橡胶树MSAP反应体系优化及其不同开割高度单株DNA甲基化分析  

Optimization of MSAP Analysis System for Hevea brasiliensis and Its Application in Plant of Different Heights of Opening for Tapping

作　　者：吴春太[1] 班硕 黎瑜[1] 曾日中[1] 

机构地区：[1]中国热带农业科学院橡胶研究所/农业部橡胶树生物学与遗传资源利用重点实验室/国家橡胶树育种中心/海南省热带作物栽培生理学重点实验室,儋州571737 [2]华中农业大学植物科学技术学院,武汉430070

出　　处：《植物研究》2016年第6期860-869,共10页Bulletin of Botanical Research

基　　金：国家自然科学基金资助项目(31270713);橡胶研究所基本科研业务费资助项目(1630022012008)~~

摘　　要：以‘热研7-33-97’橡胶树无性系幼嫩叶片为材料,通过利用单因素和正交试验相结合的方法,对酶切、预扩增和选择性扩增3个影响甲基化敏感扩增多态性(MSAP)分析的关键步骤的反应体系中关键影响因素进行了优化,建立橡胶树MSAP反应最佳体系,并用于高低割线橡胶树基因组DNA甲基化差异分析。结果表明:在50μL反应体系中,750 ng基因组DNA用EcoRⅠ20 U,HpaⅡ20 U或MspⅠ10 U于37℃恒温同步酶切10 h,酶切完全。最佳预扩增体系(20μL)为:连接产物4μL,Mg Cl_2(25 mmol·L^(-1))0.15μL,d NTPs(2.5 mmol·L^(-1))0.1μL,上下游引物E-00/HM-00(10μmol·L^(-1))各0.3μL,Taq酶(5 U·μL^(-1))0.1μL,10×PCR Buffer 2μL。最佳选择性扩增反应体系(20μL)为:稀释20倍的预扩增产物2μL,Mg Cl_2(25 mmol·L^(-1))0.1μL,d NTPs(2.5 mmol·L^(-1))0.125μL,上下游引物E+3/HM+3(10μmol·L^(-1))各0.4μL,Taq酶(5 U·μL^(-1))0.1μL,10×PCR Buffer 2μL。高低割线树DNA的甲基化比例分别为37.22%和36.43%,2种开割胶树基因组CCGG位点胞嘧啶全甲基化率明显高于半甲基化率,推测橡胶树基因组甲基化主要模式可能是Cp G型。综上表明,建立的MSAP反应体系稳定可靠且重复性好,为后续橡胶树不同胁迫(割胶)程度DNA甲基化的研究奠定了基础。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,Mg Cl_2（ 25 mmol·L^-1）0. 15 μL,d NTPs（ 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,Mg Cl_2（ 25 mmol·L^-1） 0. 1 μL,d NTPs（ 2. 5 mmol·L^-1） 0. 125μL,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,Cp G 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.

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 

分 类 号：S794.1[农业科学—林木遗传育种]