水稻叶片衰老相关蛋白磷酸酶基因OsSAPPs的克隆与功能分析

As the last stage of leaf development, senescence is a fine-tuned process regulated by interplays of multiple signaling pathways. Now the distinct pathways that transduce different signals to control the initiation and progression of leaf senescence have not been fully defined. Dephosphorylation mediated by phosphatases plays a critical role in the regulation of multiple important life processes of plant. 2C-type phosphatase (PP2C) was the largest number in plant phosphatase family, but the research about PP2C regulate leaf senescence in rice was rare. We have identified 7 PP2C encoding genes which may involve in leaf senescence of rice through bioinformatical calculation of microarray database and blast between homologous sequences. We named these 7 genes SENESCENCE-ASSOCIATED PROTEIN PHOSPHATASE (OsSAPP) 1-7. Quantitative RT-PCR was used to analysis the transcript levels of OsSAPP1-7 in the dark-induced seedlings between super rice Shen Nong 265 and senescence-delayed rice Long Sheng 04042. It was revealed that the expression of 5 genes was changed dramatically in dark-inducible senescence system. Analyses of expression levels of these 5 genes mentioned above in the natural leaf senescence process of rice will take in this program, and 2 or 3 genes which transcript level changed the mostly may be chosen as the candidate genes. Gene cloning, construction, plant transformation, and phenotype and molecular analyses of the transgenic plants will take to reveal the function of OsSAPPs, and to definite the molecular and regulation mechanisms of OsSAPPs in leaf senescence of rice.

叶片衰老是生长发育的最后一个阶段，目前关于启动和调控这一进程的分子机制及信号转导研究十分有限。蛋白磷酸酶催化的蛋白质去磷酸化在植物多种重要的生命过程中发挥作用；2C型蛋白磷酸酶（PP2C）是植物中数量较多的一类，但关于其参与水稻叶片衰老进程调控的报道较少。我们通过公共Microarray芯片数据分析、生物信息学计算结合序列同源性比对，筛选了7个可能参与水稻叶片衰老进程调控的PP2C基因，命名OsSAPP1-7。利用实时定量RT-PCR检测他们在超级稻沈农265和抗衰老水稻龙生04042人工黑暗诱导衰老过程中的表达情况，发现其中的5个表达变化较显著。本项目将进一步检测上述基因在水稻自然衰老过程中的表达情况，筛选2-3个变化最强烈的基因；从基因克隆、双元表达载体构建、转基因植物的获得及表型观察和分子生物学分析入手，解析OsSAPPs基因的功能，探讨其参与水稻叶片衰老进程调控的分子机理。

叶片衰老是生长发育的最后一个阶段，目前关于启动和调控这一进程的分子机制及信号转导研究十分有限。蛋白磷酸酶催化的蛋白质去磷酸化在植物多种重要的生命过程中发挥作用；2C型蛋白磷酸酶（PP2C）是植物中数量较多的一类，但关于其参与水稻叶片衰老进程调控的报道较少。我们通过公共Microarray芯片数据分析、生物信息学计算结合序列同源性比对，筛选了7个可能参与水稻叶片衰老进程调控的PP2C基因，命名OsSAPP1-7。利用实时定量RT-PCR，我们筛选了在人工黑暗诱导处理中表达变化比较显著的2个基因，分别是OsSAPP-2、OsSAPP-3作为进一步通过自然衰老进程筛选的目标基因。对其进行表达区及启动子区域进行分析、克隆候选基因全长CDS和启动子序列、构建植物过表达和启动子-GUS报告基因的双元表达载体，进一步，通过农杆菌对野生型拟南芥和水稻进行遗传转化，获得转基因植物。得到过表达OsSAPP-2/3转基因拟南芥纯合株系后与野生型拟南芥相比，过表达OsSAPP-2/3转基因拟南芥抽薹开花进程提前，莲座叶变小，叶片提前黄化，叶绿素含量显著下降等早衰表型。POsSAPP3-GUS转基因拟南芥在Stage1.02、Stage1.04、Stage1.10三个发育时期的叶片中均能检测到POsSAPP3-GUS的表达过活性，且活性依赖于叶龄。POsSAPP3-GUS转基因拟南芥极度响应外源激素ABA和IAA，促进POsSAPP3-GUS的表达。过表达OsSAPP-2水稻也表现出叶片提前黄化，叶绿素含量明显下降等早衰表型。综上所有研究结果显示OsSAPP2/3参与植株叶片衰老调控过程，是叶片衰老的正向调控因子。