高效液相色谱法测定红树林放线菌DNA的(G+C)mol%含量

The Identification of the G+C Content of DNA by HighPerformance Liquid Chromatography for Mangrove Actinomycetes

应用高效液相色谱法测定了放线菌的(G+C)mol%,并就有关分析条件和DNA水解条件进行了优化选择。以Escherichia.coli DH5α作为标准菌株,实验室分离鉴定的8株红树林放线菌作为待测菌株,采用流动相为20mmol/LKH_2PO_4缓冲液(pH 5.6):甲醇(体积比90:10),检测波长为260 nm,流速为1 ml/min,柱温为35℃,分析时间15 min对4种碱基进行分离。结果表明:标准碱基溶液的pH值、流动相的组成和pH值是影响各碱基色谱峰分离和峰形的主要因素。在优化选择的条件下测定,DNA碱基分离效果好,无杂峰干扰,以峰面积计算得到标准菌株Escherichia coli DH5α的(G+C)mol%为54.9694%与已经报道的差异不显著;待测菌株的(G+C)mol%均在菌株所在科或属下的(G+C)mol%范围内;采用酸水解放线菌的DNA可缩短鉴定时间,步骤简明。实验结果充分显示高效液相色谱法测定放线菌(G+C)mol%快速、准确、结果稳定,是放线菌分类鉴定的一种可靠方法。

High performance liquid chromatography was used to establish a method for analyzing the(G+C) mol%of actinomycetes.Escherichia coli DH5αwas used for the standard strain and eight isolated mangrove actinomycetes were used for test strains.Various bases were separated on a C18 column by using 20 mmol/L KH2PO4(pH 5.6):methanol(90:10,v/v) with a flow rate of 1 ml/ min.The detection wavelength was 260 nm,the column temperature was 35℃and the analysis time was 15 min.The results demonstrate that the pH value of standard base solution and mobile phase and composition of mobile phase were the main factors for the base peak separation and peak shape.The(G+C)mol%of Escherichia coli DH5αand eight mangrove actinomycetes were closely match to the(G+C)mol%of the documental values after the analysis.Using of acid hydrolysis can shorten time and make steps simple.This method is rapid,accurate and reliable for actinomycetes classification.