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目的:建立并优化多伞阿魏简单重复序列区间(ISSR)-聚合酶链反应(PCR)体系。方法:通过单因素试验确定影响多伞阿魏ISSR-PCR扩增结果的Mg2+、dNTPs、引物、Taq DNA聚合酶、DNA模板最佳水平范围。对Mg2+、dNTPs、引物、Taq DNA聚合酶、模板DNA进行5因素4水平的正交试验,优选最佳ISSR-PCR体系条件。ISSR-PCR扩增结果用SPSS 18.0统计软件分析。结果:不同水平的因素对PCR均有明显影响,其中Mg2+、dNTPs、引物影响最大。多伞阿魏ISSR-PCR最佳反应体系为:在50μl的反应体系中含有10×Taq Buffer 5μl、Mg2+2.0 mmol/L、dNTPs 0.4 mmol/L、引物0.4μmol/L、Taq DNA聚合酶1.75 u/50μl、DNA模板0.25 ng/μl。在此基础上,从100条引物中筛选出15条扩增稳定、多态性丰富的ISSR引物。结论:建立的多伞阿魏ISSR-PCR体系具有较高的稳定性和重现性,可为进一步利用ISSR技术进行多伞阿魏遗传多样性分析、不同种源鉴定及亲缘关系分析提供基础。
OBJECTIVE: To establish and optimize the multi-parasitic ferulic simple repeat sequence interval (ISSR)-polymerase chain reaction (PCR) system. METHODS: The single factor test was used to determine the optimal level of Mg2+, dNTPs, primers, Taq DNA polymerase and DNA template that affected the results of ISSR-PCR amplification. For the Mg2+, dNTPs, primers, Taq DNA polymerase, and template DNA, 5 factors and 4 levels of orthogonal test were performed to optimize the ISSR-PCR system conditions. ISSR-PCR amplification results were analyzed using SPSS 18.0 statistical software. RESULTS: Different levels of factors had significant effects on PCR, among which Mg2+, dNTPs, and primers had the greatest effect. The optimal reaction system for ISSR-PCR of Pleurotus ferulae was as follows: 50 μl reaction system containing 10 μl Taq Buffer 5 μl, Mg2+ 2.0 mmol/L, dNTPs 0.4 mmol/L, primers 0.4 μmol/L, Taq DNA polymerase 1.75 u/50 μl, DNA template 0.25 ng/μl. On this basis, 15 ISSR primers with stable amplification and polymorphism were screened out from 100 primers. Conclusion: The established ISSR-PCR system has high stability and reproducibility, which can provide basis for the further use of ISSR technology for genetic diversity analysis, identification of different provenances and analysis of genetic relationship.