小黑杨PnsICE1基因启动子克隆及瞬时表达分析

Cloning and Transient Expression Analysis of Promoter of PnsICE1 from Populus simonii×P. nigra

ICE1基因是能够编码类似MYC的b HLH转录因子,在低温条件下能够特定地结合CBF3启动子中的MYC作用元件,继而诱导CBF3调控的下游基因的表达,在植物逆境胁迫应答中具有重要的功能。本研究通过PCR扩增技术从小黑杨(Populus simonii×P.nigra)基因组DNA中克隆得到1 247 bp ICE1基因启动子序列,在线预测分析发现,该段序列中含有典型的真核生物核心启动子区域,除了包含多个TATA-box、CAAT-box等基本顺式作用元件外,还存在其它反应元件,如ABRE、DOFCOREZM、MYBCORE、W-box和MYCCONSENSUSATHSE等,这些元件有的与赤霉素、脱落酸等激素相关,有些与逆境胁迫诱导相关,这表明Pns ICE1基因启动子可能与逆境胁迫相关,在小黑杨抵御逆境胁迫的生理过程中具有重要功能。将克隆得到的Pns ICE1启动子取代p BI121中的Ca MV35S启动子,构建植物表达载体p BI121-ICEPro,由Pns ICE1启动子驱动报告基因gus表达,并用农杆菌介导的瞬时侵染法转化拟南芥。对瞬时侵染后的拟南芥进行GUS染色分析。结果表明,在Pns ICE1启动子的驱动下,报告基因gus在花及根部中特异表达,Pns ICE1启动子具有启动活性。

ICE1 encodes a MYC-like bHLH transcriptional activator, which could specially bind to active domain of CBF3 promoter and induce transcriptal expression of cold-responsive genes downstream of CBF3, play important roles in stress response. A 1 247 bp Sequence of PnslCE1 promoter was cloned from DNA of Populus simonii × P. nigra by PCR amplification method. By sequence analysis, it consisted of a typical core promoter region of eukaryotic, beside the basic elements： TATA-box and CAAT-box, and there were some hormone responsive elements and multiple stress-induced elements of ABRE, DOFCOREZM, MYBCORE, MYCCONSENSUSATHSE and W-box. PnsICE1 promoter may associated with adversity stress and play important roles in response to various stresses in P. simonii x P. nigra. To verify the biology function, recombinant vector was designated as pBI121-ICEPro through replacing CaMV35S promoter in pBI121 by cloned PnslCE1 promoter fragment. In the vector, gus reporter gene was driven by PnsICE1 promoter. GUS histochemical assay in Arabidopsis thaliana after transient infection showed that gus expressed in both flower and root.