The Function Verification And Regulation Pathway Of Two Rice Genes TCD10 And OsCRM Related To Chloroplast Development

There are two rice leaf coloration mutant MR31 and MR71,which genes have been cloned. MR31 is a Rice thermo-sensitive leaf coloration mutant,its phenotype is caused by the mutation of gene TCD10.The protein prediction website shows that TCD10 encodes a Pentatricopeptide repeat(PPR) protein. MR71 is a green-revertible yellow-green mutan,its phenotype is controlled by gene Os CRM.The protein prediction website shows that Os CRM encodes a CRM domain protein. This research mainly uses the method of RNA interference(RNAi) to verify the function of two rice chloroplast development related genes,by constructing p TCK303 binary vector and transferring into agrobacterium,infecting callus of the wild type rice Jiahua 1 and obtaining the RNAi transgenosis seedlings to verify the function of genes.Moreover,the organizational positioning analysis,subcellular localization and Real-time PCR quantitative analysis to analyze the regulation of genes,the following conclusions can be drawn:1.According to the bioinformatics website NCBI forecasts,TCD10 encodes a PPR protein that may be related to the process of RNA transcription,shear,edit and stability,Os CRM encodes a CRM domain protein that may participate in the class I and class II introns splicing.2.RNA interference experiments prove the mutation of TCD10 can lead to MR31 low temperature sensitive phenotype;3. The tissue specific expression and subcellular localization analysis on TCD10 and Os CRM,the result shows that both of them are specific expression in leaves. In addition,their encoding protein are all in the chloroplast.4.According to the result of mutant MR31 real-time quantitative analysis,the expression of the chloroplast development related genes V1,V2 and RNRL which control the early development of plastids are significantly down regulation at low temperature. Because RNRL1 and RNRS1 form a functional RNR enzyme in rice,and RNR is the key to open the first step in chloroplast development. Furthermore,these genes and others related to chloroplast development almost restore expression at high temperature. This result shows that the mutation of TCD10 may inhibit the expression of genes related to the plastids early development at low temperature. Then the development of plasmid is retarded,and influence the chloroplast development andthe accumulation of photosynthetic pigment.5.The result of mutant MR71 real-time quantitative analysis shows that the expression of most chloroplast development related genes is normal.Only the expression of V2 and RNRS related to the plastids early development and the expression of rps20 which encodes the small subunit of chloroplast ribosome are down regulation.This suggests that the mutation of Os CRM may mildly inhibit the plastids early development and then influence the development of plasmid and the accumulation of photosynthetic pigment.But it remains to be further researched whether Os CRM involved in the splicing of class I and class II introns.