单链DNA结合蛋白Whirly2对叶片衰老调节的分子细胞学研究

Plant senescence is a gene-controlled natural decay and death procedure that are affected or induced by internal and external environmental factors. Senescence occurs early or late and its duration is closely related to the crop yield, quality and so on, which has great significance to actual agricultural production. Research on the mechanisms of senescence has become one of the most hot spot in plant life science. In this project we intend to characterize the cellular and molecular functions of Whirly2 on plant leaf senescence. Whirly2 belongs to the single-stranded DNA binding protein Whirly family, which is specific and widely exists in the plant kingdom. In this study, we will detect the existence and content changes of Whirly2 in mitochondria, chloroplasts and nucleus in different periods of leaf senescing, and establish the relationship of Whirly2 distribution changes in organelles to leaf senescence. After that we will overexpress Whirly2 specifically localized to mitochondria, chloroplasts or nucleus and observe the influence of Whirly2 specific distributions to leaf senescence compared to WT and the whirly2 mutant plant, also the changes of expression of senescence-related genes in these plants will be examined. Finally we will assess the interaction of Whirly2 with the already indentified senescent-related protein Whirly1 in vitro and in vivo, and the influence of their interaction on the expression of senescence-related genes will be determined, this will help to decide the relationship of Whirly2 and Whirly1 in regulating leaf senescence. These experiments will give us a new insight to understand the regulating mechanism of whirly proteins on leaf senescence, and provides some important clue for us to control the senescence process of economic crops such as rice, corn, barley, Chinese cabbage and so on by regulating the expression of Whirly genes.

植物衰老是由基因控制并受内外环境影响和诱导的一种自然衰退和死亡过程，其发生早晚对农作物生产影响巨大。本项目拟对植物中特异和广泛存在的一类单链DNA结合转录因子Whirly小家族成员Whirly2在叶片衰老调节中的分子和细胞功能进行分析。通过检测Whirly2随叶片衰老不同阶段在细胞器和细胞核中的分布和含量变化，建立Whirly2在不同细胞器中的分布水平与衰老之间的关系；同时通过转基因植株表型的变化鉴定不同亚细胞定位的Whirly2对衰老表型的影响以及对下游衰老基因表达的调控；最后通过鉴定Whirly2和Whirly家族另一成员Whirly1之间的蛋白互作对下游衰老相关基因表达的影响来确定Whirly2和Whirly1在调控叶片衰老之间的关系。这一研究将有助于对植物中Whirly家族调控叶片衰老的机制提出新的见解，为一些重要农作物如水稻、玉米、大麦和白菜等利用Whirly调控衰老提供依据。

WHIRLYs（WHYs）单链DNA结合蛋白转录因子小家族已经被报道在植物叶片衰老的调控中发挥作用，但是它们具体的作用机制还不是很明确。WHY2是WHY家族中一个非常特殊的成员，它的亚细胞定位与其它成员之间存在很大差异，而且调控叶片衰老的模式也与已经发表的WHY1蛋白调控叶片衰老的模式相反。本课题基于前期的研究成果，通过制作和运用WHY2多克隆抗体以及各种细胞器特异性的标记Marker，发现WHY2在拟南芥叶片细胞中既可以定位到线粒体上，也可以定位到叶绿体和细胞核中，并且在这些细胞器中的定位也随着叶片发育和衰老的进程发生一定的变化。通过删除WHY2不同区位的亚细胞定位信号研究了不同定位的WHY2对叶片衰老进程的调节，显示只有超表达全长的WHY2才能诱导叶片早衰表型，发育受制，植株矮小。有趣的是，在3周以前小苗中超表达WHY2并未引起叶片衰老，但4周的小苗快速衰老，表明WHY2在这两个阶段中发挥不同的作用。通过GUS染色分析，发现3周以前的小苗中WHY2在所有幼嫩叶片及叶顶端分生组织中都有表达，但在4周小苗到7周成熟植株叶中的表达都很低，只在叶（4-5周）和花（7周以后）顶端分生组织中高表达。有趣的是，在7周植株的衰老叶中GUS染色相较于成熟叶有一定的上升，表明WHY2在衰老叶中的表达升高和叶片衰老存在关联，这一结果也通过定量分析WHY2在叶片中的表达模式得到了证实，以上结果也解释了为何在4周小苗中超表达WHY2会诱发叶片早衰。对WHY2在不同细胞器中定位的变化与叶片衰老表型进行关联，发现删除不同亚细胞定位信号的WHY2在细胞器中的定位与预期存在一定差异，这些WHY2超积累后对叶片衰老表型并无显著影响；给这些WHY2接上特异性亚细胞定位信号也并未引起早衰表型，它们之间相互杂交的后代也未表现出早衰症状，表明WHY2调控叶片衰老过程既存在着表达时期和水平的变化也存在着细胞器定位和含量的变化。本项目同时对WHY2和WHY1互作对叶片衰老调节的关系进行了研究。