SnRK2类蛋白基因SAPK10在水稻种子发育和休眠中的功能研究

Abscisic acid is a sort of phytohormone that plays important roles in plant development and abiotic stress response. As core protein members in ABA signaling pathway, SnRK2 proteins have been found to be involved in regulating seed size and dormancy as revealed by several studies. However, the functional study of SnRK2s in rice is still in its infancy. Our preliminary data showed that the expression level of SAPK10 increased with the time of seed development, indicating important roles of SAPK10 in seed development. Ectopic expression of SAPK10 conferred to ABA hypen-sensitivity of the transgenic lines and finally led to earlier flowering, smaller grains and deeper dormancy. This proposal aims to study the function of SAPK10, a SnRK2 type protein, in seed development and dormancy in rice. We will investigate the biological function of SAPK10 by phenotyically characterizing the mutants, RNAi and Over-expression lines, and also monitoring the physiological parameters in seeds. Meanwhile, we will examine the temporal- and spatial- expression pattern as well as the expression profile under different hormones and abiotic stress treatments. The subcellular localization of SAPK10 will also be investigated. Digital Gene Expression Tag Profiling technique will be used to find out the down-stream genes that regulated by SAPK10. Furthermore, we will employ yeast-two-hybrid technique and in-vitro phosphalase assay to indentify the phosphylation substrate proteins to elucidate the SAPK10 regulatory network. This study will greatly enhance our understanding of ABA signal transduction pathway, provide useful gene resources for rice molecular breeding, and thus put us in a good position in the international competitions in ABA signaling and rice seed development studies.

脱落酸是一类重要的植物激素，SnRK2是脱落酸信号传导途径中的核心蛋白，大量数据表明SnRK2蛋白参与调控种子的粒型大小和休眠，但水稻中SnRK2蛋白基因的功能研究仍少有报道。我们前期的数据表明SAPK10表达量随种子的发育进程逐渐升高。过量表达SAPK10可以导致植株对脱落酸敏感，出现早抽穗，粒型变小和休眠加深等表型。本项目旨在研究水稻SnRK2类蛋白基因SAPK10在水稻种子发育和休眠中的功能。我们将通过对突变体和超表达植株种子发育和休眠过程中的表型观察和生理数据检测，明确其生物学功能；检测该基因的时空和胁迫处理下的表达模式，并明确蛋白的亚细胞定位；寻找下游调控基因，并找出SAPK10直接作用的磷酸化底物蛋白，明晰调控网络。本研究的实施将会极大增强我们对脱落酸信号通路的了解，并为分子育种提供基因资源，促进我国在水稻种子发育和脱落酸信号研究的国际竞争中处于有利地位。

SnRK2是存在于植物中的一类丝氨酸/苏氨酸激酶家族,在响应ABA介导的种子发育和休眠过程中发挥着关键的作用。SAPK10是水稻中的SnRK2成员，其表达受ABA的诱导并随着种子的成熟而表达量逐渐增加。亚细胞定位显示SAPK10定位于细胞核。在自然栽培条件下，SAPK10超表达株系较野生型长势矮小，株高同时在粒型大小、结实率、千粒重、有效穗数、穗长方面均较野生型有明显的下降。种子品质分析发现SAPK10超表达株系较野生型在直链淀粉和总蛋白上有显著的差异, 而在支链淀粉和脂肪含量上的差异并不明显。通过核酵母双杂交、in vitro Pull-down和体内co-IP等实验，我们鉴定到SAPK10调控的靶基因-bZIP类转录因子A（简称bZIPA）, 并通过质谱分析鉴定到SAPK10磷酸化修饰该靶蛋白的磷酸化修饰位点，同时通过体外磷酸化实验验证了两者间的激酶-底物修饰关系。本项目组发展了该靶基因的超表达和该磷酸化位点突变非磷酸化位点的模拟非磷酸化的超表达材料，研究表明bZIPA超表达株系对脱落酸敏感，较野生型Nip的种子休眠加深，萌发延迟，而模拟非磷酸化的bZIPASXA超表达株系对ABA的敏感度下降，萌发速率提高，表明SAPK10对bZIPA X位点的磷酸化修饰是该蛋白行使功能的重要开关。上述研究有助于深入了解ABA信号途径核心蛋白SnRK2调控种子灌浆和萌发的机制，对作物品质的改善和产量的提高具有重要意义。