The Study Of The Expression Of Non-imprinted Gene Expression In OsEMF2b Regulated Rice Flower Organ Development Pathway And DNA Methylation

Part I:Study on the regulation of OsEMF2binvolved in floral organ development in RiceEMBRYONIC FLOWER(EMF)is a Polycomb-group protein(PcG)protein whose lack of gene function causes the vegetative growth process of Arabidopsis thaliana to enter the reproductive growth stage quickly,because the plant does not accumulate enough nutrients,Mustard plants are short,reproductive growth stage of flower organ abnormal development and so on.In Arabidopsis,EMF2 and EMF1 constitute PRC2 protein to regulate flower organ-specific gene expression by H3K27me3 modification.There were two homologous genes of EMF2:OsEMF2a and OsEMF2b,and OsEMF2b regulated the flower organ development of rice by the epigenetic modification of H3K27me3.Studies have shown that chromomethylase3(CMT3),a DNA methyltransferase acting on the CNG site,has only DNA methylation in H3K27me3 in Arabidopsis,suggesting that H3K27me3 provides a recognition site for DNA methylation,Further shows that DNA methylation is involved in the regulation of flower organ development.In order to explore whether DNA methylation plays an important role in the regulation of rice,the T-DNA insertion mutant of OsEMF2b was used as the experimental material.The agronomic traits,chip analysis,real-time fluorescence quantitative PCR,sulfite sequencing The experiment concludes:The plant growth of Os EMF2b T-DNA insertion mutant was higher than that of wild type.The floral organ was malformed,the plant was short,the tiller was small and the panicle was less.The floral organ development was abnormal and the floral organ had different degree of variation.The results showed that OsMADS1 and OsMADS6(195.862 times and 51.2921 times,respectively)were significantly higher than those in wild type(P<0.05).The results showed that OsMADSl and OsMADS6 were significantly higher than those in wild type OsMADS2 and OsMADS16(43.6661 times and 14.6222 times,respectively)were significantly higher than those in the wild type.OsMADS1 and OsMADS2 were used to carry out sulfite sequencing experiments.There was no DNA methylation site in OsMADS1 gene.Although there was methyl site in OsMADS2 gene,it was proved that there was DNA methylation,but wild type And the mutant material OsMADS2 gene methylation site of the same,that is to prove OsMF2b changes OsMADS2 gene DNA methylation did not have an impact,so we speculated that OsEMF2b control of rice flower organ development process is not DNA methylation is involved in the regulation of regulation,and then speculated that H3K27me3 histone methylation modification in rice and Arabidopsis in the regulation of the way there are differences.In Arabidopsis,the chromatin domain agonist DNA-binding protein 3(CHD3)regulates the meristematic activity of Arabidopsis thaliana roots by antagonizing the H3K4me2,H3K27me3 pathway and PcG protein.In rice,CHR729(homologous gene of Arabidopsis thaliana CHD3 protein)also changed the expression level of H3K27me3.The traits of this gene function are very similar to those of the OsEMF2b mutant.In order to explore the existence of the same protein interaction pattern in rice and Arabidopsis thaliana,there was no direct interaction between OsEMF2b and CHD3 homologous gene CHR729 by yeast two-hybrid experiment.Therefore,it was suggested that Arabidopsis thaliana There is a difference in the mechanism of protein interaction between OsEMF2b in rice,but needs further validation.Part ?:The effect of DNA methylation on the expression of non-imprinted genes Genomic imprinting means that alleles from the parent and the parent have been modified when passed to the offspring through spermatozoa and eggs,so that alleles with parental imprinting have different expression properties,which are often DNA methylation Modified,including histone acetylation,histone methylation modification and so on.At present,the experimental results show that the expression of imprinted genes is regulated by epigenetic mechanisms,especially the DNA methylation regulation.However,non-imprinted gene expression regulation is still no exact results.There are two kinds of DNA methylation regulation in animal and plant.One is that the expression of SNPs does not change,and the other is only one of the SNPs.We speculated that the altered SNPs could be related to the heterosis of F1,and found that the non-imprinted genes could be imprinted at different developmental stages and became the imprinted genes.The above experimental results showed that DNA methylation was involved in a certain The expression of the imprinted genes.In order to clarify whether DNA methylation is involved in the regulation of imprinted gene expression and also regulate the expression of non-imprinted genes,we choose japonica rice(Nip)and indica rice 9311 as female parent,The endosperm of the seeds of 3-5 days and the roots,stems and leaves of the parent were used as experimental materials.Four non-imprinted genes were identified by gene function annotation and literature.Four genes were used as the research target.Nip and 9311 seeds were detected by DNA methylation inhibitor 5-azadC.The expression of four non-imprinted genes and SNPs were analyzed.The results were as follows:The seeds of F1 hybrids of Japanese sunny and indica rice 9311 were slowed down by DNA methylation inhibitor 5-azadC(30?mol/L),and some plants were slowly withered.And endosperm-specific gene expression site changes,endosperm-specific gene is no longer only expressed in the endosperm,but also expressed in the leaves.Four non-imprinted genes were amplified and sequenced.Two genes were found to be imprinted and the two genes were still non-imprinted.So we can speculate that DNA methylation plays a role in the expression of non-imprinted genes,and it can be speculated that DNA methylation is one of the transforming factors of imprinted and non-imprinted genes.