哈尔滨产乙醇杆菌属AFLP反应体系的建立与优化

Establishment and optimization of AFLP techniques of Ethanoligenens harbinense

摘要:

为建立和优化适合于哈尔滨产乙醇杆菌属基因组AFLP分析的技术体系,以哈尔滨产乙醇杆菌属Ethanoligenens harbinense为研究材料,对AFLP反应体系的几个关键参数进行探讨和优化.结果表明,20μL反应体系中,用于酶切的基因组DNA的纯度(OD260/OD280)和质量分别为1.8～1.9和50～100 ng之间为宜;限制性内切酶(EcoRI/MseI)组合的最佳酶切用量和酶切时间分别为EcoRI(2.0μL)/MseI(0.7μL)和3 h;琼脂糖凝胶电泳图谱对比显示,利用EcoRI-A/MseI-G、EcoRI-G/MseI-A、EcoRI-G/MseI-C、EcoRI-T/MseI-A、EcoRI-T/MseI-C、EcoRI-T/MseI-G和EcoRI-T/MseI-T 7个选择性扩增引物组合选扩出的条带稳定、清晰、无背景干扰,能够很好地反映哈尔滨产乙醇发酵细菌间DNA指纹图谱的多态性分布,为构建发酵产氢细菌AFLP图谱和种间遗传多样性奠定基础.

Abstract:

In order to establish and optimize the technology system of AFLP for Ethanoligenens harbinense’s genome,in this study several key factors influencing AFLP were studied and optimized in detail.The results showed that the effect was better when purity and concentration of template DNA(OD260/OD280) were 1.8-1.9 and 50-100 ng respectively in 25 μL reaction system.The best dosage and time of restriction enzyme digestion of(EcoRI/MseI) were EcoRI(2.0 μL)/MseI(0.7 μL) and 3 h respectively.Furthermore,agar gel electrophoresis map analysis also showed that the bands,which were amplified by 7 different primer combinations of EcoRI-A/MseI-G,EcoRI-G/MseI-A,EcoRI-G/MseI-C,EcoRI-T/MseI-A,EcoRI-T/MseI-C,EcoRI-T/MseI-G and EcoRI-T/MseI-T,were clear and stable without disturbed background and could be used to express the polymorphism distribution of DNA fingerprinting of Ethanoligenens harbinens clearly.