长穗偃麦草EeSKOR启动子的克隆及功能分析

Cloning and Functional Analysis of the EeSKOR Promoter of Elytrigia elongata

为了研究外整流钾通道蛋白(stelar K+outward rectifier channels,SKOR)基因SKOR在长穗偃麦草中的功能,利用热不对称交错PCR(Tail-PCR)技术,克隆了长穗偃麦草EeSKOR启动子,并进行启动子顺式作用元件及基因表达分析。结果表明:(1)成功获得长穗偃麦草EeSKOR基因起始密码子上游798 bp启动子序列,命名为pEeSKOR。(2)EeSKOR启动子除必须具备的核心启动元件外,还含有特异转录因子结合位点、植物激素响应元件、光响应元件、组织特异的启动元件和胁迫响应元件。(3)成功构建植物表达载体pEeSKOR∷GUS,经农杆菌介导的瞬时转化,EeSKOR启动子驱动GUS报告基因可在拟南芥的叶、叶柄和根中表达。(4)实时定量PCR检测显示,在NaCl、PEG、ABA和SA处理下长穗偃麦草EeSKOR基因在根中呈现不同的表达模式,NaCl处理下EeSKOR的表达量呈先下调后上调趋势;PEG处理下EeSKOR的表达量呈上调趋势,且随着时间的延长显著上调;ABA处理下EeSKOR的表达受到抑制且随处理时间延长呈显著下调趋势;SA处理下EeSKOR表现出先上调后下调趋势,且在处理72 h时表达量显著低于正常表达水平。研究认为,EeSKOR基因的表达受NaCl、PEG、ABA和SA的诱导调节。该研究结果为进一步系统研究长穗偃麦草EeSKOR基因功能提供重要理论依据。

In order to study the function of the stelar K+outward rectifier channels(SKOR)gene in Elytrigia elongata,we cloned EeSKOR promoter from E.elongata by thermal asymmetric staggered PCR(Tail-PCR)technology,and analyzed the cis-acting elements and gene expression of the promoter.The results showed that:(1)the 798 bp promoter sequence of the EeSKOR gene was successfully obtained and named pEeSKOR.(2)This promoter also contains specific transcription factor binding sites,plant hormone response elements,light response elements,tissue-specific promoter elements and stress response elements in addition to the essential core promoter elements.(3)The plant expression vector pEeSKOR∷GUS was successfully constructed.Through Agrobacterium-mediated transient transformation,the GUS reporter gene driven by the EeSKOR promoter can be expressed in the leaves,petioles and roots of Arabidopsis.(4)Real-time quantitative PCR detection showed that EeSKOR showed the different expression patterns in the roots of E.elongata,under NaCl,PEG,ABA and SA treatments.The expression of EeSKOR showed a trend of first down-regulation and then up-regulation under NaCl treatment,under PEG treatment,while the expression of EeSKOR gene was up-regulated,and significantly up-regulated with time.ABA treatment caused the expression of EeSKOR to be inhibited and decreased significantly with the extension of treatment time.The expression of EeSKOR showed a trend of up-regulation and then down-regulation under SA treatment,and the expression level of EeSKOR was significantly lower than the normal expression level after 72 h of SA treatment.Studies indicated that EeSKOR gene expression was regulated by NaCl,PEG,ABA and SA.The results of this study would provide an important theoretical basis for the further systematic study on the EeSKOR gene function in E.elongata.