普通狗牙根耐盐CdWRKY2基因的功能鉴定及分子机制解析

Bermudagrass (Cynodon dactylon) is a fine lawn grass widely cultivated in China, but its growth and development could be seriously inhibited by high salinity stress. Excavating the potential salt tolerance genes of bermudagrass and breeding new bermudagrass varieties (or lines) with high salt stress tolerance through genetic engineering are helpful to promote the application of bermudagrass in the improvement of severe saline-alkali soil. Some WRKY transcription factors in plants have been shown to play important roles during response to salt stress, but there is little evidence in bermudagrass. In our previous work, some salt-resistant and salt-sensitive genotypes were screened from a panel of bermudagrass accessions collected from all over the world and CdWRKY2 gene induced by ABA and showing differential expression pattern between salt-resistant and salt-sensitive plants under salt stress will be selected for functional study. The functional analysis will be conducted by overexpressing CdWRKY2 gene in Arabidopsis wide type and mutant, followed by overexpressing it in Brachypodium distachyon and silencing it in bermudagrass to study and the effect on salt-tolerance trait and salt-related physiological and biochemical indicators of seedlings grown under control and salt stress conditions. The sensitivity to ABA and the expression of ABA-response genes in CdWRKY2 overexpression lines and mutant will be detected to study whether CdWRKY2 participate in plant response to salt stress through an ABA-dependent pathway. Further, using the wild type and overexpression lines of Arabidopsis, transcriptome analysis will be conducted to predict the possible pathways by which CdWRKY2 might be regulated during salt stress response. Based on RNA-Seq data, some stress response genes regulated by CdWRKY2 will be selected to determine whether CdWRKY2 could directly bind to their promoters in vivo using ChIP-qPCR method. Therefore, the results obtained in this study will contribute importantly to better understand the molecular mechanisms underlying the response to salt stress in bermudagrass and provide the necessary gene resource for breeding turfgrasses or other crop plants with improved salt tolerance.

狗牙根是我国广泛种植的具有盐碱地改良潜力的优良草坪草，挖掘其耐盐关键基因并培育高耐盐狗牙根新品种（系）有助于其在重度盐碱地的推广应用。WRKY转录因子已被证明在植物盐胁迫应答中发挥重要作用，但在狗牙根中尚少受到关注。前期工作表明，CdWRKY2基因的表达受到ABA的诱导，且盐胁迫下该基因的表达在耐盐品种中显著上调，但在盐敏感品种中几乎不受影响。在此基础上，拟通过超表达、突变体互补、病毒介导的基因沉默等反向遗传学手段鉴定其耐盐功能。进一步通过对盐胁迫前后拟南芥超表达株系的转录组分析结合ChIP-qPCR技术，明确CdWRKY2转录因子发挥耐盐功能所调控的主要生物学途径及直接结合并调控的胁迫响应基因。通过以上研究解析CdWRKY2基因调控植物耐盐性的功能及作用机制，可为进一步深入研究狗牙根耐盐分子机理奠定基础，同时为耐盐狗牙根分子育种提供基因资源，具有重要的科学意义和应用价值。

狗牙根耐盐性强，在盐碱地具有很好的应用潜力。然而，由于狗牙根遗传转化相对较困难，因此对其盐胁迫响应基因的挖掘与功能研究相对较少。本项目成功将盐诱导的WRKY转录因子CdWRKY2转移到狗牙根“Wrangler”中。盐胁迫下过表达CdWRKY2的狗牙根或拟南芥植株地上部及根系生长受到严重的抑制，从而显著提高了其盐敏感性。转基因株系不仅具有较高的ABA敏感性，其盐胁迫下生长素在侧根的积累也显著减少，外源施加IAA在很大程度上恢复了盐胁迫对侧根发育的抑制。酵母单杂交实验表明，CdWRKY2可以直接结合到AtWRKY46的启动子区域并抑制其表达，后者可以进一步与生长素偶联相关基因UGT84B2和ABA应答基因ABI4的启动子结合，促进其表达。此外，酵母双杂实验证明CdWRKY2可以与AtMAPK3相互作用。在拟南芥突变体mapk3背景下过表达CdWRKY2可以部分回复过表达CdWRKY2的盐抑制侧根生长的表型。这些结果表明，CdWRKY2主要通过调控ABA信号通路和生长素稳态从而负调控植物的盐敏感性，并且其功能的发挥部分依赖于AtMAPK3的功能。进化树分析显示水稻OsWRKY30与狗牙根CdWRKY2基因亲缘关系最近。因此，通过Crispr创制了水稻OsWRKY30基因敲除突变体，并对其耐盐功能进行鉴定。与对照株系相比，Oswrky30突变体叶片在盐胁迫下的电解质渗透率明显降低。盐胁迫下突变体的最长主根长、总根数、总根鲜重均高于野生型。这些结果显示Oswrky30突变体耐盐性与野生型相比有所提高，表明CdWRKY2及其同源基因可作为耐盐作物基因工程的有用靶点，以达到提高耐盐性的目的。