狗牙根CIPK5基因调控抗旱性的研究

Bermudagrass is a widely applied warm-season turfgrass species, while the calcineurin B-like protein-CBL-interacting protein kinase (CBL-CIPK) signaling pathway is a Ca2+-related pathway that responds strongly to environmental stresses. In our previous investigations, a full length of CdCIPK5 cDNA which showed an induced expression in response to ABA treatment, drought and salinity was cloned. Sequence analysis indicated that CdCIPK5 had a high identity to CdCIPK5. Overexpression of CdCIPK5 resulted in elevated tolerance to drought and salinity, while down-regulation of CdCIPK5 expression led to reduced tolerance to drought and salinity in transgenic rice plants. Based on the observations, this project is planned to investigate the role of CdCIPK5 in regulation of drought tolerance in bermudagrass. To identify the function of CdCIPK5, expression vector of CdCIPK5 driven by OsCIPK5 promoter will be introduced into rice mutant oscipk5 to analyze the phenotypic recovery of the mutant. Transgenic bermudagrass plants overexpressing CdCIK5 and RNAi plants down-regulating CdCIPK5 will be generated using the methods of Agrobacterium-mediated transformation for evaluation of drought tolerance so as to validate the essential role of CdCIK5 in drought tolerance. To elucidate the mechanisms of CdCIK5 in regulation of drought tolerance, transcriptome profiling and the relevant biochemical pathways and physiological processes will be also analyzed using the transgenic bermudagrass in comparison with the wild type. CBL members and downstream target proteins binding to CdCIK5 will be screened and identified using two-yeast hybrid systems in combination with the methods of bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP). The investigations will provide us with novel understanding of drought tolerance in bermudagrass associated with involvement CBL-CIPK sigaling pathway as well as an effective candidate gene used for transgenic improvements of crops or turfgrass species on drought tolerance.

狗牙根是重要的暖季型草坪草，CBL-CIPK信号途径是Ca2+信号系统的重要组分，在植物胁迫应答中起重要作用。在已结题项目的后续研究中，申请人克隆了受ABA、干旱和盐胁迫诱导的狗牙根CdCIPK5基因，其编码蛋白与水稻OsCIPK5最相似。过量表达CdCIPK5提高水稻抗旱、耐盐性，干涉表达则降低水稻抗旱、耐盐性。据此，项目拟将CdCIPK5导入水稻oscipk5突变体，明确其互补OsCIPK5的功能；获得过量表达CdCIPK5和RNAi干涉CdCIPK5表达的转基因狗牙根，阐明CdCIPK5对狗牙根抗旱性的调控作用；对上调和下调CdCIPK5的转基因狗牙根及其野生型开展RNA-seq分析，了解CdCIPK5调控下游基因网络和生理生化过程，阐明CdCIPK5调控抗旱性机理；筛选和鉴定与CdCIPK5结合的CBL及CdCIPK5的下游靶蛋白，为揭示CBL-CIPK调控抗旱性分子机理奠定基础。

本项目研究狗牙根CdtCIPK5及其水稻同源基因OsCIPK5调控耐逆性的功能。CdtCIPK5定位于细胞质和细胞核内，在叶片中的表达量高于根系和茎中。ABA、脱水、低温和盐胁迫处理均诱导CdtCIPK5的表达，其中盐胁迫诱导效应最大。虽然未获得上调和下调CdtCIPK5表达的转基因狗牙根来研究基因的功能，但获得了过表达CdtCIPK5转基因水稻，分析其耐旱、耐盐及耐冷性。结果表明，过表达CdtCIPK5提高耐盐性。筛选并验证CdtCIPK5与CdtCBL4互作，CdtCIPK5与OsSOS1、OsNHX1和OsNHX2等Na+/H+反向转运体不互作；过表达CdtCBL4的转基因植物提高了耐盐性。对CdtCBL4-CdtCIPK5途径调控耐盐性机理开展研究，发现表达CdtCBL4和CdtCIPK5的转基因植物比野生型在盐胁迫下积累较低的Na+，具有更高的K+/Na+比；过表达CdtCIPK5的转基因水稻中具有更高的OsNHX3、OsHKT1;1、OsHKT1;4和OsHKT1;5 表达，表明CdtCBL4-CdtCIPK5途径通过调控这些离子转运相关基因的表达，进而调控植物在盐胁迫下的离子平衡。.在上述工作基础上，进一步对CdtCIPK5同源基因OsCIPK5进行了深入研究。获得过表达OsCIPK5的转基因水稻、OsCIPK5的RNAi植株、T-DNA插入突变体及CRISPR/CAS9编辑的突变体植株；耐逆性分析表明，过表达OsCIPK5提高耐盐性，降低耐冷性，不影响株高和结实率；敲除OsCIPK5的突变体或RNAi植株都降低耐盐性，提高耐冷性，降低株高和结实率。酵母双杂交和BiFC实验证明，OsCIPK5与OsCBL1互作。敲除OsCBL1的突变体植株降低耐盐性，提高耐冷性，但株高和结实率不改变。结果表明，OsCBL1-OsCIPK5途径正调控耐盐性，负调控耐冷性；OsCIPK5影响生长和结实与OsCBL1无关，可能与其他OsCBL有关。通过酵母双杂交筛选并鉴定出CdtCIPK5互作蛋白，但需要采用其他方法进行验证，进而阐明调控耐逆性和结实的机理。