Functional Analysis Of ERG4＿ERG24 Family Gene TS1 In Rice

Rice(Oryza sativa.L)is one of staple foods,feeding more than half of population all over the world.As the important influencing factors of rice yield,Ideal plant architecture promotes rice high yield,so cloning and identification of architecture-related genes,uncovering molecular regulatory networks of rice architecture formation are critical for rice architecture improvement and high-yield breeding.We have already found a candidate architecture-related gene TS1 by microarray analysis at early stage.For determining its function,we directly edited TS1 using CRISPR/Cas9 technology.What’s more,we also conducted bioinformatics analysis,gene expression pattern analysis,sub cellular localization,screening and validation of interacting proteins,RNA＿seq analysis and so on.Detailed results are as follows:(1)There ERG4＿ERG24 family genes,TS1,Os25189 and Os22650,were identified from rice genome.They shared similar Isoelectric point,GRAVY and instability index.Gene structures and motif distribution of them were highly conserved.The results of phylogenetic tree showed that there was obvious differentiation between ERG4＿ERG24 genes in dicotyledon plants and that in monocotyledon plants.An amount of light-related and hormonerelated cis-acting elements on their promoter,suggesting ERG4＿ERG24 genes might regulate rice growth and development.(2)pYLCRISPR/Cas9-MT-TS1 vector was successfully conduced,and six T0 positive transgenic plants were generated.In all ts1 mutants,deletions were the main mutated type,small fragments less than 12 bp were the main mutated length,and mutant sites were mainly at the 3rd base upstream of the PAM sequence.ts1 mutations showed lower plant height,shorter on panicle length,less grains per panicle and fewer yield per plant than that of wild type,there was no significant change in other agronomic traits.Phenotype of mutations were heritable,and the segregation ratios of offspring were in accordance with typical Mendel’s law.(3)The results of qRT-PCR analysis showed that transcripts of TS1 could detect in roots,stems,leaves and panicles at different development stage.But transcripts in roots,stems and S6 panicle had higher expression level.The results of GUS reporter system were similar with that qRT-PCR analysis,suggesting TS1 was constitutive expression in rice.(4)TS1 was an atypical chloroplast protein according to the results of subcellular localization.TS1 located in chloroplast in rice protoplasts with chlorophyll,but it located in cytoplasm in rice protoplasts without chlorophyll.(5)Six potential interacting proteins were found using yeast two hybrid system.And further confirmed that TS1 and C1pP had protein-protein interaction relationship by Y2H,BiFC and co-localization analysis,these indicated they might have some similar functions in chloroplast.(6)The results of comparative transcriptome analysis of ts1 mutation and wild type nodes at the point from vegetative stage to reproductive stage showed many differentially expressed genes enrichment in energy reserve metabolism process,glycogen biosynthetic process and hormone responses process and so on.Cloning and functional identification of TS1 provided a sound theoretical basis for improving molecular regulatory network of plant architecture and for uncovering the mechanism of rice morphogenesis,and provided new gene resource and new breeding materials for ideal plant architecture breeding.