Expression Of Chloroplast RNA-binding Protein Genes And DNA Methylation Changes In Promoter Of Wheat Chloroplast Genes At Low Temperature

Transcriptome data revealed that chloroplast located RNA-binding protein(RBP)gene expression was significantly different in low temperature-induced Albinism Line of wheat and its control material.According to reporte,combine pre-data of microarray and transcriptome.Compares these genes in the wheat genome database and predicted subcellular localization with online-web.Screened RBP genes that were located or predicted to be located in the chloroplasts.Bioinformatics analysis was performed on them.Semi-quantitative PCR and q PCR were used to detect changes in the expression pattern of RBP genes under low temperature treatment.Further to analysis the correlation between RBP gene and leaf whitening at low temperature conditions.At the same time,in order to study the methylation changes of wheat chloroplast genes and reveal whether chloroplast gene methylation regulates the genes expression,we established a methylation detected systerm of chloroplast genes in wheat by sulfite sequencing method(BSP-seq).Selected five chloroplast genes(pet N,trn C,pet D,rrn16 S,and rrn23S)which were differentially expressed in Albinism Line and Aibian No.1,to determine promoter methylation by BSP-seq and gene expression by q PCR.Initially revealed the changes in gene methylation of chloroplasts in wheat at low temperature.Through the above studies,the following results are mainly obtained:(1)Bioinformatics analysis of thirteen RBP genes showed that most genes have a signal peptide located in the chloroplast.The homologous of amino acid sequences are poor,most of them have RNA-binding domains.The promoter element analysis showed that CK215294,Ta SGR-2AL and Ta SGR-2BL contain low temperature responsive elements(LTR).COR14,Ta CP29,Ta CSP41 A,Ta CSP41 B and Ta GRP2 contain ABA response elements(ABRE).(2)Semi-quantitative PCR and q PCR showed that the RBP genes in Albinism Line were more sensitive to low temperature than Aibian No.1.According to the expression pattern,the RBP gene can be divided into two types.Genes expression of one type are relatively low(Ta GRP2,Ta RBD1,Ta SGR-2AL,Ta SGR-2BL,Ta SGR-2DL),and there is no obvious difference between the two materials;The genes expression of the second type in the Albinism Line are slightly higher than Aibian No.1(CP31,Ta CSP41 A,Ta CSP41 B,CK215294 and RNase T2 family genes)at room temperature.During the short-term low temperature treatment(5 days to 30 days),these genes expressed in the Albinism Line were up-regulated firstly,while under the longe-term low temperature treatment(80 days)its expression began to decline,and ultimately lower than Aibian No.1.(3)Measure the promoter methylation of five chloroplast genes at low temperature.The results showed that: the low temperature treatment will increase the total methylation level of chloroplast genome,but the methylation and the trend of five genes is not exactly the same.The expression analysis show that the expression of five chloroplast genes are different,and the expression patterns of different chloroplast genes are also different.Methylation is involved in the expression regulation of pet N.The above results lay the foundation for studying the expression regulation of chloroplast genes in wheat under low temperature conditions at the transcriptional and posttranscriptional levels,and further clarifying the molecular mechanism of bleaching leaf of Albinism Line.