地龙ISSR-PCR反应体系的建立与优化

目的 建立和优化地龙ISSR-PCR反应体系.方法 以地龙药材为实验试材,采用异丙醇沉淀法提取了地龙总DNA模板,对地龙ISSR-PCR反应体系中各个主要影响因子进行了优化和筛选.结果 建立了标记位点清晰、稳定、重复性好,可用于地龙ISSR分析的最佳反应体系,50 μl体系中含有:1×Taq酶配套缓冲液,DNA模板50 ng,2.5U Taq DNA聚合酶,2 mmol/L MgCl2,0.8 μmol/L引物,0.2 mmol/L dNTPs.扩增程序为:94℃预变性5 min;94℃变性30 s,(据不同引物的退火温度)复性30 s,72℃延伸1.5 min,共35个循环;72℃延伸5 min,反应结束后,4℃保存.结论 这一优化体系的建立为今后利用ISSR标记技术进行地龙类药用动物鉴定及种质资源遗传多样性分析奠定了基础.

Establishment and Optimization of ISSR-PCR Reaction System for Earthworm

Objective To establish and optimize ISSR-PCR reaction system for earthworm.Methods Earthworm DNA was extracted by isopropyl alcohol extraction method.Single and double factors experiment methods were used to present the effect of the main reaction system elements(Taq DNA polymerase,template DNA,Mg2+,primer,dNTPs) and annealing temperature on ISSR-PCR.Results A reaction system and amplified procedure suitable for earthworm were established,that was,50 μl reaction system containing 1×PCR buffer,50ng template DNA,2.5U Taq DNA polymerase,2 mmol/L MgCl2,0.8 μmol/L primer,and 0.2 mmol/L dNTPs.The optimal amplified procedure was as follows:after a predenaturing of 5 min at 94℃,35 cycles were performed with denaturing of 30 s at 94℃,annealing of 30 s according to denaturing temperature of different primer,extension of 1.5 min at 72℃,a final extension step of 5min at 72℃ and hold at 4℃.Conclusion This optimal system lay the standardization program for the identification of earthworm and the genetic diversity analysis of its germplasm resource.