调控Pigm穗瘟抗性关键位点qPBR10-1的功能验证与分子机理研究

Rice blast is one of the most destructive diseases in rice, especially the panicle blast cause the most serious damage to the yield. The resistance gene Pigm has been proved to be broad-spectrum resistant to seedling blast and panicle blast. Our previous study found that the seedling blast resistance level between different sister lines were almost the same when the broad-spectrum resistance gene Pigm was introduced into the background of two-line male sterile line C134S, and all significantly enhanced the resistance level of recurrent parent. However, the panicle blast resistance between different sister lines was significant different, suggesting that there was a genetic factor required for Pigm-mediated panicle blast resistance. Then, a new panicle blast resistant locus, qPBR10-1, was identified by constructing segregation population and GBS analysis, and was finally mapped to a 66.6kb physical region between molecular markers RM25397 and ID4789. By sequence analysis of parental lines and expression analysis, only one ORF was initially identified as the candidate genes. On base of the previous study, we are aim to verify disease resistant function of the candidate gene through gene complementation, overexpression, and gene silencing. In addition, initially reveal the molecular mechanism of qPBR10-1 regulates the panicle blast resistance effect of the broad-spectrum resistance gene Pigm by subcellular localization, spatial and temporal expression patterns, resistance pathways, and interaction protein screening, to provide a research foundation for further revealing molecular mechanisms of the broad-spectrum resistance gene Pigm and its efficient and accurate utilization in breeding practice.

稻瘟病是水稻最主要病害之一，尤以穗瘟对产量的危害最为严重。Pigm被证实是一个对苗瘟、穗瘟均可提供良好抗性的广谱抗性基因。本课题组在前期研究中发现，同时携带Pigm的 C134S背景下的两个高世代回交姊妹系之间苗瘟抗性水平基本相当，但其穗瘟抗性水平存在显著差异，推断存在遗传因子影响了Pigm的穗瘟抗性。通过构建分离群体和GBS分析鉴定到一个新的穗瘟抗性位点qPBR10-1，利用新开发的分子标记将其限定在66.6kb的物理区间内，进而通过抗/感亲本的测序比对结合表达分析初步明确了候选基因。在此基础上，本研究拟通过基因互补、过表达及基因沉默等方法验证候选基因的抗病功能；同时通过亚细胞定位、时空表达模式分析、抗性调控路径研究及互作蛋白筛选等初步揭示其调控广谱抗性基因Pigm穗瘟抗性效应的分子机理。为进一步揭示Pigm介导的广谱抗病分子调控机理以及推进Pigm的高效、精准育种利用提供研究基础。

稻瘟病是水稻最主要病害之一，尤以穗瘟对产量的危害最为严重。Pigm被证实是一个对苗瘟、穗瘟均可提供良好抗性的广谱抗性基因。本研究进一步将qPBR10-1精细定位至60.6Kb范围内，位于分子标记ID338与K1401之间。通过对功能蛋白的cDNA克隆和测序及诱导表达分析发现编码核酸外切酶EXOIII结构域蛋白的OsEXOIII-1基因为目标候选基因。基因互补及基因编辑的转基因功能验证表明OsEXOIII-1对Pigm介导的穗瘟抗性有显著的调控功能。OsEXOIII-1定位于细胞核中，且在叶片、叶鞘及幼穗的表达量相对较高。此外信号标记基因的表达分析初步表明OsEXOIII-1参与了多条信号通路的调控来介导Pigm的穗瘟抗性。qPBR10-1的克隆及功能分析对揭示其调控广谱抗性基因 Pigm穗瘟抗性效应的分子机理及有效利用Pigm基因培育广谱抗性水稻新品种的育种实践具有理论及实践意义。