水稻垩白主效QTL Chalk7的克隆和功能验证

Chalkiness is an important trait of rice which largely determines the rice quality and the market value. So far, only one gene controlling chalkiness has been cloned, which severely restricts the understanding of the modulating mechanism. In the previous study, we detected a new and major quantitative trait locus (QTL) Chalk7 for grain white belly rate. Chalk7 has been restricted to an 80kb region, however, the molecular basis underling Chalk7 is unknown. Based on the previous results, we first plan to fine map Chalk7 to a 20kb region using the map-based cloning method. Then we will identify the candidate gene by omparative sequencing and expression analysis. Next, we will verify the biological function of Chalk7 by genetic transformation and mutant analyses. Finally, the tissue-specific expression, scanning electric microscopy imaging analyses will be performed to dissect the molecular mechanism that how Chalk7 regulate the development of chalkiness. The isolation and function analysis of Chalk7 will provide new gene for quality breeding and improve the understanding of the regulation mechanism of grain chalkiness, which contributes to both theory and practice.

垩白是影响稻米品质和市场价值的重要性状。目前，仅有一个垩白基因被克隆，人们对垩白的调控机理认识非常有限。在前期研究工作中我们在第7染色体上发现了一个新的、效应很大的、控制水稻种子垩白率的数量性状位点 Chalk7，并将其定位在约80kb区间，但其主效基因及分子基础尚未被解析。本项目在前期工作基础上，拟先用图位克隆法将Chalk7精细定位到20kb；通过比较测序、比较表达等分析确定候选基因；利用转基因、突变体等分析来验证基因的生物学功能；通过组织特异表达、扫描电镜等分析在生化、生理及细胞层面来剖析Chalk7调控垩白的分子机理。本研究的完成，将为水稻品质改良提供新基因，还将丰富人们对水稻种子垩白调控机理的认识，在理论和实践上具有重要意义。

本研究是针对一个新的、效应很大的、控制水稻种子垩白率的QTL Chalk7为研究对象进行的深入研究。在前期定位到80kb区间基础上，本研究通过进一步精细定位将Chalk7定位在一个约10kb区间；通过组织特异性表达和胚乳表达量分析明确一个基因ORF1为候选基因，该基因编码COBRA蛋白；通过比较表达分析发现Chalk7是一个垩白的正调控因子；通过比较测序和在自然群体的效果分析明确Chalk7启动子一个SNP变异导致一个CAAT-box顺式元件的丢失，从而导致表达量的下降，最终产生低垩白的表型；通过近等基因系及遗传转化证明该基因的确是Chalk7。Chalk7的克隆及功能变异的确定为稻米分子育种提供分子基础和重要基因来源。