五条蚋ISSR-PCR反应体系的建立与优化

Establishment and Optimization of an ISSR Reaction System for Simulium quinquestriatum

通过正交试验及单因素试验对五条蚋S. quinquestriatum ISSR-PCR反应体系中的引物、DNA模板、dNTP、Mg2+、BSA、TaqDNA聚合酶浓度6个关键因素进行优化,建立五条蚋ISSR最适反应体系:20μl反应体系中引物1.0μmol/L、DNA模板50.0ng/μl、dNTP0.15mmol/L、Mg2+1.50mmol/L、BSA2.00mg/ml、TaqDNA聚合酶0.155U/μl。通过对24条备选引物进行温度梯度筛选,获得8条能够稳定扩增且多态性较好的引物。为今后利用ISSR技术进行五条蚋种群遗传多样性研究奠定了良好的技术基础。

In this paper, the orthogonal design and single factor method of six critical factors （ primer, DNA template, dNTP, Mg2＋ , BSA, Taq DNA polymerase） of ISSR-PCR reaction system were used to optimize the ISSR- PCR amplification system on S. quinquestriatum. Finally, a satisfactory ISSR （Inter Simple Sequence Repeat） reaction system for S. quinquestriatum with desirable re action system contained primer 1.0 μmol/L, DNA BSA 2.00 mg/ml and Taq DNA polymerase 0.15 5 peatability and polymorphic bands was established. The 20 μl retemplate 50.0 ng/p,1, dNTP 0.15 mmol/L, Mg2＋ 1.50 mmol/L, U/μl. Eight stable and high polymorphic primers were screened from 24 reserve primers by temperature gradient method. sis for the analysis of genetic differentiation and genetic This optimized ISSR reaction system would provide the badiversity among populations of S. quinquestriatum.