山核桃AFLP实验技术体系的建立

An AFLP analysis system for Carya cathayensis

以充分展开的山核桃Carya cathayensis嫩叶为材料,利用改良CTAB(十六烷基三甲基溴化铵)法与CTAB裂解-硅珠吸附法提取山核桃总DNA,对适合于山核桃叶片的AFLP(扩增片段长度多态性)分析体系进行了摸索。结果表明:"!改良CTAB法适用于从山核桃叶中提取高质量的DNA,提取的DNA可用于AFLP的分析;#"采用EcoR I/MseⅠ酶切组合,酶切连接一步法与两步法的效果没有明显的差异,但一步法操作更为简便;&%适合于山核桃叶AFLP分析的引物组合为E-ACT+M-CAT,E-ACT+M-CTG,E-ACG+M-CAT,E-ACG+M-CTG。扩增产物经60 g.L-1变性聚丙烯酰胺凝胶电泳后进行银染,条带信号强度一致性好,条带分布均匀、清晰,分辨率较高。

Total DNA was extracted from young, fully opened leaves of Carya cathayensis （hickory） by an improved cetyl trimethylammonium bromide （CTAB） method and a CTAB-silicon method, based on which an amplified fragment length polymorphism （AFLP）analysis system for hickory was developed. An electrophoresis was used on the amplified products in a 6% denatured polyacrylamide gel, followed by silver staining, with clearly identified bands shown. Specifically： （1）the improved CTAB method was appropriate for DNA extraction from hickory leaves with extracted DNA qualified for AFLP analysis; （2）with a combination of EcoR I and Mse I for enzyme cutting, the results were the same for the one-step method combining enzyme cutting and adaptor ligation and the two-step method which separated them with the one-step method being much easier; and （3）there were four primer pairs suitable for this AFLP analysis, namely E-ACT ＋ M-CAT, E-ACG ＋ M-CAT, E-ACG ＋ M-CAT, and E-ACG ＋ M-ATG.