乌头叶面细菌、真菌DGGE分析条件的建立与优化

Establishment and Optimization of DGGE Analysis Methods for Phylloplane Bacteria and Fungi of Aconitum carmichaeli

摘要:

目的：筛选并优化适用于分析乌头叶面细菌、真菌多样性的变性梯度凝胶电泳（DGGE）实验条件。方法：采用Touch Down-PCR技术扩增乌头叶面细菌16 S rDNA V3区域和真菌18 S rDNA~5.8 S rDNA序列，利用该扩增产物对乌头叶面细菌、真菌DGGE电泳条件进行优化。采用DGGE垂直电泳法筛选凝胶变性梯度，时间间隔法优化电泳时间。结果：乌头叶面细菌DGGE最佳分析条件为变性剂梯度30%~65%（Gel 10%），电泳温度60℃，电压120 V，电泳时间7 h。乌头叶面真菌DGGE最佳分析条件为变性剂梯度25%~45%（Gel 8%），电泳温度60℃，电压120 V，电泳时间10 h。结论：利用以上优化条件能有效分离乌头叶面细菌16 S rDNA V3区及真菌18 S rDNA~5.8 S rDNA序列，为乌头叶面细菌，真菌的群落结构PCR-DGGE分析提供可靠的技术支撑。

Abstract:

Objective: To optimize the denaturing gradient gel electrophoresis(DGGE) experimental methods applicable to analyze the community composition of phylloplane bacteria and fungi sampled from Aconitum carmichaeli. Method: Touch Down-PCR experiment was conducted to amplified A. carmichaeli phylloplane bacteria 16 S rDNA V3 area and fungi 18 S rDNA~5.8 S rDNA sequences, in order to optimize DGGE electrophoresis conditions. DGGE vertical electrophoresis was used to ameliorate degeneration gradient of gels, and time interval method was used to figure out electrophoresis time. Result: For the phylloplane bacteria, the optimum analytical conditions were denaturant gradient ranging from 30% to 65%(Gel 10%), electrophoresis temperature at 60℃, voltage of 120 V and electrophoresis time of 7 h. For phylloplane fungi, the optimum analytical conditions were denaturant gradient range ranging from 25% to 45%(Gel 8%), electrophoresis temperature at 60℃, voltage of 120 V and electrophoresis time of 10 h. Conclusion: The above DGGE analysis conditions can be used to effectively distinguish A. carmichaeli phylloplane bacteria 16 S rDNA V3 area and fungi 18 S rDNA-5.8 S rDNA sequences, so as to provide reliable support for PCR-DGGE analysis of community structure of A. carmichaeli phylloplane bacteria and fungi.