毕赤酵母Δ^9-脂肪酸脱氢酶基因的克隆和表达  

Cloning and Heterologous Expression of a Novel △^9-fatty Acid Desaturase Gene from Pichia pastoris Gs115

作　　者：张昕欣[1] 魏东盛[1] 张飙[2] 邢来君[1] 李明春[1] 

机构地区：[1]南开大学微生物学系分子微生物学与技术教育部重点实验室,天津300071 [2]天津中医药大学公共教学部,天津300193

出　　处：《南开大学学报（自然科学版）》2010年第1期28-33,共6页Acta Scientiarum Naturalium Universitatis Nankaiensis

基　　金：国家自然科学基金(30771355);国家科技部"863"项目(2007AA10Z189);教育部博士点基金新教师项目(20070055061)

摘　　要：根据真菌Δ9-脂肪酸脱氢酶保守的氨基酸序列设计简并引物进行PCR,获得一个741 bp的毕赤酵母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1 689 bp的cDNA序列.序列分析结果表明,该序列具有一个长度为1 194 b p、编码397个氨基酸的开放阅读框(PPD9).与报道的Δ9-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C-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Δ9-脂肪酸脱氢酶的基因,为了验证其功能,把开放阅读框序列PPD9亚克隆到表达载体pYES 2.0,构建重组表达载体pYPPD9,并转化到酿酒酵母的Δ9-脂肪酸脱氢酶缺陷型菌株DTY-11A中进行表达。通过平板互补实验结果表明,该序列在酿酒酵母中获得表达。所编码的蛋白具有Δ9-脂肪酸脱氢酶活性,能弥补DTY-11A中由于Δ9-脂肪酸脱氢酶的缺失而引起的致死性突变。A 741 bp DNA fragment was amplified from Pichia pastoris strain Gs115 with degenerate oligonucleotide primers designed based on the sequences information from fungal△^9 fatty acid desaturase genes by 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（rapid amplification of cDNA ends） method,and this lead to a full length cDNA sequence with 1 689 bp.Sequence analysis showed this cDNA sequence had an open reading frame（ORF） of 1 194 bp coding 397 amino acids.The deduced amino acid sequence of the ORF showed similarity to those of the above△^9-fatty acid desaturaes which comprised the characteristics of membrane bound desaturases,including three conserved histidine rich boxes and hydropathy profile.A cytochrome b5 like domain was observed at the C-terminus.The full length cDNA sequence is a putative novel△^9-fatty acid desaturase gene.To elucidate the function of the protein,open reading frame of the gene was subcloned into the expression vector pYES2.0 to generate a recombinant plasmid pYPPD9,which was subsequently transformed into Saccharomyces cerevisiae△^9-fatty acid desaturase mutation strain DTY-11A for heterologous expression.The transforments can grow on the synthetic minimal medium plate with out oleic acid supplement,the pYPPD9 complement the mutation of DTY-11A and succesfully expressed.

关 键 词：毕赤酵母 Δ9-脂肪酸脱氢酶 cDNA末端扩增 酿酒酵母 表达 

分 类 号：Q78[生物学—分子生物学]