摘要
[目的]建立基于毛细管电泳的日本落叶松最佳MSAP反应体系,获得条带清晰,信号强度均一的DNA甲基化图谱,为今后开展日本落叶松表观遗传变异研究奠定基础。[方法]以日本落叶松未成熟木质部为试材,设置不同梯度的DNA模板用量以及选择扩增体系中引物浓度、Mg^(2+)浓度、dNTP浓度和exTaq DNA聚合酶浓度,确定各组分不同用量对毛细管电泳效果的影响,从而确立最适的MSAP反应体系。[结果]结果表明模板DNA以及exTaq DNA聚合酶浓度对毛细管电泳结果影响不明显,而高的引物浓度会引起条带的减少。Mg^(2+)及dNTP浓度是影响毛细管电泳结果最为关键的因素,浓度过低或过高会导致毛细管电泳图谱条带明显减少,杂峰显著增多。[结论]最终确立的反应体系为:DNA模板100 ng,引物0.75 pmol·μL^(-1),Mg^(2+)2 mmol·L^(-1),dNTP 0.375 mmol·L^(-1)和exTaq DNA聚合酶0.05 U·μL^(-1)。
[ Objective ] This study aims at constructing the excellent reaction system of MSAP technology and obtai- ning DNA methylation maps with clear bands and uniform signal intensity, so as to provide references for the study on genetic variation of Larix kaempferi in the future. [ Method ] Taking immature xylem as experimental materials, the impact of five factors （ dosage of DNA template, concentration of primers, Mg^2 ＋ , dNTP and exTaq DNA poly- merase） on the result of capillary electrophoresis was investigated. [ Result ] It was found that different concentra- tions of DNA template and the exTaq DNA polymerase did not affect the result of capillary electrophoresis. Howev- er, high concentration of primers would lead to sparse fragments in MSAP profile. Mg^2＋ concentration and dNTP concentration were the most important factors which determined the result of capillary electrophoresis. When the concentration of Mg^2＋/dNTP was unsuitable, the amount of band would significantly decrease, and the amount of impurity peak would significantly increase. [ Conclusion ] The MASP system determined for capillary electrophore- sis in L. kaempferi is： DNA template dosage 100 ng, primers 0.75 pmol · μL^-1, Mg^2＋ 2 mmol· L^-1, dNTP 0. 375 mmol · L^-1 and exTaq DNA polymerase 0.05 U · μL^-1. It can be widely used in detecting DNA methylation.
出处
《林业科学研究》
CSCD
北大核心
2017年第3期361-367,共7页
Forest Research
基金
林业公益性行业科研专项经费重点项目(20150414)
