水稻叶片衰老和产量调控基因RLG1的克隆与作用机制分析

Rapid leaf senescence seriously affect the yield and quality of rice. In the previous work, we isolated a mutant produced by the cultivation of Nipponbare callus showing rapid leaf senescence and yield reduction and named it as rlg1（regulator of leaf senescence and grain yield 1）. There was no significant difference between rlg1 and wild type at the vegetative growth stage. However, from the reproductive growth stage to the mature stage, spotted leaves were produced in rlg1 mutant, leading to rapid leaf senescence and resulting in significantly decreased panicle length, grain number per panicle, 1000-grain weight and yield per plant. Transmission electron microscopy observation showed that the starch granules and the osmium-osmotic granules significantly increased in the chloroplasts of rlg1. Using a segregating population, RLG1 was fine mapped to a 119.8 kb region on Chr1, in which there are 19 annotated genes. Furthermore, ORF8 was identified as a candidate gene by genome sequencing and expression analysis. A 983bp transposon sequence was inserted into the 5-UTR region of ORF8 in the rlg1 mutant, resulting its significant reduced expression. In this study, vector construction, genetic transformation, screening of interacting protein and haplotype analysis will be conducted to clarify the function and molecular mechanism of RLG1, as well as the potential value for molecular breeding in rice.

叶片早衰会严重影响水稻的产量和品质。在前期工作中, 申请人发现一个日本晴愈伤组织培养产生的叶片早衰和产量减少突变体rlg1（regulator of leaf senescence and grain yield 1）。rlg1和野生型在营养生长阶段没有显著差异。但是从生殖生长阶段开始，rlg1突变体叶片开始产生斑点，一直持续到成熟时期，导致叶片枯死，造成产量显著降低。透射电镜观察表明，rlg1叶绿体内的淀粉粒和嗜锇颗粒显著增多。通过构建遗传分离群体，将RLG1精细定位到1号染色体119.8kb区间内，该区间预测有19个基因。在rlg1突变体中，ORF8 5-UTR区域插入一个983bp转座子序列，从而造成ORF8表达量显著降低，因此确定ORF8为候选基因。在此基础上本研究将开展载体构建、遗传转化、互作蛋白筛选和单倍型分析等工作，从而明确RLG1的基因功能和分子作用机制及其育种利用潜力。

叶片早衰会严重影响水稻的产量和品质。在前期工作中, 申请人发现一个日本晴愈伤组织培养过程中产生的叶片早衰和产量减少突变体rlg1（regulator of leaf senescence and grain yield 1）。rlg1和野生型在营养生长阶段没有显著差异。但是从生殖生长阶段开始，rlg1突变体叶片开始产生斑点，一直持续到成熟时期，导致叶片枯死，造成产量显著降低。通过构建遗传分离群体，将RLG1精细定位到1号染色体119.8 kb区间内，该区间预测有19个基因。在rlg1突变体中，ORF8 5-UTR区域插入一个983 bp转座子序列，从而造成ORF8表达量显著降低，因此确定ORF8为候选基因。在此基础上构建互补、敲除和过表达载体并进行遗传转化。转基因植株表型验证了RLG1确实是一个控制叶片早衰和产量的基因。为了研究RLG1基因的作用机制，我们进行酵母双杂交筛库实验并筛选到互作蛋白S。S编码一个S-腺苷-L-甲硫氨酸合成酶，能够促进乙烯的合成。利用LUC、BiFC、Pull-down和Co-IP实验验证了RLG1与S蛋白之间的互作。磷酸化实验表明RLG1可以磷酸化S蛋白。这些结果表明RLG1可能通过影响乙烯含量从而调控叶片衰老速率和穗部型态建成。本研究克隆了一个新的控制水稻叶片衰老和产量的基因RLG1，明确了RLG1的基因功能和分子作用机制，，为水稻高产分子设计育种提供优异基因资源和遗传材料。