优良食味软米品种暗胚乳形成的结构学基础及其改良研究

Rice is a major staple food crop in China. The grain appearance quality especially the grain transparency has been attracted more attention by both consumers and breeding programs than ever before. It is widely accepted that amylose content is the most important factor that determines rice eating and cooking quality. In china, soft rice with very low amylose content is very popular because of its good eating and quality. Though the soft rice has a good taste performance, the grain appearance usually showed a milky or dark phenotype due to the low amylose content and thus reduced the appearance quality greatly. So far, studies have been mainly focused on the influence of chalky and floury grains; however, current knowledge on the formation of dark endosperm is still limited. In previous studies, we have proved that rice with reduced amylose level tend to have dark grain appearance due to air spaces in the starch granules. However, we have generated new soft rice with very low amylose content but transparent grain by interfering SSSII-2. We have also generated a series of Wx near isogenic line in Nipponbare background, which showed significant difference in grain transparent. In this study, we are going to characterize the structural basis of rice dark endosperm from soft rice with good sensory property by using a series of Wx NILs and transgenic rice with similar amylose level but significant grain transparency. We also plan to transform the SSSII-2 RNAi constructure to the soft rice with both high yield and good sensory property, to improve the soft rice grain transparency. Further, we plan to knock out the SSSII-2 in the soft rice by using the CRISPR/Cas9 system. The findings will provide clear information about the formation of dark endosperm and also provide guidance for breeding of novel soft rice with improved grain appearance quality.

稻米外观品质是影响其商品价值的重要因素，也是广大消费者关注的首要性状。目前，在我国南方地区的软米因其具有较好的食味品质而广受欢迎。这一类稻米由于其直链淀粉含量较低而表现为米饭适口性好，但其最大的缺陷是外观品质较差。自然干燥下，软米通常会表现出明显的暗胚乳表型。然而，有关软米暗胚乳的形成机制和改良研究却非常少。本课题组在前期研究中发现，稻米透明度与胚乳中单个淀粉粒内的孔洞数目和大小密切相关。此外，我们通过对SSSII-2基因干扰，获得了具有低直链淀粉含量的软米，其外观为透明表型。本项目以此为基础，拟围绕优良食味软米水稻暗胚乳形成的结构学基础，以具不同籽粒透明度和直链淀粉含量的常规品种、近等基因系和转基因系等为研究对象，系统开展暗胚乳形成与水分、直链淀粉含量和淀粉精细结构之间关系的研究，以阐明优良食味软米暗胚乳形成的结构学基础，并利用基因编辑等技术研究改良软米暗胚乳外观品质的途径。

优良外观兼顾优良食味稻米一直是水稻品质遗传改良的重要研究内容。然而，由于稻米品质性状遗传调控复杂，受到环境和遗传等多个因素的影响，水稻品质性状的遗传改良还面临很多难题。稻米外观品质主要涉及稻米粒形、透明度和垩白等，其中稻米透明度形成的机制较为复杂，胚乳中的水分含量与直链淀粉含量(AC)都是影响稻米透明度的关键因素。尽管高AC类稻米透明度好，但是其食味品质较差，而低AC类稻米食味品质好，但透明度差。因此，目前通过下调稻米AC来改良稻米食味品质所面临的一个重要问题是稻米外观品质下降，食味和外观难以协调。为此，本研究围绕稻米透明度形成的淀粉结构学基础开展了系统分析，通过对具有不同AC的水稻品种以及系列近等基因系等遗传材料，稻米透明度形成的影响因素、淀粉粒结构和发育时期淀粉粒结构动态分析，发现水分和直链淀粉含量与稻米透明度直接相关。此外，淀粉粒结构分析表明，存在于单个淀粉粒内部的空腔是造成稻米胚乳透明度差异的直接因素。另一方面，基于实验室前期构建的粳稻日本晴背景下，具有较低直链淀粉含量，但胚乳透明的转基因系为切入点，构建了不同Wx背景下的聚合系，基因表达分析表明，导入SSII-2RNAi结构的近等基因系中SSII-2、SSII-3和Wx表达均极显著下降。通过稻米基本理化特性和透明度等分析，发现SSII-2RNAi结构导入后，Wxmw和Wxmp背景下稻米的AC均下降约5%。这些低AC稻米尽管在中等水分条件下透明度较常规低AC稻米有一定的改善，但是在低水分条件下透明度下降较为明显，说明仅通过引入SSII-2RNAi结构，难以在Wxmw和Wxmp等位基因背景下创建更低AC且籽粒透明的新型软米。另外，通过对不同近等基因系稻米淀粉精细结构的分析，发现支链淀粉短链和中长链与稻米透明度关系密切，考虑到SSII-2RNAi结构对稻米AC的显著影响，后续通过基因编辑等方式，更精准的对支链淀粉结构的修饰，是改善低AC软米类不利暗胚乳表型的一个重要突破口。上述研究对于深入理解稻米透明度形成的结构基础以及遗传改良途径提供了依据。