Gene Mapping And Functional Analysis Of TCD8 And TCD51 In Two Rice(Oryza Sativa L.) Chloroplast Genes

Chloroplast is an autonomous organelles and chloroplasts's related gene mutations in nuclear can lead to chloroplast development abnormalities,a decline in chlorophyll,formation of rice leaf color mutant and some have obvious correlation with temperature.In this experiment,we obtained two low-temperature sensitive chlorogenic mutant tcd8 and tcd51 by using ⁶⁰Co?.Through phenotypic analysis,map-based,bioinformatics prediction and functional research,the following conclusions are drawn:1.The tcd8 mutants grown at 20?had green and white appearance at seedling stage,with slow growth and low germination rate.At 32?,the growth was the same as that of the wild type,and the tcd51 mutant at 20?showed the albino phenotype and the sustained low temperature growth would lead to the death of the seedlings.2.Through the determination of photosynthetic pigment and the microstructure observation,we found that tcd8 and tcd51 were similar,at 20?under the condition of phase 3-leaf stage photosynthetic pigment content in the leaf blade sharp decline compared with wild type;at 32?,there was not significantly different from the wild type,tcd8 and tcd51 were incomplete under low temperature conditions,The chloroplast submicroscopic structure of tcd8 and tcd51 was incomplete at low temperature,while chloroplasts developed normally under high temperature,which was consistent with the determination of pigments.3.Genetic analysis was carried out on the F₂ generation of tcd8 and tcd51 in mutants,and we found that these two mutations were controlled by a pair of recessive nuclear genes.4.By map-based cloning,TCD8 was located on the eighth chromosome molecule labeled 173kb between ID13441 and ID13582,we found it encoding GTPase protein;TCD51 was located on the fifth chromosome molecule labeled 507kb between ID5 and ID7,through the candidate gene sequencing,we found that one of the encoding hypothesis protein.5.Through homologous sequence alignment and evolutionary tree analysis,we found that TCD8 and TCD51 are highly conserved in higher plants and have high affinity with monocots plants,and low affinity with dicotyledonous plants.6.By tcd8's qPCR analysis and transcriptional sequencing,we found that the genes related to the chloroplast development phase of the tcd8 were abnormal in varying degrees at low temperature,such as?V1,FtsZ,OsRpoTp,rpoA,Cab2R,etc.?.The mutation of TCD8 gene makes a lot of abnormal gene expression in the mutant compared with the wild type,which affects the synthesis of some secondary metabolites in the cell?four pyrrole,thiamine,etc.?,the activity of oxidoreductase,the synthesis of plastids and plastids,which are closely related to the development of chloroplast.And we have cloned a gene TSV3 encoding GTPase related to the development of rice chloroplast development.It is presumed that TCD8 may be a gene related to the development of chloroplast in rice,and it plays a key role at low temperature.7.Through the qPCR analysis of tcd51,we found that the genes related to chlorophyll synthesis and photosynthesis were down regulated at different degrees at low temperature.There was little difference from the wild type.We speculated the mutation of TCD51.It may affect the early development of chloroplasts under low temperature,affect the translation and accumulation of chlorophyll proteins,and the synthesis of chlorophyll,leading to the mutation phenotype.This study provides a theoretical basis for the functional study of TCD8 and TCD51,provides a reference for the molecular mechanism of chloroplast development in rice.