水稻Wx-lsv基因调控低分子量直链淀粉合成的分子机制及其育种利用

It is widely accepted that apparent amylose content (AAC) is the most important factor that determines rice eating and cooking quality (ECQ). In many breeding programs, the improvement of rice grain quality, especially its ECQ is an important objective as rice is mainly consumed in cooked form. It is very clear that GBSSI is encoded by the Waxy gene in cereals, and it is primarily responsible for the synthesis of amylose linear chains. So far, studies have been mainly focused on the influences of AAC on rice ECQ, however, current knowledge on the roles of amylose fine structure is still limited. In previous study, we cloned and identified the ancestral allele of the Wx gene, Wx-lsv and elucidated its effect on rice ECQ. We also evaluated the evolutional relationship between this ancestral allele and other Wx alleles widely used in modern cultivars. We found that the activity of Wx-lsv-encoded GBSSI in the developing seeds was obviously higher than that of Wxa-encoded GBSSI which might be attributed to the serine-to-proline substitution at the 415rd site in the GBSSI enzyme of Wx-lsv rice. We further proved that the GBSSI protein encoded by the Wx-lsv allele tends to produce more short-chain amylose molecules compared to Wxa-encoded GBSSI, which could explain the special ECQ of Wx-lsv rice. In this study, we are gonging to characterize the amylose formation during the endosperm development and try to find the relationship between GBSSI activity and phosphorylation level and also to find the effects of high growing temperature on the grain quality and starch physiochemical and structural characteristics in Wx-lsv rice. The findings will contribute to our improved understanding of the new role of the Wx on amylose synthesis and also provide guidance for breeding new rice cultivars with improved ECQ.

直链淀粉含量是衡量稻米品质的一个最重要指标，已有研究主要关注的是不同直链淀粉含量对稻米品质的影响，而有关直链淀粉精细结构的形成调控机制及其对稻米品质的影响并没有明确结论。本课题前期克隆了一个调控高直链、低淀粉黏滞性的Wx新等位基因Wx-lsv，通过研究，证明其为Wx位点的祖先基因，并且其可能通过编码蛋白的磷酸化位点差异调控酶活性而合成具有明显精细结构差异的直链淀粉。本项目拟围绕直链淀粉精细结构形成的调控机制开展深入研究，从直链淀粉合成动态，GBSSI酶活调控，高温响应和淀粉消化特性等角度深入解析直链淀粉精细结构形成的过程并评价其特异理化特性和GBSSI功能位点的育种利用价值。相关研究对于稻米淀粉合成调控机制以及淀粉精细结构与稻米品质形成关系的深入理解具有重要的参考价值，同时也可为稻米品质改良和特异功能性种质的开发利用提供依据。

目前我国水稻生产水平较高，但是优良食味品质稻米的生产与大众消费需求还有较大差距。淀粉是稻米胚乳中最重要的贮存物质，淀粉中的直链淀粉含量是评价稻米食味品质的最重要指标，同时，其也是影响稻米理化特性的关键组分。水稻中蜡质基因Wx编码的颗粒结合淀粉合成酶GBSSI直接负责胚乳中直链淀粉的合成，该基因的自然等位变异是造成水稻品种间直链淀粉含量和食味品质差异的主要原因。在上一个国家基金的支持下，我们克隆了Wx的一个祖先等位基因Wxlv，证明该基因第10外显子的C/T变异是造成GBSSI酶活差异和直链淀粉结构差异的主要原因。同时还发现GBSSI存在多个磷酸化位点，但是这些位点的具体效应还不清楚。本研究在此基础上构建了籼稻Q11背景中的近等基因系Q11（Wxlv）、NIL（Wxa）和NIL（Wxb），系统分析了籼稻背景下不同材料的品质和淀粉精细结构以及淀粉晶体特性。结果表明Q11（Wxlv）水稻短支链淀粉组分含量相对较低，但直链淀粉组分显著增加。链长分布分析表明，Q11（Wxlv）大米含有较少的支链淀粉短链，但含有较多的中间链组分。此外，发育中的籽粒中的淀粉在三个水稻品系之间表现出不同的积累曲线。为了明确Wxlv在高温条件下对稻米品质和淀粉结构的影响，分析了粳稻日本晴背景下Wxlv, Wxa, Wxb 和wx的近等基因系对高温的影响，结果发现携带Wxlv的近等基因系对高温最为敏感。最后，为了明确GBSSI磷酸化位点对稻米品质的影响，选取了包括第十外显子C/T变异的7个位点进行碱基编辑分析，结果表明其中5个位点被成功编辑。重点分了283位点编辑后稻米品质表现，发现283位点编辑所导致的丝氨酸到苯丙氨酸的突变能够微下调稻米直链淀粉含量，进而改良了稻米食味品质。上述研究对于深入理解稻米淀粉的合成调控以及淀粉精细结构与稻米品质形成的关系提供了重要信息，同时也为稻米品质改良提供了依据。