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以根结线虫雌虫DNA为基础,建立和优化稳定的ISSR-PCR反应体系。试验采用5因素、4水平的正交试验和单因子梯度优化相结合的方法。通过试验得到适用于根结线虫雌虫的25μL ISSR-PCR反应体系中各因素的最佳浓度分别为dNTPs 0.24 mmol/L、Mg2+2.1 mmol/L、Taq酶0.4 U、引物0.3μmol/L、DNA 140 ng。经引物UBC835、UBC857对根结线虫雌虫最佳ISSR-PCR反应体系验证。该反应体系稳定、可靠,正交结合单因子试验可以建立满足后续试验要求的ISSR-PCR体系。
Based on the root-knot nematode female DNA, a stable ISSR-PCR reaction system was established and optimized. The experiment used a combination of 5-factor, 4-level orthogonal experiment and single-factor gradient optimization. The optimal concentration of 25 μL ISSR-PCR reaction system for female M. incognita was 0.24 mmol / L dNTPs, 2.1 mmol / L Mg2 +, 0.4 U Taq DNA polymerase, 0.3 μmol / L primer, DNA 140 ng. The best ISSR-PCR reaction system of root-knot nematode was verified by primers UBC835 and UBC857. The reaction system is stable and reliable. The orthogonal combined single factor test can establish the ISSR-PCR system that meets the requirements of subsequent experiments.