东乡野生稻12号染色体上耐冷主效基因的功能与蛋白互作研究

Cold stress is one of the major restraints for rice production. The most effective and fundamental way for reducing cold damage in rice production is to develop rice new varieties with cold tolerance by mining the genetic capabilities of rice, which has a profound and far reaching significance for rice improvement.. Strong cold tolerance is the most significant feature for Dongxiang wild rice (Oryza rufipogon Giff.), which has been attracted much attention. Based on our previous researches, SSR and Indel markers are used for genetic linkage map construction and quantitative trait locus (QTL) mapping in backcross inbred lines population BC1F10 and BC4F6, which will be very helpful to illuminate the genetic mechanism and the QTL effect of cold tolerance in Dongxiang wild rice at different stages. Meanwhile, the major candidate genes for cold tolerance (CTS-1Dx and CTS-2Dx) on chromosome 12 in Dongxiang wild rice at seedling stage would be verified according to the next-generation sequencing technique, QTL-seq, qRT-PCR and molecular marker analysis. Subsequently, gene cloning and function analysis of CTS-1Dxand CTS-2Dx would be investigated through combined real-time PCR, subcellular localization analysis, RNAi and over expression technique, and yeast two-hybrid method. The gene function analysis includes identification of the gene function and expression pattern, the action mechanism and protein interaction et al. These detailed results in the present study would be helpful for us understanding the genetic and molecular mechanisms of cold tolerance in Dongxiang wild rice. It would also provide some very important molecular information for further investigation and utilizations on cold tolerance genes in O. rufipogon.

低温冷害是限制水稻安全生产的主要因子。挖掘水稻遗传潜能，培育水稻耐冷新品种是减轻水稻冷害的最有效手段和根本途径，具有重要而深远的意义。.强耐冷性是东乡野生稻最显著的特点，已受到广泛的关注。本项目在前期研究的基础上，以高代回交重组自交系BC1F10和BC4F6为试材，用SSR、Indel等标记构建高密度遗传图谱，探明东乡野生稻不同生育期耐冷相关的QTL位点及其遗传规律；基于高通量测序、QTL-seq、qRT-PCR分析和分子标记验证，鉴定东乡野生稻12号染色体上苗期耐冷主效候选基因CTS-1Dx和CTS-2Dx；综合运用Real-time PCR、亚细胞定位、过量表达、抑制表达及酵母双杂交等技术，克隆并探明CTS-1Dx和CTS-2Dx的功能，揭示该基因的表达模式、作用机制及其互作蛋白网络，为阐明东乡野生稻耐冷遗传机理和分子机制提供科学依据，对挖掘与利用野生优异耐冷基因具有重要的指导意义。

低温冷害是限制水稻安全生产的主要因子，其耐冷性是由多个基因控制的数量性状，而且栽培稻中的耐冷基因比较缺乏。因此，研究东乡野生稻耐冷的分子遗传机制，挖掘东乡野生稻中蕴藏的强耐冷基因，对培育出水稻耐冷新品种具有重要的理论和应用价值。. 本项目在前期研究的基础上，以高代回交重组自交系BC1F10为试材，成功构建了包含131对SSR和22对InDel标记的遗传连锁图谱，以活苗率和非死苗率及电导率为指标，共检测到19个QTLs位点。基于高通量测序和QTL-seq分析，在耐冷基因池和冷敏感基因池中检测到55个差异基因，经荧光定量PCR分析验证获得5个候选基因，其中3个候选基因位于12号染色体。. 转录组测序分析表明，东乡野生稻冷诱导后差异倍数大于10的上调基因有24个，3个差异显著的WRKY家族基因和3个AP2/ERF家族基因位于前人的QTL区间。Lable-free蛋白质组学分析发现，变化倍数≥2、p≤0.05的差异蛋白有101个，10个差异蛋白可以映射到前人耐冷QTL区间。蛋白质组和转录组联合分析发现，有46个差异表达基因与差异蛋白相关联。基于PPI网络分析，鉴定出三个中心蛋白（Q8H3I3、Q9LDN2和Q2QXR8）。基于多组学联合分析和qRT-PCR验证，鉴定筛选出了6个重要相关基因（其中2个位于12号染色体QTL区间）。随后，分析了上述中心蛋白和重要耐冷相关基因的互作调控网络。此外，成功克隆了3个重要耐冷相关基因，并对CsQ8H3I3和CsQ5Z9Q8的功能和表达模式做了进一步分析与验证。本研究为阐明东乡野生稻耐冷遗传机理和分子机制提供科学依据，对挖掘与利用野生优异耐冷基因具有重要的指导意义。.