Preliminary Functional Analysis Of Grain Size Gene SRG And Map-Based Cloning And Functional Analysis Of Seedling White-Striped Leaf Gene GARS In Rice(Oryza Sativa L.)

This project consists of two parts:the first one is "fine-map and preliminary functional analysis of grain size gene in rice",the other one is "map-based cloing and functional analysis of seedling white-striped leaf gene in rice".Part one:Grain size not only determines rice yield,but also affects the appearance quality of rice grain.In this study,srg exhibits dwarf and small seed size,was isolated from tissue culture of japonica cultivar Dongjin.Fine-map and preliminary functional analysis of SRG reveals molecular mechanism of rugulating rice plant height and seed development,which is helpful for fine-controlled network of seed development and breeding utilization.Results are summarized as follows:The srg mutant was shorter than wild type and has smaller seeds,smaller leaf angle.We analysed the cell number and size of the stems and lemmas between wild type and srg by SEM,and founded that the cell length was smaller than wild type in srg.We further detected the genes involved in cell wall synthesis and cell expansion by qRT-PCR,and founded that most of them were downregulated.These results indicated that decreased cell length was responsible for smaller organs in srg.Genetic analysis showed that the phenotype of srg is controlled by a single recessive nuclear gene.Then we produced an F2 populations crossing between srg and indica cultivar N22,and mapped the gene to a 174 kb genomic region on chromosome 6.There is a SNP in the 7th exon of ORF4,which causes an amino acid change.Transgenic analysis of the candidate gene by CRISPR demonstrated that the ORF4 was likely corresponded to the SRG gene.Protein blast and phylogenetie analysis of the motor domain from kinesin superfamily indicated that SRG belonged to kinesin-10 family.We also investigated the expression pattern of SRG,and founded that SRG was detected in various tissues especially in the young panicles.srg is weakly sensitive to exogenous BL.Then we analysed the expression of genes invoved in BR signaling pathway and founded that the key genes inclouding OsBRI1,MDP,BBLE1 and OsBU1 were downregulated.These results implied that SRG might affect rice growth and development through BR signaling pathway.Part two:Chloroplast is a plant-specific organelle for photosynthesis in plants,and plays an important role for plant growth and development.The process of chloroplast development is very sophisticated.The mutantion of any genes invoved in this process leaded to chloroplast blocking which resulted in abnormal leaf morphology.In this study,gars was isolated from screening a regenerated plant population derived from the EMS indica cultivar Nanjing 11.Map-based cloning and functional analysis of GARS preliminary reveals that GARS1 plays a key role in early chloroplast development in rice.Results are summarized as follows:Compared to wild type,gars displays albino at the two-leaf stage and turns green from the tip of the leaf subsequently.The new-born leaf becomes green at the five-leaf stage.Chlorophyll contents are consistent with the phenotype.We investigated the chloroplast ultrastructure of wild type and srg and found that the chloroplast thylakoid layer are significantly reduced,the starch granules become smaller,and the osmium autophagy increase significantly in gars.These results indicat that early chloroplast development is abnormal in gars.Genetic analysis shows that phenotype of gars is controlled by a single recessive nuclear gene.Then we produced an F2 populations crossing between gars and japonica cultivar Dongjin and mapped the gene to a 84 kb genomic region containing 7 ORFs on chromosome 8.There is a SNP in the coding region of the first ORF and causes an amino acid change.Transgenic analysis of the candidate gene by complementation and CRISPR demonstrated that ORF1 was corresponded to the GARS gene.GARS encodes a glycinamide ribonucleotide synthetase that catalyzes the second step of purine nucleotide biosynthesis.We subsequently examined the activities of the enzymes in wild type and gars with a specifc ELISA kit.GARS activity in gars was reduced by nearly 40%relative to the wild type.The contents of ATP,ADP,AMP,GTP and GDP in gars seedlings were significantly reduced,especially ATP.We investigated the expression pattern of GARS and founded that it was detected in all rice tissues,especially in the leaves.GARS protein displayed a typical chloroplast location pattern in rice protoplasts.qRT-PCR analysis and western blot analysis of genes invoved in chlorophyll synthesis and chloroplast development demonstrated that the expression of most genes was changed.PEP-dependent genes were significantly downregulated.