Study On Epigentic And The Molecular Mechanism Underlying The Difference Between Hevea Brasiliensis Self-rooting Juvenile And Donor Clones

In our previous study, our research group foud differences in methylation polymorphism between the genome DNA of rubber tree self rooting juvenile clones and their donor clones. Additionally, differentially expressed of gene and protein existed in the latex of rubber tree self rooting juvenile clones and their donor clones, so causing these differences should be associated with epigenetic. In order to further study the relationship between epigenetic and self rooting juvenile clones and its differences with the donor, we cloned three cytosine methylase genes of the Hevea brasiliensis, and analyzed their expression in self-rooting juvenile clones and their donor clones, as well as, we analyzed the differences between the key enzyme genes promoter during (in the process of) rubber biosynthesis in self-rooting juvenile clones and the donor methylation. The research laid the foundation for the elucidation of the molecular mechanism of differences which in rubber tree self rooting juvenile and donor.The full-length cDNA encoding three cytosine methylase genes respectively designated HbMET, HbCMT, HbDRM, were isolated from rubber tree by using PCR and RACE techniques. HbMETl was4896bp in length and contained an open reading frame of4635bp. Including248bp3’-UTR, Sequence analysis revealed that the ORF of HbMET1encode1545amino acid residues with a predicted molecular mass of174.36kD. The isoelectric point is6.36. HbCMT was2697bp in length and contained an open reading frame of2556bp. Including116bp3’-UTR, Sequence analysis revealed that the ORF of HbCMT encode851amino acid residues with a predicted molecular mass of95.67kD. The isoelectric point is5.38. HbDRM was2245bp in length and contained an open reading frame of1917bp. Including145bp5’-UTR and176bp3’-UTR, Sequence analysis revealed that the ORF of HbDRM encode638amino acid residues with a predicted molecular mass of71.39kD. The isoelectric point is4.90. Quantitative RT-PCR analysis indicates that three cytosine methylase genes of the rubber tree are almost expressed in roots, bark, leaves, flowers, latex, embryos and callus, and more highly expressed in flowers and callus than in latex. In addition, the three genes are highly expressed in latex of old clones than in self-rooting juvenile clones.Bisulfite sequencing of the promoter region of HbHMGR1、HbFDP1、HbREF and HbSRPP, witch from self-rooting juvenile clones and old clones, were performed to find out the exact location, class, and extent of methylation, cytosine methylation of HbSRPP and HbHMGR1in self-rooting juvenile clones and the donor are significantly different, but HbFDPl and HbREF has no obvious difference. Quantitative RT-PCR analysis indicates that HbSRPP、HbFDP1、HbHMGR1and HbREF are all highly expressed in latex of self-rooting juvenile clones than in old clones.