香蕉RAPD分析初步研究

比较了不同提取方法对香蕉植株不同部位组织提取 DNA的质量及其 PCR扩增结果 ,对香蕉 RAPD分析中引物种类和浓度 ,复性温度 ,d NTPs,Taq DNA聚合酶浓度 ,热循环数等因素进行了比较影响分析。结果表明 ,虽然改良的 SDS法、CTAB法和 PVP法提取的植株嫩叶和吸芽 DNA提取量和纯度各不相同 ,但其 PCR扩增结果基本相同 ;相同克隆不同植株的 DNA其 PCR扩增结果也基本相同 ;建立了适合香蕉大规模 DNA多态性分析 RAPD反应体系 :2 5 μL反应液中 ,含 1倍缓冲液 ,0 .2 m Md NTPs,0 .3 2 p M随机引物 ,IUTaq酶 ,2 0 ng模板 DNA;反应循环数为 45 ,热循环条件为 94°C,1 min;3 7°C,1 min;72°C,1 .5 min;之前为 94°C,5 min;之后为72°C,1 0 min。在筛选的 2 49个随机引物中 ,有 1 8个在 7个品种上都能扩增出 3～ 1 0条比较清晰条带。

Preliminary studies on the application of RAPD in Musa

The effects of the DNAs,extracted from young leaves and buds(or sprouts)of Musa by modified SDS,PVP and CTAB methods,the primers and their concentrations,the concentrations of dNTPs,Taq DNA polymerase,annealing temperature and time,number of amplifying cycles,etc.,on RAPD analysis in Musa were studied. The results showed that the amplified results were the same on the whole,although the quantitative and qualities of the DNAs of young leaves or buds extracted by modified SDS,PVP and CTAB methods were different;and the amplified results of different plants belong to the same clone were similar;and then the optimal amplification conditions for Musa were the following:20 ng template DNA,0 2 mM dNTPs,0 32 pM primer,1 U Taq DNA polymerase,2 5 μL 10×buffer in 25 μL reaction volume. And 45 cycles of 94 °C for 1 min,37 °C for 1 min and 72 °C for 1 5 min,then 72 °C for 10 min,4 °C storing. In the 249 arbitrary primers used in experiment,only 18 primers were amplified 3～8 clear bands in all the 7 strains of Musa.