The Possible Regulatory Mechanism Of H3K27me3 During The Ripening Of Fragaria Vesca

Studies on the regulatory mechanisms during the development processof fruit provide an important theoretical basis for the breeding works of new strawberry varieties.Chromatin immunoprecipitation techique is an important tool for studying the distributionn of histone modification across genome in an epigenetic perspective.However,the application of this method in strawberry research has not been reported,due to its high content of pectin,which results in low efficientcy of immunoprecipitation.We explored the applicability of two different chromatin immunoprecipitation techniques,X-ChIP and N-ChIP,in the seedlings and fruits of woodland strawberry.And we optimised the chromatin immunoprecipitation for strawberry on the basis of existed methods.We obtained the distribution of histone modification of H3K27me3 along the genome of strawberry at white and red stages,using the established ChIP method we estabalished in strawberry.We further performed transcriptomic sequencing to obtain information on gene expression changes during the development and ripening process of strawberry fruits.Besides,genes modified by H3K27me3 in white and red fruits were screened.The possible regulation mechanism of H3K27me3 during fruit development and ripening process was discussed by ChIP-seq and RNA-seq data.The combined analysis of transcriptomics and epigenetics help to understand the role of chromatin landscape changes in the process of gene expression during fruit development and ripening process.The main results of this research are as follows:1.We applied X-ChIP method into woodland strawberry,taking seedlings and fruits as materials,to evaluate whether X-ChIP is suitable for woodland strawberry Fragaria vesca.We optimized the parameters of sonication conditions to obtain suitable chromatin fragments.As shown in our results,we got the appropriate size of chromatin when the parameters of sonication machine(high-throughput sequencer sample pretreatment system-model:USD-600TS)were set as:15s on,30s off,6 cycles at High level.We carried out ChIP experiment using antibodies of anti-H3K9me2 and anti-H3K36me3.The results were evaluated by qPCR experiment.As shown in the ChIP-qPCR results,the fold change of H3K9me2 and H3K36me3 are 9.78-62,5.32-16.11 in seedlings,3.16-17.48、3.34-8.56 in fruit.However,the enrichments of negative control were up to 3.12%,3.29%taking anti-H3K9me2 and anti-H3K36me3 as antibodies in ChIP experiment of fruit.Thus,X-ChIP method is more suitable in seedlings than fruit,where it has high non-specific background noise.2.To decresase the non-specific background in ChIP experiment of fruit,we further explored the applicability of Native-ChIP method in strawberry fruit.Fistly,we explored the appropriate digestion time of micrococcal nuclease.As shown in agarose gel electrophoresis,most of mononucleosome(～146bp)were concentrated when digeted for ten minutes.The results showed that enrichments were shown,taking H3K9me2 and H3K36me3 as antibodies,the fold change is 6.11-61.25,11.77-35.96 of H3K9me2 and H3K36me3,the background values are lower than 2%,significantly lower than X-ChIP method.3.The pectin content is very high in straeberry fruit at the early stage,which affects the application of ChIP experiment in immature strawberry fruit successfully.In our study,the method of high-salt solution was investigated on the effects of the removal of pectin in white fruit in order to get high pull down efficiency of ChIP experiment.As shown in the results,the addition of high concentration of NaCl can significantly decrease the content of pectin,and we performed ChIP experiment by using antibody of H3K27me3successfully,which is helpful for the study of epigenetics in strawberry at early development stage.4.The transcriptome data of strawberry at five ripening stages were obtained by RNA-seq.Genes related with fruit ripening were screened and we obtnined various expression levels of IAA related genes and ABA related genes during the ripening process of strawberry.11306 differentially expressed genes were discovered between white stage and red stage.6063 genes were up regulated and 5243 genes were down regulated,which indicated that a large number of genes changed during fruit development process.Gene ontology analysis showed that the differentially expressed genes were involved in cell metabolism,lipid metabolism,protein transport,cell membrane formation,secondary metabolism synthesis and tricarboxylic acid cycle metabolism.5.On account of the high background noise in X-ChIP experiment taking fruit as materials,we performed N-ChIP experiment using strawberry fruit at white stage and red stage.Combined with high-throughput sequencing technology,ChIP-seq analysis of histone modification H3K27me3 was performed by improved N-ChIP method,covering white fruit and red fruit,which represent the immatural and matural fruit of woodland strawberry.The distribution maps of H3K27me3 in the whole genome of woodland strawberry from immature to mature were obtained.Motif of H3K27me3 was found and distribution of H3K27me3 along the gene function elements shows that most of the peaks cover promoter regions.Combined with RNA-seq analysis,we discovered 4085 genes marked by histone modification H3K27me3 in white fruit,4590 genes in red fruit.And most of genes in white and red fruit were down regulated.GO and Pathway analysis showed that genes marked by H3K27me3 have the functions of protein binding,nucleic acid binding and ATP binding functions and were involved in plant hormone signal transduction、starch and sucrose metabolism pathway et al.Furthermore,seven genes most likely regulated by H3K27me3 were screened in our study.