Whole-genome Analysis Of Histone H3K27me3 And Differences In Gene Expression In Tapidor Before And After Vanalization

Oilseedrape is one of the important oil crops in the world. But compared to rice, Arabidopsis thaliana and the other traditional model plants, there is few researches about epigenetics in oilseed rape, so the current study of epigenetics inoilseed rapehas an important significance.Chromatin immunoprecipitation(Ch IP), is the indispensable means for epigenetic research. Currently, Ch IP has been widely used in animals and plants studies, but the specific method of Ch IP for oilseed rape has not been reported.Flowering is the key physiological step in plant life and vernalization is the important condition for promoting flowering for winter type plants. Oilseed rape could be divided into two types, winter and spring type, based on the need of vernalization for flowering. So, the research about epigenetic mechanism regulating the flowering time for oilseed rape has great significance for further understanding of the molecular mechanism with vernalization. In this study, the leaves before and after vernalization of a winter type cultivar Tapidor was used as experimental materials, and the Ch IP techniques were mainly explored. After doing Ch IP by using H3K27me3 antibody, Ch IP-seq was done to analyze the histone H3K27me3 modificationin the whole-genome and and RNA-seq was done to analyze the differentially expressed genes before and after vernalization. The main results are as follows:1. We taked the Ch IP experiments of arabidopsis, corn, rice and other plants as reference, adjusted the process of Ch IP experiments and specific parameters. Among them, we identified formaldehyde cross-linking time as 10 min, sonication conditions for power with 20%, time with 30 min, frequency with opening 2s/off 9s, and other immunoprecipitation processing conditions, finally. At the same time, we used Real-time PCR to verify the results show that the Ch IP process can be used to do Ch IP-seq experiments.2. Ch IP-seq analysis showed that the mapping efficiency of reads to reference genome sequence was up to 90%, while the efficiency of unique reads was only about 25%. With MACS1.4 software detecting, 1607 and 10,662 peaks before and after vernalization were got. It was showed thatthe H3K27me3 modification of whole genome had significantly increasing after vernalization compared with that before vernalization. These peaks were mainly located in intergenic and promoter regions, and the distribution on TSS region were different obviously. The results of the annotation for the peaks showed that 513 genes were modified before vernalization and 3146 were modified after vernalization, with 0.5% and 3.1% of the total, respectively, and the number of same modified genes was 245. The statistical analysis results of expression level for H3K27me3 modified genes before and after vernalization indicated thatthe gene expression level was inhibited by H3K27me3 modification in Brassica napus L.3. RNA-seq analysis showed that the mapping efficiency of reads to reference genome sequence was more than 90%. Differences in gene expression analysis indicated that in the process of vernalization, there were 6947 differentially expressed genes, with 3382 up-regulated and 3565 down-regulated genes. GO analysis revealed that the differential expression genes were mainly concentrated in organelles, and the membrane organelles with the largest percentage; the molecular function annotation of differentially expressed gene were mainly concentrated in the primary bindings and enzyme activities; differentially expressed genes mainly concentrated in metabolic pathways, stress resistance pathway to stimulate response, cells and signal transduction pathways and the other biological process. KEGG Pathway analysis showed the differential expression genes mainly enriched in the metabolism of carbohydrates, fats, amino acids, followed by energy metabolism, the biosynthesis of secondary metabolites, degradation of exogenous factors, aspects of signal transduction and the immune system so on.4. Among the differentially expressed genes, 399 genes which with H3K27me3 modification in total, including 62 up-regulated and 337 down-regulated genes, and the percentage was 84.46% and 15.54%, respectively. In the process of vernalization, FLC geneswere inhibited by H3K27me3 and down-regulated, but the changes of H3K27me3 level of the other key genes in vernalization pathway are not significantly.