierella alpina

高山被孢霉是一种富含多不饱和脂肪酸的丝状真菌,但其脂质过程中NADPH的来源还没有研究透彻。以高山被孢霉（尿嘧啶营养缺陷型）作为出发菌株,研究亚甲基四氢叶酸脱氢酶（MTHFD1）对高山被孢霉脂质合成的影响。首先构建了过表达载体pBIG2-ura5s-MTHFD1,采用根癌土壤杆菌介导转化真菌的方法,将二元表达载体转化进高山被孢霉CCFM501中,在筛选培养基SC-CS平板上进行筛选,进而得到稳定遗传MTHFD1基因的过表达菌株（MA-MTHFD1）;其次提取MA-MTHFD1菌株基因组进行PCR鉴定,并结合qPCR分析结果,表明MTHFD1基因成功在高山被孢霉中实现了过量表达;最后通过对MA-MTHFD1中的脂肪酸含量、NADPH含量及NADPH合成途径中相关基因转录水平进行分析,研究MTHFD1基因过表达对脂质合成的影响。实验结果表明,过表达MTHFD1基因可以提高高山被孢霉脂质合成能力。与原养型高山被孢霉相比,MA-MTHFD1菌株中脂肪酸含量提高了40.13%,NADPH的含量提高了26.45%,而且NADPH合成途径中其他相关基因苹果酸酶（ME）和异柠檬酸脱氢酶（IDH）的转录水平也发生了上调。这一系列研究结果表明,在高山被孢霉脂质合成还原力形成中,MTHFD1基因起到了关键作用。这为解析高山被孢霉中NADPH来源及深入研究脂质合成机制,从而对其胞内脂肪酸代谢通路进行分子水平上的改建提供了一定的理论依据。

Mortierella alpina is a filamentous fungus with a high polyunsaturated fatty acids content,but its sources of NADPH needed for lipid synthesis are not been studied completely. The binary vector pBIG2-ura5s-MTHFD1 was constructed for the transformation of the methylenetetrahydrofolate dehydrogenase（ MTHFD1） into the M. alpina（ uracil auxotrophic） strain by Agrobacterium tumefaciens-mediated transformation（ ATMT）. PCR analysis identified the presence of the MTHFD1 overexpression cassette in the genome,and q PCR analysis showed that the transcript levels of the MTHFD1 gene in MTHFD1 overexpression strain（ MA-MTHFD1） were significantly increased compared to controls. The results showed that MTHFD1 overexpression influenced lipid synthesis significantly. In comparsion to prototrophic M. alpina,the total fatty acid（ TFA） level increased by about 40. 13% and NADPH level simultaneously increased by 26. 45% in MA-MTHFD1. Moreover,two key enzymes ME and IDH in NADPH synthesis were up-regulated. Based on the results,MTHFD1 plays an important role in NADPH generation during lipid synthesis in M. alpina. A foundation for uncovering mechanism of lipogenesis in M. alpina was established.