水稻心白主效QTL Chalk6 的图位克隆和功能验证

Grain chalkiness in rice negatively affects appearance quality, cooking and eating quality, head yield and market values, and so it is one of the most important quality traits in rice. So far, only one gene controlling white-belly grain in rice has been cloned. The molecular mechanism of chalky formation is severely limited to known. In the previous study, we detected a new and major QTL Chalk6 for white-core grain in 3 population, simultaneously and independently. Chalk6 has been restricted to 58 kb region, but the gene underlying the QTL and its regulating functions are unknown. In this project based on the previous work, we first plan to fine map Chalk6 to 20 kb region using the map-based cloning method. Meanwhile, we will identify the candidate genes in 58 kb region by the comparative sequencing analysis coupled with comparative expression analysis, and then confirm the functional Chalk6 gene by mutant and genetic complement transformation analysis. Finally, the tissue-specific expression, scanning electric microscopy imaging analyses will be performed to dissect the molecular mechanism that how Chalk6 regulate the development of chalkiness. The functional analysis of Chalk6 will make us have a better understanding on the location of chalky formation and molecular regulation mechanism. In addition, we will use the genetic diversity and association analyses to identify the favorable alleles of Chalk6 for rice genetic improvement using 533 rice mini-core accessions with a wide genetic variation word wide, to provide help for breeding new rice varieties with both high yield and high quality.

垩白直接影响稻米的外观品质、蒸煮食味品质、精米产量和市场价值，是水稻最重要的品质性状之一。目前只有一个控制腹白的基因Chalk5被图位克隆，对自然变异垩白调控机理认识很有限。前期研究中，我们同时在3个遗传群体的第6号染色体，发现了一个新的、效应很大的、控制心白的QTL Chalk6，并将其定位在58 kb区间，但其分子基础尚不清楚。本项目拟在其工作基础上，将Chalk6精细定位至20 kb；同时通过比较表达、比较测序确定58 kb区间的候选基因；利用突变体和遗传互补验证Chalk6生物学功能；通过组织特异表达、扫描电镜等在生化及细胞层面剖析Chalk6调控心白的分子机理。Chalk6功能的解析，将使我们更好的了解垩白位置形成和分子调控机制。另外，将利用533份水稻微核心种质对Chalk6进行遗传多样性和相关分析，确定适合于水稻遗传改良的优良等位基因，为培育出兼具高产和优质的新品种提供帮助。

垩白直接影响稻米的外观品质、蒸煮食味品质、精米产量和市场价值，是水稻最重要的品质性状之一。前期研究中，我们在9311/Saa、R644/Shadra和MH63/FAZ这3个遗传群体的第6号染色体，分别发现了一个新的、效应很大的、控制心白的QTL Chalk6，但其分子基础尚不清楚。本项目在其工作基础上，进一步的对这3个遗传群体进行精细定位和克隆目标基因，构建互补载体和CRISPR基因敲除载体，转化相应亲本的水稻遗传材料。主要研究结果如下：（1）9311/Saa遗传群体QTL Chalk6精细定位，通过比较测序确定候选基因。构建互补载体转化Saa、构建基因敲除载体转化9311，目前在遗传转化过程中。构建了近等基因系，并对近等基因系的株型、粒长、粒宽、千粒重、整精米率、垩白率、垩白度和总蛋白等性状在近等基因系NIL(9311)和NIL(Saa)中进行考察。同时对NIL(9311)和NIL(Saa)的14天胚乳进行扫描电镜观察，发现高垩白品种NIL(Saa)胚乳中积累的蛋白体数量减少，淀粉体和蛋白体填充不均匀、不紧密，最终导致心白的出现；（2）R644/Shadra遗传群体精细定位，通过比较测序和比较表达分析，确定候选Chalk6基因。构建互补载体转化Shadra和基因敲除载体转化ZH11(基因型与R644一致)，都得到了阳性转基因T0代材料，垩白表型观察显示，阳性互补转基因种子的垩白率无明显变化，但垩白度明显上升。基因敲除转基因阳性材料的种子垩白率显著高于阴性材料；（3）MH63/FAZ遗传群体的精细定位，通过比较测序和比较表达分析，确定候选Chalk6基因。构建互补载体转化FAZ和基因敲除载体转化MH63，目前在遗传转化过程中。本研究的完成，不仅将增进对稻米垩白调控机制和胚乳发育的认识，还将为水稻优质稻分子育种提供理论基础与技术保障，在理论和实践上具有重要意义。