受钙调素蛋白激酶调控的水稻叶片早衰基因ESL4分子机制研究

As a main photosynthesis organ, leaf blade acts a special role in rice production. Leaf senescence has been paid widely attentions, while the studies mainly focus on Arabidopsis, few involved in oryza sativa, much less natural senescence. These deficiencies restrict our knowledge how rice leaf senescence occurs in molecular level. A rice mutant showing leaf senescence from booting to mature stage was discovered from our EMS-induced library, temperately designed as esl4. The characteristic was controlled by one recessive gene mapped on chromosome 4. Among the restricted region, a gene encoding calmodulin-binding protein kinase showed one base substitution and was regarded as candidate. Physical and chemical analysis suggested that the senescence traits should be related with oxidative stress. Based on these researches, the project aims to clone ESL4 gene by map-based strategy and then unveils its molecular mechanism in regulating leaf senescence by conducting experiments of QPCR, sub-cellular localization, scanning electron microscopy, RNAi, over-expression and so on, involving technical methods of bioinformatics, transcriptomics, cytology, proteomics etc. The results provide a foundation of both leaf senescent molecular mechanism and super high yield breeding in rice, which can also been used for reference in other crops' senescence, especially for cereal crops.

叶片是植物最重要的光合器官，在水稻超高产育种时代，叶片的衰老特性研究尤为重要，目前在拟南芥上已有较多的研究，在水稻中研究较少，对自然早衰研究更少，到目前为止，仅报道了少数自然早衰突变体，这严重制约了人们对水稻叶片早衰分子机制的认识。我们在开展突变体诱导时发现了一个孕穗期叶片提前早衰的突变体esl4，表现单基因控制，已将该基因定位在第4染色体，生理和生化分析表明衰老表型与氧化胁迫相关，一个钙调素蛋白激酶编码基因发生突变引起叶片早衰。在此基础上，拟通过图位克隆分离ESL4基因，进行QPCR、亚细胞定位、扫描电镜、RNAi、超表达等分析，通过生物信息学、转录组学、细胞学、蛋白质组学等手段分析基因在叶片衰老过程中的调控机理，其结果对探讨水稻叶片衰老的分子机理和高产（超高产）育种均具有十分重要的理论和实践意义，对其它作物（尤其是禾本科植物）叶片衰老机制研究也具有重要的指导作用。

钙依赖性蛋白激酶（CPKs）参与多种生物过程，包括激素反应、生长发育、非生物胁迫应答、疾病抗性和氮代谢等。在这项研究中，我们通过图位克隆发现了一种CPK编码基因的新突变体esl4，这是一种氮缺陷突变体。esl4突变体中氮代谢相关基因表达下调，酶活性下降。 ESL4主要表达在根、茎、叶和鞘的维管束中表达。 ESL4蛋白定位于细胞膜。酶活性和生理指标分析以及氮代谢和衰老相关基因表达分析表明，ESL4参与了氮代谢。过表达转基因纯合T2植株与对照相比，低氮下两个ESL4过表达株系的结实率、单株产量、单株籽粒数、籽粒氮含量比和单株总氮含量显著或极显著地增加，表明过表达提高了低氮条件下氮的利用效率，从而在低氮条件下增加了产量。这些结果表明ESL4可能在氮代谢基因的上游起作用。此外，本研究结果为ESL4在低氮条件下培育新品种的应用奠定了基础。