水葫芦EcGS1b基因启动子的克隆及表达分析

Cloning and Expression Analysis of EcGS1b Gene Promoter of Water Hyacinth(Eichhornia crassipes)

利用染色体步移技术从水葫芦基因组DNA中扩增得到谷氨酰胺合成酶EcGS1b基因5’端上游约1.7 kb的一段序列,经PlantCARE序列分析表明,该序列中除了包括TATA-box和CAAT-box等基本元件外,还含有TATC-box、HSE、LTR和多种光反应相关的元件,初步推测其为EcGS1b基因启动子,命名为pEcGS1b。进一步通过PCR扩增了4个5’缺失的pEcGS1b片段,命名为p1200、p900、p600、p400。以pBI121为基础,构建了4个以GUS为报告基因的植物表达载体,命名为p1200::GUS、p900::GUS、p600::GUS、p400::GUS。利用农杆菌介导的叶盘法将4个植物表达载体转化烟草叶片,进行瞬时表达。结果表明4个缺失片段均具有启动功能,但弱于组成型表达的花椰菜花叶病毒(CaMV) 35S启动子,其中p1200的启动活性最强。

A 5’flanking sequence of EcGS1 b gene which is about Glutamine Synthetase was amplified by genome walking from the genomic DNA of Water hyacinth(Eichhornia crassipes).The length of fragment is approximately1.7 kb.The results derived from PlantCARE analysis showed that the sequence contained a TATA-box core element,a CAAT-box,and other cis-acting elements,including an TATC-box,HSE,LTR and a variety of light-responsive elements.Therefore,it was speculated preliminarily to be the promoter of EcGS1 b gene.The 5’end different deletion fragment of pEcGS1 b was cloned by PCR,called p1700,p1200,p700 and p300 respectively.To investigate function of this promoter,four fragments were fused to GUS reporter gene of pBI121,generating four plant expression vector,named p1200::GUS,p900::GUS,p600::GUS,p400::GUS.With tobacco leaves for the receptor,the results of transient expression that Agrobacterium-mediated that showed all 5’deletion fragment can drive the expression of GUS reporter gene,but their activities were weaker than that of cauliflower mosaic virus(CaMV)35 S promoter,and the activation activity of p1200 was the strongest.