水稻长穗颈基因qPE-11的克隆和功能验证

The phenomenon of panicle enclosure exists in all the male sterile lines, which seriously affects the hybrid reproduction and seed production. It will be helpful to elucidate the molecular mechanism of panicle extrusion for solving this problem. The chromosomal single fragment substitution line 107 (CSSL107), which shows a significant length of panicle extrusion compared with 9311, was screened. qPE-11, a major QTL in CSSL107 was initally mapped. Our data also showed that: 1, ratio of long and short panicle neck traits in the F2 derivative segregating population was 3:1; 2, qPE-11 was tightly linked with the panicle neck trait; 3, the physical area of qPE-11 was narrowed into 1.2Mb. Based on these studies, we will fine map qPE-11 by the strategy of map-based cloning. By sequencing candidate genes and using the bioinformatics technology, target its mutated gene and identify gene structure, protein structure, and the relationships in evolution; By means of PCR and sequencing, analyze allelic differences in several rice varieties; Using trangenic tests of RNAi, OVER and GUS/GFP vectors, verify the function of the targeted gene. Alao, determine the correlation between its expressional levels and phenotypes of panicle extrusion, and clear its patterns in spatial and temporal expression and subcellular localization. This project will reveal the molecular regulation mechanism of the elongation of rice panicle neck and provide strong evidence.

不育系包穗问题是制约杂交水稻繁殖和制种产量的重要因素，研究穗抽出度（穗颈）伸长的分子机制，为解决该问题提供科学依据。申请人及研究室成员前期筛选到一个与亲本93-11差异极显著的长穗颈单片段置换系CSSL107，并定位了主效QTL qPE-11；而后研究结果表明，其衍生分离群体的穗颈长度性状符合3：1，且与qPE-11区间内的分子标记紧密连锁；现已将qPE-11定位在1.2Mb的物理区域内。本项目拟利用图位克隆技术对qPE-11进行精细定位，通过候选基因测序和生物信息学技术，获得目标基因，明确基因结构、蛋白结构及物种进化关系；利用PCR及测序技术分析目标基因在多个品种中的等位性差异；通过干涉、超表达、GUS/GFP等转基因试验，明确基因的表达量与表型之间关系及其时空表达特性和亚细胞定位模式。本项目将为阐明水稻穗颈伸长的生物学机制奠定基础。

水稻是重要的粮食作物，其产量与穗部性状发育直接相关，其中，穗抽出度（穗颈长度）与穗部性状形成密切相关。穗颈长度的遗传研究对水稻穗颈长度形成及对产量相关性状的影响具有重要理论和实践意义。利用“9311/日本晴”染色体片段置换系（CSSLs），将长穗颈表型置换系C107的控制基因qPE-11定位到第11条染色体4Mb区域内。对另一个短穗颈C115中的控制基因qPE-12进行精细定位和候选基因分析，通过图位克隆技术将qPE-12精细定位至130kb区域内，该区间包含5个候选基因。调查了“TD70/Kasalath”重组自交系（RILs）群体的穗颈长度及产量相关性状等12个性状，发现穗颈长度与株高、穗长和千粒重表现为显著正相关性，与穗部性状如一次枝梗数、每穗粒数等表现出显著负相关性，而与谷粒产量、结实率表现出低或无相关性。在RIL群体中12个农艺性状共定位到38个QTLs，检测到4个QTL簇，鉴定到5个穗颈长度QTLs。穗颈长度qPE9和qPE11携带长穗颈亲本Kasalath来源的等位基因，并且在两年中稳定存在，可用于后续TD70及其他短穗颈水稻品种的育种改良中。本项目获得了一个控制穗颈伸长的候选基因qPE-12，鉴定了2个长穗颈QTLs qPE9和qPE11，这些新遗传位点的发现将为后续水稻穗颈伸长的分子机理研究奠定基础，为短穗颈水稻品种的遗传改良及解决不育系包穗问题提供基因资源，因此该项目具有重要的理论和实践意义。