摘要
为了研究杜氏盐藻双拷贝碳酸酐酶(DCA1)启动子中高度重复的GT序列在盐诱导表达时的调控作用,设计不同的引物,通过PCR法获得6条不同长度的DCA1启动子片段,分别与gus报告基因融合后构建6个表达载体;电击法转化杜氏盐藻细胞。组织化学染色和荧光定量法检测GUS在不同盐浓度下的瞬时表达。结果显示,DCA1启动子内高度重复的GT序列无论与其上游、下游或上下游片段同时结合均能驱动gus基因的表达,并且其表达受氯化钠浓度调控,其中和上下游均结合时活性最强;无GT重复序列的融合片段及GT重复的下游片段也能驱动gus基因的表达,但其表达不受氯化钠浓度调控;而GT重复的上游片段不能驱动gus基因的表达。结果提示:盐藻DCA1启动子中高度重复的GT序列在盐诱导调控中起重要作用,可能为一种新型的盐诱导元件。
To identify whether a highly repeated GT sequence from DCA1 promoter from Dunaliella salina, which have been proved to be a salt-inducible promoter in our previous study, would be a salt-inducible regulation element, different primers were designed to amplify 6 different-length fragments of DCA1 promoter from D. salina by PCR. After these fragments were respectively inserted into the Hind Ⅲ -BamH I sites of the vector pUΩGUS, serial expression vectors containing the gus gene were generated. D. salina cells transformed with these recombinant plasmids by electroporation were grown in liquid media containing different concentrations of sodium chloride respectively revealed that 3 fragme GUS enzyme activity was measured histochemieally and fluorometrically. The results nts containing GT repeated sequence drove the external gus gene expression and the expression pattern of the gus gene was regulated by the concentrations of sodium chloride. Additionally, the 2 fragments without tandem GT sequence drove the gus gene expression, but the expression pattern of the gus gene wasn＇ t regulated by the concentration of sodium chloride; Also, the upstream fragment of the tandem GT sequence wasn＇ t able to drive the gus gene expression. In conclusion, the highly repeated GT sequence from the DCA1 promoter plays an important role in the salt-inducible regulation of DCAI promoter from D. salina and might be a novel salt-inducible element.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2009年第7期50-55,共6页
China Biotechnology
关键词
杜氏盐藻
碳酸酐酶
GUS基因
GT重复序列
D. salina duplicated carbonic anhydrase Repeated GT sequence gus gene
