水稻营养生长阶段致死突变体vsl的基因克隆与功能研究

Vegetative growth contributes one of the most important stages for rice development, of which, any defect would lead to loss of rice yield. We obtained a novel rice vegetative senescence lethal mutant (vsl) from tissue cultured rice Kitaake. The mutant vsl starts to exhibit its senescence at the four-leaf stage accompanied with lesion mimics, programmed cell death (PCD), reactive oxygen species (ROS) occurring, and up-regulated expression of pathogenesis related genes. This senescence then expands in all leafs and entire plant until plant death before flowering. The chloroplast starts to be damaged seriously at the very beginning stage of senescence in the mutant vsl suggesting that the gene VSL1 might be required for chloroplast development. By using map-based cloning and sequencing approaches, we successfully cloned the gene VSL which encodes a protein with unknown function. Sequencing analyses revealed that a deletion of 590 bp occurs in the promoter region of VSL which results to a deeply reduced expression of VSL leading to lethal senescence in the mutant vsl. In this study, we intend to explore the molecular function of VSL in regulation of rice vegetative development using genetic, cell biological and molecular biological strategies. Our study will provide the basic knowledge for developing rice with high yield. Through the successful implementation of this study, we aim to get one to two papers published and one or two graduate students well trained.

营养生长是水稻发育的重要阶段，该阶段的发育缺陷会直接影响水稻的能量积累，从而导致减产甚至绝收。申请者获得了一个营养生长发育阶段衰亡致死的水稻隐性突变体vsl。该突变体在四叶期开始出现叶片细胞非正常坏死，并不断蔓延直至在营养盛期死亡。该过程伴随着细胞的程序化死亡、活性氧迸发以及PR基因的明显上调。细胞学研究显示，该突变体表型的发生可能与叶绿体功能异常相关。申请者利用分子标记结合图位克隆策略，鉴定得到了VSL的候选基因，其编码了一个水稻特异的功能未知的新蛋白。通过RNAi 策略在正常水稻中抑制VSL的表达，能获得类似突变体vsl突变的性状。本项目通过遗传、细胞生物、分子生化等技术手段分析基因VSL的功能，揭示其在调控水稻营养生长过程中的分子作用机理，为水稻高产育种提供重要理论依据和实践指导。通过该项目的成功实施，将在国内和国际权威杂志上发表论文1-2 篇，培养硕士研究生1-2 名。

衰老是植物发育的最后阶段，是植株为了繁育后代的一种生理表现。对于一些农作物例如水稻而言，衰老的进程直接关系到了产量，是育种中的一个关键因素。但是水稻衰老的相关分子作用机制，尚不清楚。vsl是一个营养生长发育阶段衰亡致死的水稻隐性突变体, 该突变体在四叶期开始出现叶片细胞非正常坏死，并不断蔓延直至在营养盛期死亡。该过程伴随着细胞的程序化死亡、活性氧迸发以及PR基因的明显上调。本研究以vsl突变体为研究对象，综合使用遗传学、细胞生物学、生物化学等多学科技术手段，对VSL基因在水稻营养生长阶段的功能进行了深入解析，取得了以下主要研究结果：VSL定位在水稻第8号染色体上，基因编号为LOC_Os08g19320，其编码了蛋白含有两个Kinetochor_Ybp2结构域，是拟南芥aberrant lateral root formation 4 (ALF4)的同源蛋白；VSL表达量下调的转基因材料表现出了与vsl突变体类似的早衰致死表型；VSL在六叶期的叶片中表达量最高；与拟南芥的ALF4基因功能不同，vsl突变体并未表现出明显的侧根发育异常，这说明VSL基因在遗传学功能上有别于ALF4；VSL-GFP蛋白定位在水稻叶片原生质体细胞的细胞核和细胞质部分；通过酵母双杂交的方法，筛选得到了1个与VSL互作的蛋白VIP1，并得到了验证；VIP1编码了一个ADP-核糖基化因子，为后期对VSL蛋白功能的解析提供了基础。本项目揭示了Kinetochor_Ybp2类蛋白在水稻生长发育过程中的重要性，丰富了对水稻衰老机制的认识，可为水稻育种提供新的理论和依据。