DNA Methylation Of ASL8 Gene Encoding RNA-binding Protein Inhibits Chloroplast Development In Rice(Oryza Sativa L.)

Chloroplast development has an important influence for growth and development of plants.At the same time,it is regulated by the nuclear genes and plastid genes together,mutations of any gene can lead to loss of its function,which will affect the growth and development of the chloroplast,cause the leaf color change,even lead to the plant death.In addition,gene expression is controlled by the DNA sequence and epigenetics.A rice leaf color mutant asl8 derived from japonica variety Jiahua No.1treated by ⁶⁰Coγ-rays was used in this study.Characterizations of the asl8 mutant could be conclused as follows:1.Compared with the wild type（WT）,the asl8 was a yellow lethal mutant,which not affected by temperature control.The mutant of photosynthetic pigment content significantly decreased than those in WT plants,which no complete chloroplast structure.2.Genetic analysis on individuals from cross of Pei’ai 64S and asl8 revealed that the phenotype was controlled by a single recessive nuclear gene.The mutant gene was localized on chromosome 8 of LOC＿Os08g37700 gene encoding RNA binding protein,which could occur methylation.3.By predicting methylation,demethylation experiment and BSP sequencing found which LOC＿Os08g37700 gene belongs to DNA methylation mutation which found the gene had two methylation change at initiation codon ATG downstream+223and+268 bp loci.4.The gene was further confirmed by complementation and knockout tests,the results suggested that LOC＿Os08g37700 is responsible for the phenotype of asl8mutants.5.Semiquantitative assay was performed to study the expression profile of ASL8.Results show that ASL8 was expressed with a significantly high level of expression detected in leaves,especially in young leaves.In tobacco cells,the ASL8 protein displayed a typical chloroplast location pattern.Real-time PCR analyses showed that the expression levels of the genes associated with chloroplast development were seriously affected in asl8 mutants.6.Conservative analysis on amino acid sequence encoding by ASL8 revealed that they share high sequence similarity to different higher plants.Further analysis of the homologous evolution relationship with more species,showing that ASL8 is more conserved in monocotyledons,rather than dicotyledons.This study verified the mutant gene ASL8,it belonged to methylation of epigenetics,meanwhile did the function analysis.In conclusion,the aim of this study is to provide new theoretical basis and reference for later study of chloroplast development and its related gene expression regulation mechanism.