匍枝根霉（Rhizopus stolonifer）As 3.38△6-脂肪酸脱氢酶基因的克隆与酵母表达

Cloning and expression of a △6-fatty acid desaturase gene from Rhizopus stolonifer in Saccharomyces cervisiae

摘要:

通过气相色谱法（GC）快速分析8种真菌的脂肪酸成分，发现匍枝根霉（Rhizopus stolonifer）具有较高的γ-亚麻酸含量，利用RT-PCR和RACE方法获得了全长为1475bp的匍枝根霉△6-脂肪酸脱氢酶基因的cDNA序列，其中开放阅读框为1380bp，编码459个氨基酸。生物信息学分析所克隆的基因具有△6-脂肪酸脱氢酶的典型结构：N端具有细胞色素b5结构、具有3个保守的组氨酸区序列和跨膜结构；把该基因的开放阅读框序列连接到表达载体pYES2.0，构建重组表达载体pYRnD6D，并将其转入缺陷型酿酒酵母INVScl中进行表达。GC分析表明，该序列在酵母中获得了表达，表达产物表现出△6-脂肪酸脱氢酶的酶学活性，能将底物亚油酸转化为γ-亚麻酸。新生成的γ-亚麻酸占酵母细胞总脂肪酸的12.25%。

Abstract:

Fatty acid composition of fungi is analysed through the gas chromatography(GC) technique. With specific activity a novel enzyme △6-fatty acid desaturase was screened and isolated from Rhizopus stolonifer. In this study R.stolonifer was identified as a fungal species that produced plentiful γ-linolenic acid. A 1475bp full-length cDNA, designated as RnD6Dhere, with high homology to fungal △6-fatty acid desaturase genes was isolated from R. stolonifer using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1380bp encoding a deduced polypeptide of 459 amino acids. Bioinformatics analysis characterized the putative RnD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, hydropathy profile and a cytochrome b5-like domain in the N-terminus. To elucidate the function of his novel putative desaturase, the coding sequence was expressed in Saccharomyces cerevisiae strain INVScl. A novel peak corresponding to γ-linolenic acid(GLA) methyl ester standards was detected with the same retention time,which was absent in the cell transformed with empty vector. The percentage of this new GLA was 12.25% of total fatty acids. The result demonstrated that the coding produced △6-fatty acid desaturase activity of RnD6D which led to the accumulation of γ-linolenic acid.