杜氏盐藻GAPDH cDNA的克隆鉴定及其启动子功能表达载体的构建

Identification of GAPDH Gene from Halotolerant Alga Dunaliella salina and Construction of Expression Vector Driven by Its Promoter

根据藻类甘油醛3-磷酸脱氢酶(Glyceraldehyde-3-phosphate dehydrogenase,GAPDH)氨基酸高度保守序列设计简并引物,采用5′,3′-RACE和巢式PCR的方法,得到了杜氏盐藻(Dunaliella salina)GAPDH cDNA全长序列。序列同源性比较和进化树等生物信息学分析结果表明,根据获得的盐藻GAPDH的核酸序列推导的氨基酸序列与其他已知物种的GAPDH有较高的同源性。定向克隆于原核表达载体的盐藻GAPDHcDNA在大肠杆菌中以融合蛋白的形式得到了高效表达,并成功构建了由盐藻GAPDH基因启动子驱动的cat(氯霉素乙酰转移酶)基因表达载体pUCGCat,为进一步研究杜氏盐藻GAPDH基因和启动子功能奠定了试验基础。

Increasing evidence suggests that glyceraldehyde-3-phosphate dehydrogenase（GAPDH）plays diverse activities such as energy production,apoptosis and microtubule assembly in mammalian and plants.Despite its importance,there is little information available about the GAPDH gene in the halotolerant,unicellular green alga Dunaliella salina.To understand and elucidate the function of D.salina GAPDH,a GAPDH cDNA was identified from D.salina by 5′,3′ rapid amplification cDNA of end（RACE）and nested PCR.The cloned D.salina GAPDH gene（1 394 bp）had an open reading frame of 1 128 bp encoding 376 amino acid residues.Analysis of bioinformatics revealed that it shared high homology with that of other organisms.The full-length cDNA of D.salina GAPDH was heterologously expressed in E.coli as a fusion protein.Furthermore,in order to identify promoter activity of the 5′flanking region of D.salina GAPDH,expression vector pUCGCat containing the GAPDH promoter of D.salina and the coding sequence of the cat gene was successfully constructed.The results reveal that the cloned sequence is a GAPDH cDNA from D.salina and the findings are useful for further functional study of the GAPDH gene in D.salina.