Cloning And Functional Analysis Of A Leaf Tip Senescence Gene LTS2 In Rice

Early failure of rice leaves seriously affected rice yield and quality,although many rice early decay genes have been reported,the early decay molecular mechanism is still unclear,therefore,this study used a stable genetic leaf tip dead early aging mutant obtained by Wuyunjing21,a EMS mutagenic japonica rice variety（WYJ21）,compared found the phenotype similar to rice early decay mutant lts1 muotype,so named lts2（leaf science）.This study identified lts2 phenotypes,mapped cloning and candidate gene prediction of genes controlling lts2 premature senescence phenotype,and analyzed chlorophyll content,senescence-related indicators and expression levels of genes affecting premature senescence.The main findings are as follows:1.Phenotypic analysis of the mutant lts2.At the early tillering stage,the tip of the lts2began to wither,and then it was found that the withered area gradually expanded with the growth of the plant,and the rust-colored spots appeared at the same time;Different from wild type lts2 the leaf tip began to wither and brown.Compared with wild type,the spike length of lts2 was shorter,the effective spike number and grain number per spike were also decreased,and the seed setting rate was slightly lower but the difference was not significant.2.LTS2 genetic analysis and map cloning.Construction group analysis shows that lts2is controlled by the mono-recessive nuclear gene.The target gene was located in the physical interval of 42 kb on chromosome 10 by using the localization population constructed by Huazhan×lts2.There are 5 predicted ORF,in this region.We sequenced and compared all the genes（including promoters and introns）lts2 and WYJ21,and found that single base mutation G→A the 1242 bases on exon 1,From the original tryptophan process to Terminator,the gene coding terminates in advance,and finally leads to the loss of coding protein function.3.LTS2 encodes a LRR-like protein consisting of 1130 amino acids and is predicted to be a leucine-rich receptor-like protein kinase.the N end of the protein consists of 10 LRR and the C end is a catalytic domain of a S＿TKc protein kinase.Construction of GFP fusion protein vector,expressed in protoplasmic,shows that LTS2 protein is localized on the cell membrane.4.Mutation of LTS2 resulted in the decrease of chlorophyll content and photosynthetic ability of lts2 leaves,the number of chloroplast in leaf cells was less than that in wild type,the chloroplast was significantly atrophy and shriveled,and the number of ordered inner sac matrix became less and the arrangement was disordered.The RT-PCR showed that chlorophyll synthesis related genes（Os PORA、Os PORB）,photosynthesis related genes（Os Psa A、Os Psb A、Os Cab2）,chloroplast transcription/translation related genes（Os YGL、Os CHLH、Os HEMA）were significantly decreased.5.Mutations in the LTS2 lead to higher accumulation of peroxide,H₂O₂,and malondialdehyde（MDA）content in lts2 plants that indirectly reflect lipid oxidation levels than in wild type.The enzyme activity of SOD、CAT、POD with scavenging reactive oxygen species and H₂O₂function decreased significantly,resulting in the decrease of total antioxidant capacity（T-AOC）of the enzyme.The catalase gene（Os CATB）,peroxidase gene（Os POX1）,ascorbic acid peroxidase gene（Os APX1 and Os APX2）,polyamine oxidase gene（Os PAO）is shown by RT-PCR.It turns out,The expression of Os APX1、Os APX2、Os POX1、Os CATB genes in mutant lts2 was down-regulated,Causes peroxide and other ROS to be removed,The up-regulation of Os PAO gene expression in H₂O₂production of spermine further weakens oxide clearance,The accumulation of ROS in lts2 plants.The results showed that the deletion of OsLTS2 gene induced the premature senescence phenotype of leaf tip and its expression product was receptor-like protein kinase（LRR-RLK）,which was involved in the regulation of aging.This study provides a reference for exploring the physiological and biochemical changes in leaf senescence and further revealing the role of LRR-RLK in the aging regulatory network...