淀粉富集限制因子α-葡聚糖，水双激酶（GWD）参与木薯淀粉代谢的研究

Starch metabolism is the basis of crop yield and the regulatory function of starch phosphorylation, the only naturally occurring covalent modification of starch, has largely unknown. Functional elucidation of the key gene α-glucan, water dikinase 1 (GWD1) in the regulation of starch metabolism in cassava leaf and storage root can clarify the mechanisms for their role in yield development. In this proposal, we will focused on the expression characterization of GWD1 in wild-type and RNAi transgenic cassava, in addition to the cassava mutants with other starch biosynthetic enzymes, and their changes of starch metabolism and carbon flux in order to verify the biological function of cassava GWD1 in starch phosphorylation and starch metabolism. Using the analysis tools co-IP, GC-MS, HPAEC-PAD etc., the changes of sugars, starch phosphorylation level and physico-chemical properties of starch granules will been confirmed. Furthermore, in combination of isotope tracing and fluorescence labeling to test the carbon flux fluctuation, the physiological parameters and economical traits will be analyzed by the use of.field-grown transgenic cassava in order to elucidate the GWD1 function in carbon transport and partitioning of cassava development. The study will provide the fundamental knowledge and technology for breeding novel germplasms with high yield and better starch.

淀粉代谢是作物产量形成的基础，磷酸化是淀粉代谢过程中唯一的共价修饰，但其对淀粉代谢的调控功能尚未完全阐明；解析淀粉磷酸化关键基因α-葡聚糖，水双激酶1（GWD1）在木薯源（叶片）和库（块根）淀粉代谢中的作用，可阐明其参与木薯产量形成的调控机制。本研究拟通过对野生型和GWD-RNAi转基因木薯开展基因表达特性分析，检测淀粉代谢及碳流转运的变化，结合实验室已有的其它淀粉合成酶突变木薯，验证木薯GWD1在淀粉磷酸化及淀粉代谢中的生物学功能。利用co-IP、GC-MS、HPAEC-PAD等技术分析木薯叶片及块根的淀粉合成酶互作模式、糖分、淀粉磷酸化程度及淀粉粒理化性质的变化，结合13C同位素示踪验证碳流的动态及木薯田间生理生化和产量等农艺性状的变化，解析GWD1在木薯发育过程中对库源碳运输及碳分配的调控机理，为下一步培育高产、优质淀粉新品种提供理论指导和技术支持。

源头强度对储藏根发育的调控在很大程度上是未知的。木薯储藏根延迟（srd）T-DNA插入突变体推迟储藏根的发育，但地上部分表现为与野生型植株相同的正常生长。SRD基因被鉴定为α-葡聚糖，水二激酶1（GWD1）的同源基因，其表达在叶片中受光/暗节律的调节，并与储藏根发育时期相关联。 GWD1-RNAi转基因木薯植株显示出延迟的植株和储藏根生长，这是由于叶片淀粉过量表型引起的光合能力下降和可溶性糖水平降低所导致的结果。其叶片淀粉颗粒直链淀粉含量显著增加，形成具有短链增多的C型半结晶结构，显示为储藏型淀粉。在GWD1-RNAi株系的储藏根中，麦芽糖含量显著降低，淀粉磷酸化水平下降并表现出显著降低的β-淀粉酶分解率。这些结果表明GWD1通过减少从源头到库的光同化分配以及块根作物中的淀粉动员来调节临时型淀粉形态建成和储藏根生长。