Identification And Expression Analysis Of Chloroplast RRM-Containing Proteins（cpRNPs） In Arabidopsis And Rice As Well As Functional Study Of The Rice Oscp31B Gene In Chilling Tolerance

Temperature stress could occur at any time during the life cycle,causing adverse effects on plant growth and development.In order to adapt the changes in temperatures,plants have evolved response mechanisms which help them to survive and grow.For instance,cold acclimation can improve the freezing tolerance of plants and its mechanism has been well researched.However,the molecular mechanism of chilling adaption is not clear.Studies have shown that the process of protein translation in chloroplasts is particularly sensitive to cold stress.Many genes associated with chloroplasts show a certain degree of response to low temperature at the transcript level.Before chloroplast precursor RNA becomes a mature product,it often needs to be processed by many nuclear coding factors,including terminal deletion,intron cutting and base editing.Most of these factors are RNA-binding proteins（RNP）.This study intends to annotate and characterize expression patterns of the cpRNP members in Arabidopsis and rice.In addition,a functional study was carried out on Oscp31B,one a cpRNP gene in rice,in chilling tolerance.1.Annotation of cpRNP genes in Arabidopsis and riceWe used bioinformatics tools to perform genome-wide identification and sequence analysis of cpRNP proteins in Arabidopsis and rice.Based on the presence of two RRM domains and an N-terminal chloroplast targeting signal peptide,ten cpRNP genes were identified in each of the Arabidopsis and rice genomes.Comparison of intron/exon justions,relative molecular weight,and isoelectric point of the protein revealed that most of the cpRNP genes in Arabidopsis have apparent orthologous gene in rice.These cpRNP proteins,either from Arabidopsis or rice,are localized in the chloroplast.The cpRNP genes have different tissue expression patterns.Most genes are highly expressed in green tissues.Their expressions respond to abiotic stresses such as cold,heat and drought.Our study thus provides a comprehensive annotation of the cpRNP protein family and their expression patterns in Arabidopsis and rice,and it will assist further study of the role of the cpRNP genes in plant growth and stress response.2.Functional analysis of the rice Oscp31B gene in cold tolerancePrior to this study,53 Quantitative Trait Loci（QTLs）related to cold tolerance at the bud stage were detected from 187 rice natural accessions through Genome wide association study（GWAS）.This study functionally analyzed one candidate gene Oscp31B which is located in a major QTL interval on chromosome 9.We generated knockout mutants of the Oscp31B gene using the CRISPR/Cas9 gene editing technology.Under low temperature stress but not at normal growth temperature,the Oscp31B mutant exhibited a faster germination than the wild type.The mutant also exhibited a higher chilling tolerance at the seedling stage compared to the wild type.Quantitative real-time PCR revealed that the expression of the Oscp31B gene was decreased after low temperature treatment.The Oscp31B protein was localized on chloroplasts,and the mutant had reduced chlorophyll content compared to the wild type.These results indicate that the Oscp31B gene is a negative regulator of chilling tolerance in rice and it may do so by affecting chloroplast function.