水稻叶片淀粉累积早衰突变体pls5的鉴定及基因定位

Identification and gene mapping of a premature leaf senescence 5 mutant with starch accumulation in rice leaves

[目的]本研究旨在对水稻叶片淀粉累积早衰突变体pls5进行表型分析及基因定位,探讨水稻叶片早衰的分子机制。[方法]对pls5突变体进行田间农艺性状调查和光合速率测定;于抽穗期对倒2叶(无明显早衰表型)、倒3叶(出现早衰)和倒4叶(严重早衰表型)进行色素和活性氧含量测定,观察叶绿体超微结构;利用Real-time PCR对淀粉代谢和衰老相关基因进行表达分析,以及候选基因的图位克隆与测序。[结果]pls5突变体早期大田生长正常,早衰表型始于分蘖后期,至乳熟期所有叶片衰老。与野生型相比,pls5突变体色素含量和净光合速率极显著降低,同时每穗总粒数、结实率和千粒质量显著下降。倒3叶叶绿体中积累大量的淀粉粒,类囊体片层由于淀粉粒的大量积累被挤压到细胞膜周围;倒4叶叶绿体中积累大量嗜锇体,类囊体片层解体。同时,pls5中淀粉代谢和衰老相关基因的表达显著上调,活性氧积累。遗传分析表明,pls5的突变表型是由隐性单基因突变引起的;利用图位克隆将目标基因定位在第5染色体的长臂标记P7和P4之间,物理距离154 kb,共18个ORF;对区间内已报道基因ES5(Os05g0554400)测序,发现在第11个外显子存在437 bp的插入,导致移码突变翻译提前终止,推测其为候选基因。[结论]pls5是早衰突变体es5的等位变异,在衰老叶片中存在淀粉累积,这些有利于进一步理解叶片早衰的内在机制。

[Objectives]In this study,identification and gene mapping based cloning of pls5(premature leaf senescence 5),a mutant of premature leaf senescence with starch accumulation in rice leaves,were conducted to explore the molecular mechanism of leaf senescence.[Methods]Agronomic characters and net photosynthetic rate of pls5 were measured in the field.A series of analysis,including the contents of pigment and active oxygen,the ultrastructural observation of chloroplast and the expression of genes related to starch metabolism and leaf senescence through Real-time PCR,were conducted on the second(noobvious premature senescence phenotype),the third(emergence of premature senescence phenotype)and the fourth(serious premature senescence phenotype)leaf from the top at heading stage.Meanwhile,map-based cloning and sequencing of candidate genes were conducted.[Results]The pls5 plants grew normal in the field before the late tillering stage.Afterwards,the early senescence of the pls5 leaves emerged relative to the wild type,and all leaves showed necrotic yellow-leaf symptom at the milk-filling stage.Compared to the wild type,the number of spikelets per panicle,1000-grain weight,seed setting rate,pigment content and net photosynthetic rate were all significantly reduced in the pls5.A large amount of starch granules were accumulated in the chloroplasts of the third leaf from the top in pls5 mutant,which resulted in the extrusion of the thylakoid lamellae around the cell membrane.Lots of osmiophilic bodies were accumulated and thylakoid lamellae was disintegrated in the fourth leaf from the top in pls5 mutant.In agreement with leaf color,the pigment content of the third leaf and the fourth leaf from the top were both reduced in pls5 mutant and the content of ROS(reactive oxygen species)was increased.Real-time PCR analysis showed that the expression of some starch metabolism related genes and leaf senescence associated genes were up-regulated significantly in pls5 mutant.Genetic analysis and gene mapping revealed that the premature leaf senescence of pls5 was controlled by a single recessive nuclear gene and the mutant gene was mapped in a 154 kb region on chromosome 5 between marker P7 and P4,containing 18 putative open reading frames.Of them,a reported gene ES5(Os05g0554400)was identified as a candidate gene based on a 437 bp insertion in its 11th exon.The insertion resulted in premature termination of transcription due to the frameshift mutation.[Conclusions]The pls5,leading to starch accumulation in senescent leaves,is the allelic variation of es5.Our results will be beneficial to deepening the understanding of the mechanism of premature leaf senescence.