毛白杨MSAP体系优化及DNA甲基化的初步分析  

Optimization of MSAP System and Preliminary Analysis of DNA Methylation in Populus tomentosa

作　　者：马开峰[1] 张志毅[1] 王斯琪[1] 宋跃朋[1] 张德强[1] 

机构地区：[1]林木育种国家工程实验室(北京林业大学),北京100083

出　　处：《东北林业大学学报》2012年第12期1-7,共7页Journal of Northeast Forestry University

基　　金：国家林业公益性行业科研专项经费项目(201004009)

摘　　要：首次利用MSAP技术开展毛白杨DNA甲基化研究。在优化选择性扩增体系的基础上,对毛白杨亲子代的CCGG位点甲基化相对水平、CCGG位点胞嘧啶甲基化模式的遗传变异进行了初步分析。结果表明:①引物H/M+3、引物E+A+2、dNTP、Taq酶、预扩增产物稀释倍数分别为0.03 nmol、0.03 nmol、0.20 mmol/L、1 U(或1.5U)、40倍时组成的20μL反应体系经PCR扩增后电泳,可得到品质最佳的银染谱带。②毛白杨CCGG位点甲基化相对水平约为26.75%～29.39%,CNG甲基化相对水平低于CG甲基化相对水平,子代的甲基化相对水平低于亲本的甲基化相对水平。③子代中发生遗传变异的CCGG位点数之比为1∶1。遗传自亲本的CG甲基化位点数高于遗传自亲本的CNG甲基化位点数,遗传自父本的甲基化位点数高于遗传自母本的甲基化位点数;子代甲基化位点的变异以去甲基化为主,发生CG甲基化变异的位点数与发生CNG甲基化变异的位点数之比为1∶1。Methylation-Sensitive Amplified Polymorphism （MSAP） technique was used firstly to detect DNA methylation in Populus tomentosa, and the selective amplification system was optimized. The relative level of DNA methylation in CCGG sites of P. tomentosa-1, P. tomentosa-2 and their offspring Pxtomentosa-151 was studied. Also, heredity and variation of CCGG cytosine methylation patterns between parents and their offspring were analyzed. Results showed the best quality silver straining bands would be got from the 20 L selective amplification system, including 0.03 nmol/L H/M3 primer, 0.03 nmol/L E＋^＋2 primer, 0.2 mmol/L dNTP, 1 U （ or 1.5 U） Taq enzyme and 4 IxL 40 times dilution of the pre-amplification product, respectively. In both parents and offspring, relative level of methylated CCGG was 26.75% -29.39%. The relative level of CNG methylation was lower than that of CG methylation. The relative level of methylated CCGG sites in offspring was lower than that in its parents. There was a ratio of 1 ： 1 between heredity and variation in offspring. CG methylation sites inherited from parents were more than that of CNG methylation. And the methylated loci inherited from the male parent were more than that inherited from the female parent. In offspring, the variation of complete demethylation mainly occurred. The variation sites of CG methylation and CNG methylation were equal.

关 键 词：毛白杨 甲基化敏感扩增多态性(MSAP) DNA甲基化 相对水平 遗传变异 

分 类 号：S722.34[农业科学—林木遗传育种]