Cloning And Function Analysis Of Rice Premature Senescence Gene LPS1

Rice is one of the main food crops to solve the problem of food and clothing for the world’s population.When rice leaves have premature aging,it will seriously affect the photosynthesis rate of the leaves,and then affect the formation and accumulation of photosynthetic products.Therefore,in-depth study of the regulation model and molecular mechanism of rice leaf premature senescence has important theory and practice for improving rice yield and the reduction in hybrid rice’s premature senescence.In this study,we used EMS to mutate the japonica rice cultivar’Longjing 31’to obtain a mutant lps1 with a stable inheritance of the premature aging phenotype.Then conducted phenotypic investigation,physiological and biochemical index determination,histological and cytological analysis of lps1,completed genetic analysis,map-based cloning,functional verification,subcellular localization and related gene expression analysis of lps1,and the main research results obtained are as follows:（1）Phenotype analysis of lps1:The leaf edge of lps1 appeared yellowing from the three-leaf stage.When the tillering stage was reached,the yellowing part of the leaf extended to the middle and upper part,and the edge and tip of the leaf begin to die,but the base of the leaf was still green.The main agronomic traits such as plant height,effective tiller,seed setting rate and thousand-grain weight decreased significantly during the mature period.（2）Physiological analysis of lps1:By comparing the chlorophyll content of wild type and lps1 at tillering stage,it was found that the content of chlorophyll a and chlorophyll b in the leaves of different parts of lps1 decreased significantly,while the content of carotenoids increased significantly.Quantitative results showed that the expression level of genes that related to chlorophyll synthesis in lps1 was significantly reduced,while most of the chlorophyll degradation-related genes had no significant changes It showed that LPS1 affects the expression of rice chlorophyll synthesis and metabolism-related genes,and then induced premature senescence of rice leaves.（3）Electron microscopic analysis of lps1:Electricity test results at the tillering stage showed that the surface of the lps1 blade is relatively smooth,and the number of silicified protrusions around the stomata is reduced;At the same time,the number of chloroplasts in the leaves of lps1 was significantly reduced,the osmophilic granules increased and became larger,and a large number of starch granules appeared.Dyeing and content determination of the starch in the leaves of wild-type and lps1 found that the starch content of lps1 was significantly higher than that of wild-type both day and night,indicating that there is a process of starch accumulation in lps1.The results of qRT-PCR showed that the expression level of genes related to triose phosphate transport was significantly down-regulated at night,indicating that mutations in the LPS1 gene made triose phosphate more used for starch synthesis,which resulted in a large accumulation of starch.（4）Histochemical analysis of lps1:Collected the leaves at the tillering stage for DAB and NBT staining,it was found that superoxide anion（O₂^-）and hydrogen peroxide accumulated（H₂O₂）in large amounts in the leaves of lps1.The detection of physiological indicators related to aging found that the content of H₂O₂ and malondialdehyde（MDA）in the leaves of lps1 increased significantly,while the activities of catalase（CAT）,peroxidase（POD）and superoxide dismutase（SOD）were significantly reduced and the content of soluble protein were extremely significant decline.Using TUNEL to detect the cell death of lps1,it was found that a large number of DNA breaks appeared in the leaves of lps1,which indicated that LPS1 mutation would induce large-scale cell death.（5）Genetic analysis and localization of lps1:Through the hybridization between lps1and ZF802,the separation ratio of the representative type after the hybridization was counted and the chi-square test showed that the premature senescence phenotype of lps1 is controlled by a single recessive nuclear gene.Then use map-based cloning to locate LPS1 in the 77.5 kb region of rice chromosome 5.The candidate genes in this region were predicted and analyzed and sequenced and compared.It was found that the 566th nucleotide G in the coding region of the ubiquitin-conjugating enzyme gene LOC＿Os05g48390 was mutated to A,which resulted in premature stop codon generation and premature termination of the coding.（6）Expression analysis of LPS1:By constructing PAN580-LPS1-e GFP expression vector,injecting tobacco to make it transiently expressed,the result found that LPS1 has an expression signal in the cytoplasm and nucleus The qRT-PCR test results of various tissues showed that LPS1 was more expressed in roots and leaves,followed by sheaths and panicles,and relatively less expressed in roots The expression results of LPS1 in different periods indicated that the mutation of LPS1 may be the cause of the decrease in the number of tillers in the mutant lps1.（7）Bioinformatics analysis of LPS1:Using online software Plant CARE analysis,it was found that the upstream promoter region of LPS1 not only contains common core function elements and light-responsive elements,but also many action elements related to hormones such as Me JA,ABA,IAA,GA.Therefore,we speculated that the LPS1 gene may play an important role in the growth and development of rice and the hormone-mediated senescence of rice leaves.（8）The response of LPS1 gene to exogenous stress:LPS1 mutation can not only accelerate the senescence process of lps1 under dark conditions,but also respond to the stress of exogenous ABA and Me JA by changing its plant height or root length.The results of qRT-PCR showed that after ABA treatment,the expression levels of Os ABA3,Os NCED1,and Os ZEP in lps1 were significantly up-regulated.It showed that LPS1 mutation can promote the synthesis of ABA,which in turn affects rice senescence.In addition,the lps1 can change the root structure under P-conditions to enhance the absorption of phosphorus.