Cloning And Functional Analysis Of Chloroplast Mutant Gene Rps4 In Chinese Cabbage (Brassica Campestris Ssp. Pekinensis)

In higher plants,leaf color mutants are considered to be ideal materials for studying mechanisms of photosynthesis,chlorophyll synthesis,development and differentiation of chloroplast,gene function identification and nuclear-cytoplasmic interactions.Leaf color mutants can be divided into three types: controlled by nuclear genes,controlled by cytoplasmic genes,and controlled by nuclear-cytoplasmic interaction.Chinese cabbage(Brassica campestris ssp.pekinensis)is a vegetable crop of the genus Brassica.In this study,the Chinese cabbage yellowing mutant cdm was screened by EMS mutagenesis combined with isolated microspore culture.The physiological,biochemical and genetic analysis of cdm was carried out,and the yellowing gene was mapped by chloroplast whole genome resequencing,and the proteomics analysis of the yellowing mutant cdm and the corresponding wild type ?FT? was obtained by iTRAQ-labeled proteomics.A large number of differentially expressed proteins.The main results are as follows:1.Morphological and physiological characteristics of cdmThe mutant cdm in the seedling stage showed yellow inner young leaves,and the growth rate was slower than that of the wild type ?FT?.Chlorophyll deficiency and reduced growth rate in cdm were more apparent under lower temperature conditions,and gradually recovered.The mutant cdm was the total chloroplly deficiency type,and its photosynthetic parameters were significantly lower than wild type ?FT?,among them,Fv/Fm and ETRII in cdm was lower,and qN was higher,indicating that photosynthesis and photosynthetic efficiency of cdm are affected.The chloroplast grana lamellaes in cdm were loosely arranged,irregular and uneven,and lower degree of folding,the mitochondrial membrane boundary cdm was clear and the number of internal mites was normal,which is not different from ?FT?.2.Genetic characteristics of cdm and chloroplast genome sequencing resultsGenetic analysis of the mutant cdm showed that the chlorophyll deficient trait of the mutant cdm was controlled by cytoplasmic gene.The chloroplast genomes of wild-type 'FT' and mutant cdm were re-sequenced separately,then inter-compared sequencing data with the reference genome.A SNP(A-C)was found in the rps4 coding region with a mutation rate higher than 99%,resulting in Val substitution for Gly at residue 193 in RPS4 protein.qRT-PCR results showed that the expression level of the rps4 gene was down-regulated in cdm.3.Chloroplast rRNA processing characteristics in cdmChloroplast 16 S rRNA and 23 S rRNA were significantly reduced in cdm and cdm×?FT?,and their accumulation patterns were consistent by using formaldehyde agarose gel electrophoresis.The results of northern blot analysis showed that the mature 23 S,5S,4.5S and 16 S rRNA in cdm were significantly reduced,while the 23 S,4.5S and 16 S rRNA precursors were over-accumulated in cdm.This indicates that the processing of chloroplast rRNA in cdm has been affected.4.Analysis results of differential proteome based on iTRAQ technology6245 proteins were identified from the leaves of Chinese cabbage by using the iTRAQ technology.After comparing cdm and ?FT?,a total of 540 differential proteins were obtained,of which 233 were up-regulated and 307 were down-regulated.Significant enriched GO terms and metabolic pathways were obtained for GO annotation and KEGG Patheway analysis of total and differentially expressed proteins,respectively.The down-regulated proteins were mainly localized in the chloroplast,and significant enriched GO terms and metabolic pathways were associated with photosynthesis and carbon fixation,which may be the main cause of the mutated yellowing phenotype.Moreover,a class of proteins was associated with cold stress found in down-regulated proteins,suggesting that the more serious mutated phenotype of cdm under lower temperature conditions may be associated with these proteins.In the up-regulated proteins,some proteins were found to be involved in methylated,transcription and translation terminated,indicating that epigenetics may be associated with chlorophyll deficiency in cdm.5.Expression analysis of chloroplast gene encoding proteinAmong the 6245 total proteins,14 chloroplast gene-encoded proteins were found by annotation,12 of which were down-regulated,and the other two proteins were not significantly different.Analysis of the expression patterns of these 12 genes revealed that the differential expression level was small.This result suggests that down-regulation of these proteins encoded by the chloroplast gene may be due to reduced transcription levels or translational level in the chloroplast.The secondary maps were used to search the Arabidopsis database(Tair-10-pep-20101214),and 2346 proteins were identified.47 chloroplast genes-encoded proteins were down-regulated proteins,and only 3 were up-regulated.The results showed most of the proteins encoded by the chloroplast genes were down-regulated.