Cloning And Functional Analysis Of The Essential Gene TSV13 For Chloroplast Development In Rice Under Low Temperature Conditions

Ribonuclease II,a nucleic acid hydrolase commonly found in organisms,acts to regulate the type and quantitative distribution of RNAs and is involved in RNA shearing,modification and degradation.In plants,it affects chloroplast r RNAs processing factors and is involved in the maturation of the transcription products of chloroplast r RNAs.In this study,a low temperature albino mutant strain tsv13 with stable genetic ability was obtained by mutagenesis of japonica rice"Jiahua 1"with EMS(ethyl methanesulfonate),and its leaf color phenotype,chloroplast development,gene localization cloning,tissue specificity and subcellular localization were analyzed,and the following conclusions were obtained:1.At a low temperature of 20°C,the rice mutant tsv13 showed whitish leaf color,started to wilt and die at the four-leaf stage,and had significantly down-regulated chlorophyll content and incomplete chloroplast development,whereas at 24°C and above,the leaf color was green phenotype,the chlorophyll content of seedlings was basically the same as that of the wild type,and there was an intact chloroplast structure.This indicated that the tsv13 mutant showed a major defect in its chloroplast development at a low temperature of 20°C.2.Genetic analysis of the F₂ generation of the tsv13 mutant showed that two leaf color traits segregated in the F₂ generation,green(wild type)and white(mutant),with a 3:1 ratio of trait segregation,indicating that the mutant trait was controlled by a recessive nuclear gene(tsv13).3.The map cloning localized the mutated gene TSV13 to an interval of 186 kb on rice chromosome 1,which has 25 genes,and sequencing revealed a mutation of a"G"base in the coding region of a ribonuclease II gene(LOC?Os01g62440)to"A".A"in the coding region of a ribonuclease II gene(LOC?Os01g62440),resulting in premature translation termination.Meanwhile,both knockout and complementation experiments verified that this gene is the target gene TSV13,which affects chloroplast development in rice seedlings at low temperature,and the TSV13 protein is localized in rice chloroplasts.4.The m RNA transcript level analysis of chloroplast-related genes in tsv13 mutant showed that almost all NEP-related genes appeared to be more than 5-fold highly expressed at 20°C,while almost all PEP-related genes were again not expressed;at32°C,the expression of both NEP and PEP-related pathway genes were restored to different degrees.This indicates that this gene is involved in the developmental process of rice chloroplasts and plays an irreplaceable role for the normal development of rice under low temperature environment.