柽柳WRKY转录因子应答外源ABA的分子调控机制

WRKY transcription factor is a class of new plant-specific transcription factor that are found in the recent years. The expression of WRKY is found to be induced by various of environmental factors, and is closely related with the biotic and abiotic stress responses. In this project, a WRKY gene is cloned from Tamarix hispida which is extremely resistant to stresses. The WRKY gene is transferred to Arabidopsis. The transgenic line with the strongest resistance to ABA is screened for further study. The differentially expressed genes between WRKY transgenic Arabidopsis and wild-type Arabidopsis will be monitored by using cDNA microarray technology. Consequently, the potential downstream target genes that may be regulated by WRKY transcription factor can be confirmed. Some cis-acting elements in common in these target promoters are confirmed by sequence analysis using bioinformatic program. Cis-acting elements potentially bind to WRKY transcription factor are identified by yeast one-hybrid method. Promoter segments which play an important role in the stress response process is constructed into reporter vector pHis2, and the genes bind with this promoter segment are determined by using yeast one-hybrid system . Further validation is performed to determine the upstream regulatory genes of WRKY in the stress resistance process. Ultimately, the molecular regulation mechanism of the WRKY transcription factor response to ABA stress is clarified through all the studies above.

WRKY转录因子是近年来发现的一类植物特有的新型转录因子。WRKY基因受多种环境因子诱导表达，与植物的生物和非生物胁迫应答密切相关。本研究从抗逆植物柽柳中克隆WRKY基因，将其转入拟南芥中，筛选对ABA抗性较强的株系进一步研究。利用cDNA微阵列技术筛选转WRKY基因拟南芥与野生型拟南芥的差异表达基因，从而确定可能受WRKY转录因子调控的下游靶基因。分析这些靶基因的启动子序列，通过酵母单杂交鉴定出可能与WRKY作用的顺式作用元件。将在胁迫响应过程中起重要作用的启动子区段构建到pHis2报告载体中，利用酵母单杂交技术研究在这段序列上起调控作用的基因。通过进一步验证，确定在抗逆过程中调控WRKY表达的上游基因。通过上述研究阐明WRKY转录因子应答ABA胁迫的分子调控机制。

WRKY转录因子是近年来发现的植物中特有的新型转录调控因子家族。研究表明，WRKY转录因子参与多种生物过程，如发育、代谢和胁迫应答。然而，它们在非生物胁迫耐受过程中的作用很多是未知的。在此，我们阐述了柽柳中一个WRKY基因（ThWRKY4）的功能在胁迫应答过程中的一种作用模式。在胁迫初期（胁迫3、6、9h），ThWRKY4被脱落酸（ABA）、盐和干旱高度诱导，而且受ABF（ABRE结合因子）和Dof调控，也可以受其他的WRKY转录因子交互调控以及自我调控。ThWRKY4能够形成同源二聚体，也能与其他WRKY基因如ThWRKY2和ThWRKY3互作形成异源二聚体。ThWRKY4基因的过表达赋予转基因植物对盐、氧化胁迫和ABA处理的耐受力。ThWRKY4通过提高超氧化物歧化酶（SOD）和过氧化物酶（POD）的活性、降低O2- 和H2O2水平、减少电解质渗漏、保护叶绿素免受损失、保护细胞免于死亡来提高对盐和ABA处理的耐受力。cDNA微阵列分析显示，ThWRKY4基因在拟南芥中过量表达导致了165个基因显著上调表达和100个基因显著下调表达。启动子筛查分析显示，ThWRKY4通过与启动子区域的W-box元件结合来调控基因的表达。这项研究表明，ThWRKY4起着转录因子作用，调节非生物胁迫耐受，并参与调节活性氧（ROS）。