少根根霉Δ~6-脂肪酸脱氢酶基因的克隆和表达  

Cloning and Heterologous Expression of a Novel Δ~6- Desaturase Gene from Rhizopus arrhizus NK030037

作　　者：张琦[1,2] 李明春[1] 孙颖[1] 马海庭[1] 任勇[1] 邢来君[1] 

机构地区：[1]南开大学微生物学系 [2]云南大学微生物研究所,教育部微生物资源开放研究重点实验室,昆明650091

出　　处：《Acta Genetica Sinica》2004年第7期740-749,共10页

基　　金：国家自然科学基金 (No .3 0 2 0 0 176);天津市自然科学基金 (No .0 13 80 2 5 11)项目~~

摘　　要：根据真菌Δ6 脂肪酸脱氢酶保守的氨基酸序列设计简并引物进行RT PCR ,获得一个 5 93bp的cDNA片段 ,再根据获得的部分序列设计基因特异性引物 ,通过cDNA末端扩增技术 (RACE)获得该cDNA的 3′和 5′序列 ,从而得到全长为 14 82bp的cDNA序列。序列分析结果表明 ,该序列具有一个长度为 1377bp、编码 4 5 8个氨基酸的开放阅读框 ,所编码蛋白质的大小为 5 2kD。与报道的Δ6 脂肪酸脱氢酶一样 ,推测的氨基酸序列具有膜整合脂肪酸脱氢酶特异性的 3个组氨酸保守区和疏水结构 ,在其氨基酸序列的N 末端具有类似于细胞色素b5的血红素结合区。该序列为一个新的编码Δ6 脂肪酸脱氢酶的基因 ,为了验证其功能 ,把开放阅读框序列RAD6亚克隆到表达载体 pYES2 0 ,构建重组表达载体pYRAD6 ,并转化到酿酒酵母的缺陷型菌株INVScl进行表达。通过气相色谱(GC)和气相色谱 /质谱 (GC MS)分析表明 ,该序列在酿酒酵母中获得表达。所编码的酶具有Δ6 脂肪酸脱氢酶活性 ,能将外源性的底物亚油酸转化为γ 亚麻酸 ,γ 亚麻酸的含量占酵母总脂肪酸的 3 85 %。A 593 bp DNA fragment was amplified from Rhizopus arrhizus NK030037 with degenerate oligonucleotide primers designed based on the sequences information for fungi Δ~6-fatty acid desaturase genes by RT-PCR and sequenced.Gene specific primers derived from this partial sequence were used for the amplification of the 3′- and 5′-ends of this cDNA by RACE method,and this lead to a full-length cDNA sequence of 1 482 bp was amplified.Sequence analysis showed this cDNA sequence had an open reading frame(ORF) of 1 377 bp coding 458 amino acids of 52 kD.The deduced amino-acid sequence of the ORF showed similarity to those of the above Δ~6-fatty acid desaturases which comprised the characteristics of membrane-bound desaturases,including three conserved histidine-rich boxes and hydropathy profile.A cytochrome b 5-like domain was observed at the N-terminus.The full-length cDNA sequence is a putative novel Δ~6-fatty acid desaturase gene.To elucidate the function of the protein,two specific primers corresponding to the nucleotide sequences of start and stop codons were used to amplify the coding sequence.The amplified cDNA RAD6 was subcloned into the expression vector pYES2.0 to generate a recombinant plasmid pYRAD6,which was subsequently transformed into Saccharomyces cerevisiae strain INVScl for heterologous expression by lithium acetate method.Grown to logarithmic phase at 30℃,the transformed cells were supplemented with 0.5 mmol/L Linoleic acid and induced by 2% galactose for a further 48 h of cultivation at 20℃.Total fatty acids were extracted from the induced cells and subjected to methyl-esterification.The resultant fatty acid methyl esters(FAME) were analyzed by gas chromatography(GC).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 fatty acid to total fatty acids was 3.85%.Gas chromatography-mass spectrometry(GC-MS) analysis of this fatty acid methyl deriva

关 键 词：少根根霉 △^6-脂肪酸脱氢酶 Γ-亚麻酸 cDNA末端扩增 酿酒酵母 表达 

分 类 号：Q933[生物学—微生物学]