冬虫夏草ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System for Cordyceps sinensis

为进一步研究冬虫夏草的遗传多样性,试验采用改良的CTAB法提取高质量DNA模板,克服了冬虫夏草中富含多糖、蛋白、盐类和色素的影响。建立并优化了冬虫夏草ISSR-PCR最适反应体系,即在25μL体系中反应物的含量分别为:10×PCRBuffer 2.5μL,模板DNA20 ng,引物0.9μmol/L,dNTP 0.12 mmol/L,Mg2+1.5 mmol/L,Taq DNA聚合酶0.5 U,引物835号的最适退火温度为50.8℃。结果表明此反应体系标记位点清晰、稳定、重复性好。

For further research on the genetic diversity of Cordyceps sinensis,the improved method of CTAB was adopted to extract high quality total DNA template,and it can conquer the influences that Cordyceps sinensis contains abundant kinds of polysaccharides,proteins,salts and pigments.The ISSR-PCR optimal reaction system was established and optimized,which in 25 μL reaction mixture,the content of each reactant was as follows： 2.5 μL of 10×PCR Buffer;20 ng of genomic DNA;0.9 μmol/L of ISSR prime;0.12 mmo1/L of dNTP;1.5 mmo1/L of Mg2＋;0.5 U of Taq DNA polymerase;the suitable annealing temperature for primer 835 was 50.8℃.The results showed that this reaction system was clear,stable and repeated for markers loci.