冷胁迫诱导柽柳ThCAP基因表达的分子机制研究

A new gene （GeneBank accession:AY587773）encoding cold acclimation protein, named ThCAP belongs to COR413 gene family. Our previous research showed that the ThCAP gene was involved in temperature response and played important roles in stress responses. In this study, The sequence of the ThCAP gene promoter was used as research base, and the key regulation region of ThCAP promoter to cold response was identified by its seried of 5'-deleted promoter technology. The upstream transcription factor（TF）which regulate the cold responsive region of ThCAP promoter was selected thought a yeast one-hybrid experiment, and identified by electrophoretic mobility shift assay（EMSA）. The over-expression vector of the TF gene was constructed and transformed into Arabidopsis to study its biological function according to cold-resistance physiological indicator and expression analysis of related gene.The molecular mechanism of ThCAP gene expression from Tamarix hispida in response to cold stress was obtained.These results will help to provide theoretical basis for revealing the expression regulation mechanism of COR413 gene.

柽柳冷适应蛋白ThCAP基因（基因登陆号：AY587773）属于COR413基因家族的一个新基因，申请者前期研究结果表明其在植物抗冷胁迫过程中起到重要作用。本项目拟以克隆的ThCAP基因启动子为基础，通过启动子5'系列缺失技术鉴定出柽柳ThCAP基因启动子中冷响应的关键调控区域；采用酵母单杂交体系筛选出调控该区域的候选上游转录因子（TF），进一步通过凝胶阻滞试验验证TF的正确性；通过转基因技术在拟南芥中过表达TF，研究转基因植株的抗冷相关生理指标和基因表达分析，明确TF在低温胁迫条件下的生物学功能，从而获得冷胁迫诱导柽柳ThCAP基因表达的分子机制，为全面揭示COR413基因表达的调控机制奠定基础。

本研究利用Tail-PCR染色体步移策略从刚毛柽柳的基因组中扩增出 一段长度为1538bp的柽柳ThCAP基因上游序列。经生物信息学预测发现,该序列中含有TATA-box, CAAT-box及多种与逆境反应相关的顺式作用元件，表明该序列具有启动子的一般生物学功能。进而对ThCAP基因启动子进行系列片段缺失分析，经试验验证全长启动子及各缺失启动子片段（除P5外）均能够驱动GUS基因的表达，表明所述启动子均属于诱导型启动子。4℃低温处理条件下，GUS组织化学染色结果表明ThCAP基因启动子1538bp-900bp为冷响应的关键调控区域。在该区域之间存在DREB/CBF、bZIP、MYB类转录因子的顺式作用元件。为了进一步证实DREB/CBF转录因子和DREB/CBF元件以及bZIP 转录因子和bZIP元件的特异性识别，分别对bZiP元件及CBF元件进行碱基突变，利用酵母单杂交技术，验证两者之间的相互作用。结果显示bZIP蛋白无法识别突变后的bZIP元件m-element元件结合，但可能与WT-element元件结合。由此推测，bZIP蛋白能够通过识别柽柳ThCAP基因启动子中的bZIP元件来实现对ThCAP 基因表达的调控。