OsGSK1调控水稻盐胁迫响应的分子机制研究

Salt stress has serious adverse effects on rice growth and development. One of the effective ways to further expand rice planting area and increase yield is to elucidate salt stress regulatory mechanism and improve rice salt tolerance through genetic improvement. By screening the T-DNA mutant library of rice, we identified a mutant tas tolerance to salt stress. Compared with the wild type, tas was tolerance to salt stress. Molecular identification showed that T-DNA was inserted into the coding region of OsGSK1, resulting in the down-regulation expression of OsGSK1. We further verify the function of OsGSK1, including measuring the proline content and enzymatic activity of reactive oxygen species scavenging enzymes, gene transformation, gene expression pattern and protein subcellular localization, and to identify the function of OsGSK1 through RNA-seq, proteomics and other techniques, and to screen OsGSK1 interacting proteins through yeast two-hybrid system, to elucidate the molecular mechanism of OsGSK1 regulating rice response to salt stress. This study can provide gene resources for further improving rice salt tolerance, and has important theoretical significance and application value for innovation and utilization of rice salt tolerance germplasm resources.

盐胁迫对水稻的生长发育有着严重的不利影响，阐明其调控机制，通过遗传改良来提高水稻耐盐能力是进一步扩大水稻种植面积和提高产量的有效途径之一。本研究通过筛选水稻T-DNA突变体库，鉴定出一个耐盐胁迫的突变体tas(tolerance salt)。相比野生型，tas对盐胁迫敏感性降低，耐盐性增强。分子鉴定表明，T-DNA插入到基因OsGSK1编码区，导致其表达水平下调。我们进一步通过测定脯氨酸含量与活性氧清除酶活性，转基因验证OsGSK1的功能，明确OsGSK1组织表达特性与蛋白亚细胞定位，并通过转录组测序、蛋白质组学等技术解析OsGSK1参与的分子调控路径，进一步通过酵母双杂交系统筛选OsGSK1的互作蛋白，最终阐明OsGSK1调控水稻响应盐胁迫的分子机理。本研究能为进一步提高水稻耐盐性提供基因资源，对水稻耐盐种质资源创新与利用具有重要的理论意义与应用价值。

土壤盐渍化是世界农业生态环境退化的重大问题之一，严重制约着水稻生产。全球约有8亿公顷的土地受到高盐的影响，并在不断扩大。水稻耐盐性是一个由多基因控制的数量性状，遗传基础较为复杂，通过遗传改良来提高耐盐能力是进一步扩大水稻种植面积和提高产量的有效途径之一。在本研究中，我们筛选T-DNA插入突变体分离出一个盐胁迫耐受性增强的突变体osgsk1。用CRISPR-Cas9技术敲除和过表达验证OsGSK1负调控水稻耐盐性。在盐胁迫处理下，相比野生型，突变体中过氧化氢和丙二醛含量显著降低。OsGSK1在水稻不同组织中都有表达，且受ABA、低温、盐胁迫的诱导表达。OsGSK1定位在细胞质和细胞核内。RNA-seq结果显示OsGSK1突变影响许多胁迫相关基因表达，如OsHAK5、OsHOX22、OsHOX24、OsDREB1G、OsWRKY71。OsGSK1在体内和体外与OsbZIP72和OsSAPK9相互作用。突变体crbzip72和ossapk9对盐胁迫敏感。OsSAPK9在体内和体外与OsbZIP72相互作用，且能磷酸化OsbZIP72。OsbZIP72可以直接结合OsNHX1的启动子，激活其表达。在水稻原生质体中，相比单独表达OsbZIP72，同时表达OsGSK1、OsSAPK9与OsbZIP72，LUC活性显著降低。相比野生型，突变体osgsk1主要农艺性状未发生显著变化。上述结果揭示了OsGSK1-OsSAPK9-OsbZIP72通路在调控水稻盐胁迫中重要的作用，加深了人们对水稻耐盐调控网络的认识，为水稻耐盐育种提供重要的基因资源。