杜氏盐藻甘油醛-3-磷酸脱氢酶基因启动子驱动氯霉素乙酰转移酶基因的表达及其活性检测

目的为建立稳定高效的盐藻生物反应器寻找合适的内源性启动子驱动表达外源基因。方法克隆鉴定了盐藻甘油醛-3-磷酸脱氢酶(GAPDH)基因5'上游区序列并成功构建由盐藻GAPDH基因启动子驱动的氯霉素乙酰转移酶(CAT)基因表达载体pUC-Gcat。利用构建的表达载体电击转化盐藻并在含有氯霉素的培养基中筛选转化藻株。随机挑选稳定转化的盐藻藻株进行CAT酶联免疫吸附测定分析。结果获得3株稳定转化的盐藻藻株。聚合酶链式反应鉴定和CAT酶联免疫吸附测定分析结果表明,CAT基因已整合到了转化的盐藻基因组中。结论本研究所克隆的内源性盐藻GAPDH基因启动子能够驱动CAT基因在盐藻中表达。

Expression and activity detection of chloramphenicol acetyltransferase gene driven by the glyceraldehyde-3-phosphate dehydrogenase gene of Dunaliella salina

Objective To explore expression of foreign gene driven by a strong endogenous promoter in order to construct stable and high-performance bioreactors in Dunaliella salina. Methods In the present study,the upstream sequence of glyceraldehyde phosphate dehydrogenase of Dunaliella salina was cloned and identificated.Using electroporation,the alga was transformed with a plasmid pUC-Gcat containing glyceraldehyde-3-phosphate dehydrogenase(GAPDH) gene promoter of Dunaliella salina and chloramphenicol acetyltransferase(CAT) gene as a seletable gene.Using the expression vector,the Dunaliella salina cell was translated and the transformational strain was screened in nutrient medium containing chloramphenicol.The stable transformational strain was selected randomly to undertake CAT enzyme linked immunosorbent assay(ELISA). Results Three stable transformational strain were obtained.The results of polymerase chain reaction and CAT ELISA indicated that the CAT gene had been transferred to the alga. Conclusion The results of this paper suggest that the GAPDH gene promoter can work for genetic transformation of Dunaliella salina.