| Rice is one of the most important food crops and an excellent cereal model plant.Leaf senescence is one of the restrictive factors for yield and quality,therefore,it has important theoretical and practical significance to study rice leaf senescence mechanism.Recently,more and more studies have proved that epigenetic modifications play important roles in gene expression reprogramming during rice growth and development,yield,quality,response to stresses,and crop breeding.However,the relationship between epigenetic modifications and leaf senescence in rice remains largely unknown.In this study,the abundance changes of H3K9ac during rice flag leaf senescence was measured on a genome-wide scale using ChIP-seq for the first time,and the relationship between histone acetylation and leaf senescence in rice has been revealed.The results are as follows:1.The chromatin immunoprecipitation technique system in rice was optimized,and the optimal conditions for two types of sonicator machine were:(1)Bandelin Sonoplus HD2070:16 times×30 s,40%duty cycle,20%power,1 min interval.(2)Covaris M220 Focused-ultrasonicator:10%duty cycle,75 Watts Intensity Peak Incident power,200 cycle per Burst,7℃ Bath Temperature,25 min.The appropriate dosage of two antibodies(H3K4me3 and H3K9ac)were verified to be 3 μL,which was convenient for subsequent experiments.2.The changes of leaf color,morphology,chlorophyll content and chlorophyll fluorescence(Fv/Fm)were analyzed and the expression pattern of senescence-related genes were tested to confirm the accuracy of the material.At the same time,the changes of histone modifications(H3K4me3 and H3K9ac)during the different aging phases of two S40 genes were detected by chromatin immunoprecipitation technique.Then the histone modifications(H3K9ac)with more significant changes at different times relative to H3K4me3 were selected for a whole genome ChIP sequencing.3.A genome-wide analysis of H3K9ac modifications during flag leaf senescence in rice was performed.(1)The abundance level of H3K9ac was found to be increased during flag leaf aging.The peak of read counts were mostly located at the 500 bp region downstream of the transcription.start site(TSS),indicating that H3K9ac modification may play important roles in regulating gene transcription initiation.Then the H3K9ac modification significant difference related genes(≥8 pair-wise comparisons)were screened for the following experiments.(2)GO and KEGG enrichment analysis were performed on differential H3K9ac modified genes of all comparisons.It was found that they were involved in various biological processes that were related to senescence.Genes related to chlorophyll degradation,photosynthesis,protein degradation and synthesis of sugars were enriched.These biological processes play the center role of rice senescence regulation,showing that H3K9ac modification should be involved in regulating rice leaf senescence process through regulating those genes that were mentioned above.(3)Gene expression information in RiceXPro database was used to analyze the relationship between H3K9ac modification and gene expression.By identifying genes including TF that show positive correction in H3K9ac modification and gene expression,our study provides important gene resources for further study on the epigenetic regulatory mechanism during senescence in rice.4、The ChIP-qPCR technique was used to detect the changes of H3K9ac modification in OsGDCH,bHLH and OsS40-7 during different leaf aging period.The results of ChIP-PCR showed consistence with the ChIP-seq result,confirming the reliability of the ChIP-seq.Further,the expression pattern of the modified differential genes at different developmental stages were detected using qRT-PCR.The results verified a relationship between gene expression and H3K9ac modification.In summary,a significant different changes in H3K9ac modification level was observed at different aging stages of rice flag leaf We proposed that H3K9ac modification plays important roles in the regulation of rice leaf senescence.Further study is also necessary for the histone modification mechanism on specific genes. |
