盐藻的一种盐诱导碳酸酐酶基因转录起始点的确定及表达载体的构建

Identification of transcription start site of Dunaliella salina salt inducible carbonic anhydrase gene and construction of its expression vector

采用5′RACE和巢式PCR的方法确定盐藻(Dunaliella salina)一种盐诱导碳酸酐酶基因的转录起始位点,通过序列分析得出转录起始位点为A,从而确定核心启动子及上游调控区的位置。应用巢式PCR技术分别扩增盐藻这种碳酸酐酶基因上游调控区的2个片段,长度大约分别为0.8和1.2kb,序列经测定无误后分别与带β-葡糖苷酸酶(GUS)报告基因的载体相连接,经酶切鉴定这两种表达载体构建正确。用基因枪法将这两个载体导入盐藻细胞中,经染色检测,GUS基因在转化藻株中得到瞬时表达。

The enzyme activity of carbonic anhydrase (CA) from Dunaliella salina can be stimulated by high salinity, so the 5′ franking region of carbonic anhyd rase gene can be used as a potential inducible promoter to regulate heterogeneou s gene expression in D. salina, an extremely halotolerant alga which would be us ed as a new type bioreactor to produce valuable materials. To understand and elu cidate the regulation of CA, the transcription start site was identified by 5′ rapid amplification of cDNA end (5′ RACE) and nested PCR. The expression vector s containing two 5′ franking regions of CA and the coding sequence of the Esche richia coli beta-glucuronidase (GUS) reporter gene were also constructed. The t ranscription start site was defined as A. DNA sequence analysis and restriction enzyme digestion revealed that 5′ franking regions of CA were not only identica l to those published sequences but also contained in the recombination vectors. The two vectors were then transformed into D. salina by gene gun bombardment. Hi stochemical analysis demonstrated that the GUS reporter gene could be expressed transiently in transformed D. salina.