菜心ISSR-PCR反应体系的优化  

Optimization of an ISSR-PCR Reaction System in Flowering Chinese Cabbage

作　　者：孙雪梅[1] 乔爱民[2] 孙敏[1] 桂腾琴[1] 

机构地区：[1]西南大学生命科学学院,重庆400715 [2]仲恺农业技术学院农业与园林学院,广州510225

出　　处：《西南大学学报（自然科学版）》2007年第10期129-133,共5页Journal of Southwest University(Natural Science Edition)

基　　金：广东省自然科学基金资助项目(B1010412)

摘　　要：以菜心（Brassica campestris L．ssp．Chinensis Var．utilis Tssen．et Lee．）为材料，对影响ISSR-PCR扩增结果的因素如Mg^2＋、Taq DNA聚合酶、dNTPs、Primer、模版DNA的浓度及引物退火温度、延伸时间和循环次数进行了探讨，确立了适合菜心ISSR-PCR分析的最佳反应体系和PCR扩增参数：在25pL反应体系中含10×buffer 2．5μL，2．0mmol／LMg^2＋，0．5U Taq DNA聚合酶，0．2mmol／LdNTPs，0．5μmol／L引物，30ng模板DNA．PCR扩增程序：94℃预变性3min；94℃变性1min，49．7～56℃退火（退火温度随引物不同而定）1min，72℃延伸45s，40个循环；72℃延伸5min．Factors which affect the ISSR-PCR analysis of flowering Chinese cabbage （Brassica campestris L. ssp. Chinensis vat. utilis Tssen. et Lee. ）, such as concentration of Mg^2＋ , Taq DNA polymerase, dNTPs, primer, template DNA, and the annealing temperature, number of cycle and extension time, were studied. The optimal ISSR-PCR reaction conditions were determined： in a reaction systen with a total volume of 25 μL, 10 × buffer 2.5 μL, 2.0 mmol/L Mg^2＋ , 0. 5 units of Taq DNA polymerase, 0. 20 mmol/L dNTPs, 0.50 μmol/L primer and 30 ng template DNA should be contained. The optimal procedures for PCR amplification were： initial denaturation at 94 ℃ for 3 min, at 49.7-56 ℃for 1 min and extension at 72 ℃ for 45 s, 40 cycles of denaturation at 94 ℃ for 1 min, annealing and a final 5 min extension at 72 ℃.

关 键 词：菜心 简单序列重复间区(ISSR) 反应体系 

分 类 号：Q789[生物学—分子生物学]