粳稻耐碱主效基因挖掘及其功能鉴定与分子育种利用

Saline–alkaline stress is a major abiotic stress that limits rice production worldwide. Alkaline salt stress(Na2CO3 and NaHCO3) are much more destructive for rice growth than neutral salt stress (NaCl and Na2SO4), and saline–alkaline tolerance molecular breeding in rice has no valuable alkaline tolerance genes. Therefore, mining of main genes for alkaline tolerance in rice, breeding improved varieties of alkaline tolerance by molecular breeding method are important for developing rice cultivation in saline–alkaline soil. In our previous study, 2 main QTLs for alkaline tolerance in rice were detected based on the genome-wide association study (GWAS) in a natural population, the traditional QTL analysis in an F2:3 primary population and verification in an BC2F2:3 secondary population. Next, we would use the strategy of combining high-precision linkage analysis of recombinant plants and RNA-Seq of near-isogenic lines to fine mapping the 2 main QTLs and determine candidate genes, then the candidate genes would be cloned, and gene functions would be indentified via the combination with gene knock down and over expression. Furthermore, the functional molecular markers of candidate genes would be developed and verified to breed the new rice varieties of alkaline tolerance through the marker-assisted selection (MAS). The completion of this project would lay the technical, material and theoretical basis for improving rice alkaline tolerance by molecular design breeding and rice cultivation in saline–alkaline soil.

土壤盐碱化是世界范围内限制水稻生产发展的重要逆境因子之一，Na2CO3、NaHCO3等碱性盐胁迫对水稻生长的损害程度比NaCl、Na2SO4等中性盐胁迫更为严重，水稻耐盐碱分子育种方面至今尚无具有利用价值的耐碱性基因。因此，挖掘水稻耐碱性主效基因，通过分子育种手段提高品种耐碱性，对开展盐碱地水稻种植具有重大意义。本项目在前期自然群体的GWAS分析及F2：3初级群体和BC2F2：3次级群体的传统QTL分析检测到的2个“一致性"水稻耐碱主效QTL基础上，拟采用交换单株的高精度连锁分析和近等基因系转录组测序相结合的策略，完成2个主效QTL的精细定位及候选基因的确定，克隆候选基因，并通过CRISPR/Cas9技术和过表达技术鉴定候选基因的功能，进一步开发功能性标记、验证准确性后进行育种利用，为最终通过分子设计育种改良水稻耐碱性及盐碱地水稻种植奠定技术、材料和理论基础。

土壤盐碱化是世界范围内限制水稻生产发展的重要逆境因子之一，然而目前缺少可利用的水稻耐碱性新基因。项目在前期定位到的2个水稻苗期耐碱性QTL qASH7和qSNC3基础上，本研究结合交换单株的高精度连锁分析、候选基因单倍型、qRT-PCR和序列差异分析，分离、克隆了qASH7和qSNC3的候选基因AT1和AT2，AT1编码一个NAC家族的转录因子，AT2编码一个bHLH家族的转录因子。构建了AT1的基因敲除、过表达及AT2的基因敲除、过表达、RNAi转基因纯合株系，在正常和碱胁迫条件下，分别测定野生型和转基因株系的存活率、碱害级别、苗高、根长等形态指标及脯氨酸、丙二醛、叶绿素含量、Na+/K+等生理指标，结果表明，AT1和AT2均可以负调控水稻苗期耐碱性。AT1pro:GUS转基因植株的GUS染色和qRT-PCR分析表明，AT1在水稻的多个组织中均有不同程度的表达，其中在水稻成熟的根中表达量最高，而在水稻成熟叶片中几乎不表达；AT1受NaHCO3、NaCl、H2O2三种非生物胁迫和ABA、JA、GA、IAA、KT五种激素的诱导表达；AT2在不同浓度的Na2CO3处理下均可诱导表达。通过构建GFP共表达载体瞬时转化烟草和水稻原生质体将AT1蛋白定位在细胞核；前人研究表明，AT2同样定位在细胞核。结合双荧光素酶试验和前人研究结果，发现AT1和AT2分别作为转录激活因子和抑制因子调控下游基因表达，进一步利用野生型和突变体的转录组测序初步解析了AT1和AT2可能参与的基因调控网络。此外，本研究基于AT1和AT2的自然变异开发了2个功能性分子标记用于标记辅助育种。本项目研究结果不仅为水稻耐碱性分子机理解析提供重要基础，而且为利用分子育种途径提高水稻品种耐碱性提供理论支持。