转译起始密码子周边序列的改变对Δ~6-脂肪酸脱氢酶基因表达的影响  

Influence of Sequence Modification Flanking AUG Codon on Δ~6 -fatty Acid Desaturase Gene Expression

作　　者：张琦[1,2] 李明春[1] 孙颖[1] 马海庭[1] 邢来君[1] 孙红妍[1] 任勇[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：《微生物学报》2004年第4期536-539,共4页Acta Microbiologica Sinica

基　　金：国家自然科学基金 ( 3 0 2 0 0 176) ;教育部高等学校骨干教师资助计划项目~~

摘　　要：将少根根霉中Δ6_脂肪酸脱氢酶基因 (RAD6 )的起始密码子周边序列作适当的修改 ,并把修改后获得的片段(RAD6_1 )亚克隆到表达载体pYES2 0 ,构建重组表达载体pYRAD6_1。经测序验证 ,把pYRAD6_1转化到酿酒酵母的缺陷型菌株INVScl进行表达分析 ,同时以空载体pYES2 0和出发序列所构建的pYRAD6作为对照。通过气相色谱 (GC)和气相色谱 质谱 (GC_MS)分析表明 ,在pYRAD6和pYRAD6_1转化的酿酒酵母中生成γ_亚麻酸 ,而pYES2 0中没有检测到。其中 ,pYRAD6_1转化的酿酒酵母γ_亚麻酸表达量占细胞总脂肪酸含量的 5 2 3% ,而对照pYRAD6转化的酿酒酵母中表达量只占 2 6 4 %。With modification of sequences flanking AUG codon, a novel fragment( RAD6_1 ) of Rhizopus arrhizus Δ 6 fatty acid desaturase gene (RAD6 ) was subcloned into expression vector pYES2 0.A recombinant vector pYRAD6_1 was constructed and sequenced for conformation. The resultant recombinant vector was then transformed into Saccharomyces cerevisiae strain INVScl for expression, along with the yeasts transformed with vacant vector pYES2 0 and pYRAD6 derived from original sequence as controls. Yeast cultures, grown to logarithmic phase at 30 ℃, were supplemented with 0 5mmol/L LA and 2% galactose and cultivated at 20 ℃ for a further 48h. Total fatty acids were extracted from the induced cells and eserified. Gas chromatogram (GC) and gas chromatogram mass spectrum (GC_MS) analysis of the resultant fatty acid methyl esters (FAME) showed thatγ_lenolenic acid was detected only in pYRAD6 and pYRAD6_1 transformed yeasts. Furthermore, the percentage of γ_lenolenic acid to total fatty acids in pYRAD6_1_transformed yeasts was 5 23%, and only 2 64% in pYRAD6_transformed yeast was detected. The results demonstrated that the expression level of Δ 6 _fatty acid desaturase gene was increased by the modification of sequences flanking AUG codon.

关 键 词：△^6-脂肪酸脱氢酶 基因 表达 酿酒酵母 少根根霉 转译起始密码子 

分 类 号：Q78[生物学—分子生物学]