Study Of Epigenetic Mechanisms Of OsBBM1 Resilence And Functionalanalysis Of OsPIE1 Gene

Epigenetics, including DNA methylation, histone modification, chromatin remodeling, small RNA and epigenome derived from high-throughput sequencing techniques, has become the hot field and forefront of bioscience. Rice is a main food crop and a classic monocot model plant, therefore, studying rice epigenetic regulation mechanisms is of great importance.In higher organisms, the gene expression pattern and epigenome are reprogrammed during the process of reproduction. Rice callus cell, like the embryonic stem cell in animals, possess the ability to regenerate a new plant, its regeneration process also includes the reprogramming of gene expression pattern and epigenome. In this study, we want to discover a novel epigenetic modification mechanism by focusing on a callus-specific expressing gene. Additionally, histone variants regulatory mechanism is rarely reported in rice. By sequence homology blasting, we found Os PIE1, a homologous protein of At PIE1 in Arabisopsis, and preliminarily analyzed the mechanisms of Os PIE1 regulating rice growth and development. The main results are as follows:1. The yeast-two-hybrid vectors, prokaryotic expression vectors and Os BBM1::LUC vectors were constructed. Os BBM1 over-expression, Os BBM1 RNAi and Os BBM1::LUC transgenic plants were obtained, and will provide access in finding the epigenetic suppressor of Os BBM1 through EMS mutation.2. Profile analysis found that Os BBM1 was callus-specific expression gene, and could be induced by plant hormone. During callus regeneration, DNA methylation level remained unchanged, while H3K4me3 and H3K9 ace level were down-regulated, and H3K27me3 level was up-regulated.3. Os BBM1 was silenced in the mutants or transgenic plants of some DNA methylation related factors such as ddm1 b, dng701, dmt704 RNAi, dmt706 RNAi,chromatin related factor chr729, histone modification related factors such as LHP1 RNAi,ox SDG711, SDG728 RNAi and other transgenic plants, indicating that Os BBM1 expression is not directly regulated by these epigenetic modification factors.4. Os PIE1 RNAi, PIE1 CRISPR/Cas9,INO80 RNAi、HTA705 RNAi,ox ARP6,PIE1 RNAi:: HTA705 RNAi, PIE1 RNAi::jmj705 and other H2 A.Z related transgenic and hybrid materials were obtained.5. Os PIE1 RNAi plants showed significant phenotype in spikelet, grain, flowering differences and lesion-mimic compared with transgenic negative plants. Western Blot demonstrated that, compared with transgenic negative plants, the total amount of histone H2 A.Z in Os PIE1 RNAi plants was significantly reduced.6. Expression level of some ABA pathyway related inducible genes were significantly higher in Os PIE1 RNAi materials than in transgenic negative plants, while H2 A.Z enrichment around TSS of these genes was down-rugulated. Expression level of some oxidative stress-related genes and hydrogen peroxide content by DAB staining were found increased in Os PIE1 RNAi materials, suggesting that PIE1 not only regulate ABA pathyway related genes by assembling H2 A.Z, but also oxidative stress related genes.