水稻苗期耐盐基因qSTS8的克隆及功能鉴定

Soil salinity is a major abiotic stress harmful to rice production. As a common phenomenon in Ningxia, salinization has caused soil degradation and a prominent reduction of rice yield. In our previous study, a major salt-tolerant QTL, designated qSTS8, with the contribution rate to the total phenotypic variance reached to 22 % was delimited to a 30-kb region on the long arm of chromosome 8 using a BC2F2 population derived from a salt-tolerant rice variety, Faguodao, as the donor, and a salt-sensitive rice variety, Nipponbare, as the recurrent parent. There were three candidate genes in the region. In this project the candidate gene was obtained by comparing the predictive gene sequences of the parents, analyzing the expression pattern difference under salt stress and association analysis with SNP markers. Constitutive expression vector and RNA interference vector were constructed and functional characterization of qSTS8 was completed by genetic complement and RNAi analysis through agrobacterium-mediated rice transformation. Gene structure was analyzed using molecular biological method. The expression pattern and response to salt, drought, ABA, jasmonic acid, BR stress treatment of the target gene were analyzed by RT-qPCR. Subcellular localization of the target protein was achieved by introducing a qSTS8-GFP fusion gene co expression vector into onion inner epidermal cells. A qSTS8-GUS fusion gene co expression vector was constructed to analyze the tissue expression characteristics and salt stress response of the promoter from salt tolerant parent. Cluster and haplotype analysis were carried out by sequencing the target gene of the rice natural population to explicit genetic diversity and molecular evolution. The results will give important reference for studies of molecular mechanisms of salt-tolerance in rice, and to provide theoretical support for rice breeding for salt tolerance using molecular methods.

土壤盐渍化是危害水稻生产的重要非生物胁迫之一。宁夏土壤盐渍化非常严重，水稻生产受其影响极为明显。申请人利用染色体代换定位策略将1个新的耐盐主效QTL qSTS8（贡献率22%）精细定位到1个30Kb区间，包含3个预测基因。本项目拟通过亲本预测基因序列比对，盐胁迫表达量差异结合SNP关联分析获得候选基因。通过构建组成型表达载体、 RNA干扰载体，利用农杆菌介导的水稻遗传转化开展遗传互补和RNAi分析，鉴定其功能。采用分子生物学方法进行基因结构分析。利用RT-qPCR技术分析该基因时空表达特性及对盐、旱、ABA、茉莉酸、BR等胁迫处理的响应。构建GFP共表达载体转化洋葱表皮细胞进行亚细胞定位。构建GUS共表达载体分析耐盐亲本启动子的组织表达特性及盐胁迫响应活性。利用水稻天然群体该基因序列进行聚类和单倍型分析，明确其遗传多样性和分子进化特点，可为水稻耐盐分子机理研究和耐盐水稻品种选育提供参考。

土壤盐渍化是危害水稻生产的重要非生物胁迫之一。宁夏位于黄河中上游，降水稀少，蒸发强烈，耕地盐渍化率高，严重限制了水稻生产的发展。本研究在前期完成耐盐基因qSTS8精细定位的基础上，结合盐胁迫下日本晴和法国稻转录组测序，将精细定位区间与差异基因表达谱和SNP信息整合，确定LOC_Os08g33160为候选基因，命名为STS1（Salt Tolerance of Seedlings1）。该基因属于茉莉酸信号通路阻遏蛋白家族，亚细胞定位结果显示，其编码蛋白位于细胞核。STS1表达受ABA、盐、旱和低温胁迫诱导，通过不同耐盐性水稻种质资源靶基因测序和盐胁迫下不同缺失启动子片段GUS染色分析发现，其差异表达主要来源于启动子区域的3个SNP和2个Indel位点。STS1超表达转基因水稻材料显著降低了苗期耐盐性，表明该基因可能是调节水稻盐胁迫响应的负调控因子。通过转录组学结合表达量分析发现，该基因可能是通过与OsJAZ4和OsMYC2转录因子互作，抑制了超表达株系调节钠钾离子平衡能力，从而降低其耐盐性。本项目研究结果不仅为水稻耐盐分子机理研究提供重要参考，而且为利用分子育种途径提高水稻品种耐盐性提供理论支持。