关键词: 高低割线橡胶树, DNA甲基化, 甲基化敏感扩增多态性(MSAP), 体系优化

Abstract: Using the tender leaves of Hevea brasiliensis variety‘CATAS 7-33-97’as materials, several key influencing factors in leaf genomic DNA restriction enzyme digestion, pre-amplification and selective amplification system which affected the system quality of MSAP were optimized through the single factor and orthogonal design method in order to obtain the optimal MSAP reaction system of rubber tree. The optimized reaction system of MSAP was used for difference analysis of DNA methylation in two kinds of H.brasiliensis of high and low tapping cuts. The results showed that 750 ng genomic DNA in a reaction volume of 50 μL could be fully digested by EcoRⅠ 20 U, HpaⅡ 20 U or MspⅠ 10 U at 37℃ for 10 h. MSAP-PCR pre-amplification was performed in 20 μL of reaction volume with the following mix:ligation products 4 μL, MgCl₂(25 mmol·L^-1)0.15 μL, dNTPs(2.5 mmol·L^-1)0.1 μL, Taq polymerase(5 U·μL^-1) 0.1 μL, 10×PCR Buffer 2 μL, primer E-00/HM-00(10 μmol·L^-1) 0.3 μL, respectively. The 20 μL selective reaction mixture contained 20 times diluted amplification products 2 μL, MgCl₂(25 mmol·L^-1) 0.1 μL, dNTPs(2.5 mmol·L^-1) 0.125 μL, Taq polymerase(5 U·μL^-1) 0.1 μL, 10×PCR Buffer 2 μL, primer E+3/HM+3(10 μmol·L^-1) 0.4 μL respectively. The total methylation rates of two samples were 37.22% and 36.43%, respectively. The full methylation rates were more than the hemi-methylation rates in H.brasiliensis of different tapping cuts. Therefore, CpG methylation may represent the major form of DNA methylation in rubber tree. Our data indicated that this MSAP reaction system with high stability and reliablity and good reproducibility have provided the foundation for different degree tapping stress of rubber tree associated research with the MSAP technology.

Key words: Hevea brasiliensis of high and low tapping cuts, DNA methylation, methylation sensitive amplified polymorphism(MSAP), system optimization