Studies On DNA Methylation During Dedifferentiation Of Mature Wheat Embryos

In order to study the change of DNA methylation in wheat mature embryos dedifferentiation, this paper researched the expression of related genes of DNA methylation using oligonucleotide microarray, and determined the percentage of 5-methylcytosine in mature wheat embryo genome dedifferentiation using HPLC (High Performance Liquid Chromatograph), and screened the change situs during DNA methylation of in mature wheat embryos in dedifferentiation using molecular marker MSAP. So that revealed the change rule during DNA methylation in mature wheat embryo dedifferentiation, and ultimately knew the function of DNA methylation in wheat dedifferentiation. The main results were as follows:1. The expression changes of genes related to DNA methylation and histone modification were researched using Affymetrix GeneChip? Wheat Genome array in mature wheat embryo dedifferentiation,and then definited some significant genes in dedifferentiation.This study estimated the expression situation of genes involved in DNA methylation and histone modification in mature wheat embryo dedifferentiation(0,2,6,12,24 and 72 h)using Affymetrix GeneChip? Wheat Genome array, and finally indicated that there were 36 genes related to DNA methylation and histone modification. Of 36 genes, at least 16 genes were expressed significantly at any point in wheat dedifferentiation; 9 genes, CA500212 and so on, were upregulated; 6 genes, CD373978 and so on, were downregulated and only BJ301005 was upregulated sometimes and downregulated sometimes(to call for short up/down -regulated expression). Among the total, in the SST(0-2h), 3 genes, BJ213871 and so on, were upregulated initially and CD373978 and CD912490 were downregulated initially; in the SSR(2-12h),8 genes, CA500212 and so on,were upregulated initially and 5 genes, CD373978 and so on,were downregulated initially;in the SCR(12-24h), BQ166624 and BJ308556 were upregulated initially and BQ161896 and CA600655 were downregulated initially;in the SCF(24-72h),only one gene, BQ162603 was upregulated initially and BQ161896 and CA600655 were downregulated initially. The whole situation of the genes expression was that the total frequencies of up and down-regulated expression were respectively 44 and 23, i.e. four phases above-mentioned of wheat dedifferentiation, the number of up-regulation and down-regulation was separately 3 and 2, 18 and 9, 12 and 7, 12 and 5. Range of up-regulation was from 2 to 34 times higher than control, and that of down-regulation was also 2-17.2. Using HPLC (high Performance Iiquid Chromatograph) measured percent of 5-methylcytosine of mature embryo genome during dedifferentiation in wheat, and compared with the percent of 5-methylcytosine of no 2,4-dichlorophenoxy acetic acid treatment mature embryo genome.The result indicated at 0 h the percent was 21.53%, and reduced to 2.75% culturing 2 h in 2mg/L 2,4-D MS, along with the temporal continue, the contain gradually hoist,12 h up to tiptop (24.30%), and then fell(72 h was 14.97%). Compared with no 2mg/L 2,4-D treatment, they presented abruptly fall in 0-2 h and patencily descend in 24-72 h.3. MSAP(Methylation Sensitive Amplification Polymorphism)analysed during mature wheat embryos in the dedifferentiation.The study screened the changes situes of DNA methylation in wheat mature embryos dedifferentiation using MSAP. The results indicated that 4 primer pairs of MspI,Hpaâ…¡/ EcoRI combinations produced 23 polymorphic bands. The results of selective amplification indicated that Polymorphism of bands represented the change of DNA methylation because that Mspâ… , Hpaâ…¡/ EcoRI combinations could distinguish the changeable situs of DNA methylation.Bands sorted into 4 groups according to the appearance or disappearance of bands:the first group was these bands showed up gradually in dedifferentiation ,with hemimethylation of C5mCGG or hemimethylation of 5mCCGG; the second was the band disappeared gradually.with full methylation of C5mCGG or hemimethylation of 5mCCGG; the third was that the band appeared primal and then disappeared,with hemimethylation of 5mCCGG; the last was the band disappeared after appeared and appeared at last, what deteced the full methylation of sequence of C5mCGG and then there being no methylation at 2-6h, at last hemimethylation of C5mCGG at 24-72h. In the all, there was a dynamic chang in the whole dedifferentiation.