粳稻芽期耐盐候选基因挖掘与分析

Soil salinity is a major abiotic stress that limits rice production worldwide. The rice salt-tolerance at the germination stage directly affects the rate of survival seedings in directing seeding of rice and the final yield. Therefore, mining of candidate genes for salt tolerance at the germination stage, breeding improved varieties of salt tolerance by molecular breeding method are important for improving salt tolerance and developing directing seeding of rice in salt-affected soils. Transcriptional regulation is the most important component of bio-regulation in beings, and transcriptome is the base for the genetic research of genes’ function and structure. Based on the 2 main QTLs for salt tolerance in japonica rice at the germination stage by QTL analysis of traditional linkage mapping, GWAS and regional mapping, analyzing of genes differential expression of the samples in two parents and two bulked RILs consisting of ten lines grown under control and salt-stress conditions by RNA-Seq, combining the results of QTL mapping and RNA-Seq, and the validation of RT-PCR to mining the candidate genes, revealing the molecular genetic mechanism of salt tolerance in japonica rice the germination stage. Furthermore, gene knock-down technology and physiological-biochemical analysis are applied to research the gene function. The results can provide theoretical basis for the breeding application of salt-tolerance genes in japonica rice at the germination stage.

土壤盐渍化是世界范围内限制水稻生产发展的重要逆境因子之一。水稻芽期耐盐性直接影响盐渍土水稻直播的成苗率及最终的产量，因此，挖掘水稻芽期耐盐相关基因，通过分子育种手段改良品种耐盐性，对提高该时期耐盐性及开展盐渍土水稻直播具有重要意义。基因转录水平上的调控是生物最重要的调控形式，转录组是基因功能和结构研究的基础和出发点。本研究在前期传统QTL定位与GWAS定位及目标区域关联分析检测到的2个水稻芽期耐盐性主效QTL基础上，利用转录组测序技术对RIL群体2个亲本及2个极端混池在正常和盐胁迫条件下进行差异基因表达分析，结合QTL定位和基因差异表达结果，以及RT-PCR技术的验证，获得相关候选基因，揭示粳稻芽期耐盐性的分子遗传机理，进一步通过基因敲除、生理生化分析等手段开展候选基因的基因功能研究，为粳稻芽期耐盐基因的育种应用提供理论基础。

土壤盐渍化是世界范围内限制水稻生产发展的重要逆境因子之一，水稻芽期耐盐性直接影响盐渍土水稻直播的成苗率及最终的产量。因此，挖掘水稻芽期耐盐相关基因，通过分子育种手段改良品种耐盐性，对提高该时期耐盐性及开展盐渍土水稻直播具有重要意义。本研究利用东农425（盐敏）/长白10（耐盐）构建的含有1000个单株的F2:3群体为试验材料，筛选极端混池进行QTL-Seq分析，通过ΔSNP-index和ED算法将1个主效QTL qRSL7定位在水稻第7染色体20160000-24330000bp的4.17Mb范围内，进一步利用25个KASP标记进行局部QTL分析，将qRSL7缩小在22877768-23099509bp的222kb的物理区间内。同时利用295份粳稻构成的自然群体，结合重测序获得的778,396个高质量SNP和芽期耐盐表型数据进行了全基因组关联分析，得到耐盐相关SNP位点43个，其中在第7染色体检测到与相对苗高显著关联的SNP位点3个（同1个LD内只列出TOP SNP），分别为Chr07_15425074、Chr7_22804363和Chr07_29133783，Chr7_22804363距离qRSL7 80kb左右，两者为同一个QTL。利用东农425和长白10为试验材料，在对照和1.5%处理条件下进行转录组测序分析，东农425检测到5921个差异表达基因，长白10检测到1519个差异表达基因。结合QTL-Seq、GWAS及转录组测序结果，提取QTL区段内的差异表达基因，共预测到5个候选基因，经过RT-PCR分析及双亲序列差异分析，将Os07g0569700（OsSAP16）确定为影响粳稻芽期耐盐性的候选基因。进一步采用CRISPR-Cas9对候选基因进行基因编辑，长白10的T1纯合植株在芽期1.5% NaCl处理条件下，芽期耐盐性显著降低。研究结果为粳稻芽期耐盐基因的育种应用提供了理论基础。