水稻生育后期早衰基因lps1的分子调控机制及利用

Premature senescence occurs mainly in late growth stage of rice production, which shortens leaf function period and grain filling time, seriously affecting the yield and rice quality. However, no late-stage premature senescence gene has been cloned so far in rice. Here we isolated lps1, a late-stage premature senescence mutant of rice, using large-scale mutagenesis. The lps1 mutant exhibited normal growth in early stage. But its leaves gradually turned yellow after heading and accelerated senescence after the start of grain-filling. Most leaves of the lps1 mutant died in the middle of grain-filling, which caused almost all grains to become an abortive grain. Genetic analysis suggested that the phenotype of the lps1 mutant was caused by a recessive mutation in a nuclear gene. In the preliminary study, the lps1 mutant gene had been mapped on 38.3-kb region of rice chromosome 10, and a candidate gene had been identified in this region through DNA sequencing, which revealed that it is a novel gene for premature senescence. In this study, we will construct expression vector of the wild-type LPS1 gene, and confirm its function through genetic complementation test. At the same time, we will further identify its biological function through analysis of spatial and temporal expression patterns of LPS1 gene, subcellular localization of LPS1-GFP fusion protein, investigation of phenotypic and physiological characteristics, and detection of related enzymes and active substances. In addition, we will also evaluate potential application of LPS1 gene in delaying leaf senescence and restraining premature senescence through the overexpression of the wild-type gene.

水稻生产上早衰现象主要发生在生育后期，它缩短叶片功能期和籽粒灌浆时间，严重影响产量和稻米品质，然而水稻生育后期早衰突变基因克隆迄今鲜有报道。我们通过化学诱变获得一份水稻生育后期早衰突变体lps1，该突变体主要特征为前期生长发育正常，抽穗前后叶片逐渐变黄，灌浆开始后叶片衰老加速，在灌浆中期叶片基本枯死，导致籽粒多为秕粒，遗传分析表明该突变表型由1对隐性核基因控制。前期研究已将该基因精细定位在第10染色体上38.3kb区域内，并通过基因预测和DNA测序确定了一个候选基因，且为新基因。本项目拟构建该基因表达载体，通过转基因互补验证该基因功能，同时，分析该基因时空表达模式和编码蛋白亚细胞定位，系统观察检测该突变体衰老过程中表型特征、生理特性以及相关酶类和活性物质的变化，探讨该基因调控水稻衰老的分子机制，并通过过量表达转基因等途径，探明该基因对延缓水稻衰老、防止早衰、促进高产稳产等方面的应用潜力。

水稻生产上早衰现象主要发生在生育后期，它缩短叶片功能期和籽粒灌浆时间，严重影响产量和稻米品质，然而水稻生育后期早衰突变基因克隆迄今鲜有报道。本项目通过化学诱变获得一份水稻生育后期早衰突变体lps1，该突变体主要特征为前期生长发育正常，抽穗期叶片逐渐变黄，灌浆开始后叶片衰老加速，在灌浆中后期叶片焦黄干枯，整个植株基本枯死，导致籽粒多为秕谷，产量极显著降低。随着衰老的发展，该突变体光合色素含量快速下降，叶肉细胞中叶绿体等细胞器遭到破坏，直至几乎完全降解。遗传分析表明该突变表型由1对隐性核基因控制。分子标记定位和图位克隆结果表明，lps1突变基因位于第10染色体长臂，是一个新的早衰基因，其候选基因DNA和cDNA全长分别为8839bp和3060bp，含有16个外显子和15个内含子，编码一个与拟南芥TIC蛋白同源的位于叶绿体内膜上的转运蛋白。lps1突变体中该基因第287位T→A突变导致一个错义突变，使得编码蛋白序列第96位的亮氨酸(Leu)突变为谷氨酰胺(Gln)。转基因互补实验表明野生型基因能够恢复突变体表型，从而验证了候选基因功能。基因时空表达模式分析表明LPS1基因为组成型表达，但在叶片中表达很高，而在根中表达很低，亚细胞定位结果显示LPS1蛋白定位于叶绿体上，这与预测的该基因发挥功能的组织和细胞器相一致。进一步，我们采用qRT-PCR分析了抽穗期突变体和野生型中编码叶绿体外膜转运蛋白的OsToc159、OsToc75和OsToc34，编码叶绿体内膜转运蛋白的OsTic40，编码叶绿素生物合成途径中原叶绿素酸酯氧化还原酶的POR和1,5-二磷酸核酮糖羧化酶小亚基的rbcS，以及LPS1基因本身等7个基因的表达水平，结果显示在lps1突变体中，LPS1基因本身以及OsTic40、POR和rbcS等基因的表达量显著下调，而OsToc159和OsToc75基因的表达量显著上调，只有OsToc34基因的表达量基本不变，表明LPS1基因突变显著影响叶绿体外膜、内膜以及核基因编码的叶绿体蛋白基因的表达，暗示该基因可能通过参与叶绿体膜上的蛋白转运从而调控水稻早衰。该研究结果表明LPS1基因对延缓水稻衰老、防止早衰等方面可能具有潜在应用价值。