Cloning And Functional Analysis Of PPR Gene TCD34 In Rice (Oryza Sativa L.)

More and more genes encoding PPR protein have been reported in rice.As one of the largest protein families in plants,PPR protein mainly affects the development process of chloroplasts,leading to premature or delayed senescence.In this study,the mutant tcd34 was obtained by mutagenesis of japonica rice.variety Jiahua 1 with60Co?-ray irradiation,and its character analysis,map cloning,complementation verification,gene editing verification,subcellular localization experiment,bioinformatics analysis and related functions leads to the following conclusions:1.At 20?,the entire plant of the mutant tcd34 showed a complete albino phenotype,gradually withered and died after the four-leaf stage;while at 32?,the third leaf newly grown in the three-leaf stage is green,and the leaves in the four-leaf stage are basically green,and the plant was able to grow normally in the later stage,indicating that the mutant tcd34 is a low temperature sensitive lethal leaf color mutant.2.Consistent with the mutant tcd34 phenotype,the photosynthetic pigment content of the mutant decreased significantly at a low temperature of 20?,and the chloroplast did not show thylakoid structure required for photosynthesis;while at32?,its photosynthetic pigment the content and chloroplast structure gradually returned to normal levels.3.Through genetic analysis of the F₂ generation obtained from the cross between Pei'ai 64S and tcd34,it was found that the leaf color phenotype of the F₂generation plants appeared the phenomenon of green and white separation.After chi-square test,it was found that the result meet the 3:1 separation ratio.This indicates that the cold-sensitive leaf color mutation traits are controlled by a pair of single recessive nuclear genes?tcd34?.4.The mutant gene?tcd34?was located within 143 kb on chromosome 3 by using F₂ population obtained by crossing Pei'ai 64S with tcd34.The deletion of a base G in exon of a gene encoding PPR protein?LOC_Os03g40020?was found by DNA sequencing,which resulted in the early termination of translation.5.At the same time,through genetic complementation experiments and gene editing experiments,it was found that the gene encoding the PPR protein,LOC_Os03g40020,was the target gene TCD34.6.Performed homology analysis on the amino acid sequence of the protein encoded by the TCD34 gene,and found that it has high conservation in different higher plants;and it was found that TCD34 gene was most closely related to maize,which belonged to monocotyledonous plants.7.Analysis of tissue-specific expression showed that TCD34 was expressed in all tissues of plants,and the expression level in leaves was relatively high;subcellular localization analysis showed that TCD34 protein acts on chloroplasts.8.Analysis of mRNA transcription level,found that mutations in the TCD34gene directly caused significant down-regulation of genes related to photosynthesis and chloroplast development;and it was concluded that the TCD34 gene is mainly involved in regulating genes related to ribosomes and chloroplast division in the first stage of chloroplast development.In summary,the results show that the TCD34 gene is essential for the development and growth of chloroplasts in rice in a low temperature environment.At the same time,the gene encodes a PPR protein,and the preliminary exploration of the TCD34 gene provides a theoretical basis for a deeper understanding of the role of PPR protein in rice growth and chloroplast development.