Cloning And Functional Analysis Of Essential Gene TCM8 For Chloroplast Development Under Low Temperature Conditions In Rice (Oryza Sativa L.)

The chlorophyll synthesis of plants is involved in a series of related genes.If one of the genes is mutated,its function loss will block the normal synthesis of chlorophyll and affect the normal development of chloroplasts,and it will result in the mutations of the color and affect the photosynthesis.On the other hand,it can even caused the death of plants.In this study,A rice low temperature sensitive mutant tcm8 was derived from japonica variety Jiahua No.1 treated by ⁶⁰Co?-rays.Characterizations of the tcm8mutant could be concluded as follows:1.Through analyzing the phenotype of the two mutations under different temperatures,in the condition of 20?,tcm8 always presents an albino phenotype until death.However at 32?,leaf phenotype of tcm8 always consistent with that of the wild type?WT?.In addition,it was found by transmission electron microscopy that there was no significant difference between tcm8 and wild type at 32°C,there were complete chloroplast and thylakoid fold structure in the plant.At 20°C,the chloroplast of tcm8was destroyed and there was no complete chloroplast in the plant,besides it contains a lot of bubble structure.2.By analyzing the photosynthetic pigment content of 3-leaf-stage seedlings grown at 20?and 32?we can see that this phenotype was associated with the accumulation of photosynthetic pigment.At 20°C,the photosynthetic pigment contents in tcm8 mutants at the 3-leaf stage were drastically lower than those in WT plants.However,the pigment levels were similar to WT plants at 32°C.3.Genetic analysis on individuals from the cross of Pei'ai 64S and tcm8 revealed that the phenotype was controlled by a single recessive nuclear gene.4.A map-based cloning of the mutant gene was conducted.The mutant gene was localized between the Indel markers ID00607 and ID00785 on chromosome 8.A three-base mutation in the LOC_Os08g01870?TCM8?gene encoding the PPR protein was finally determined,resulting in a change in the protein sequence that ultimately affected the leaf color phenotype.5.This gene was validated by complementary experiment and CRISPR/Cas9genome editing experiment,which successfully demonstrated that the mutation of this gene resulted in an albino trait of the mutant.6.Conservative analysis on amino acid sequence encoding by TCM8 revealed that it shares high sequence similarity to different higher plants.Further analysis of the homologous evolution relationship with more species,showing that TCM8 is more conserved in monocotyledons,rather than dicotyledons.It was found by subcellular localization that TCM8 acts mainly in chloroplasts.7.The analysis of transcript levels of chlorophyll synthesis,photosynthesis and chloroplast development in tcm8 mutants showed that the gene TCM8 may affect the early development of chloroplasts,mainly in the first stage of chloroplast development,thus causing slow chloroplast development and affecting chlorophyll.Accumulation and photosynthesis,resulting in a mutant with a whitened phenotype.This study reports a chloroplast development essential gene TCM8 which encodes a PPR protein under low temperature conditions,and it provides a new idea for studying the function of PPR protein,photosynthesis metabolism and chloroplast development mechanism in plants.