链格孢属小孢子种ISSR和RAPD-PCR最佳反应体系的建立

Optimization of ISSR and RAPD-PCR Reaction Systems for AlternariaSmall-spored Species

以13个链格孢属小孢子种菌株为试材,采用正交实验设计的方法,研究Mg2+、dNTPs、Taq DNA聚合酶和引物4个因素在4个水平上对链格孢属小孢子种的ISSR和RAPD反应体系的影响。结果表明:链格孢属小孢子种ISSR和RAPD的最佳反应体系为25μL的ISSR-PCR反应体系中,10×PCR Buffer 2.50μL,Mg2+浓度为2.50mmol/L,dNTPs浓度为0.10mmol/L,引物浓度为0.30μmol/L,Taq DNA聚合酶用量为1.25U;在RAPD-PCR反应体系中,10×PCR Buffer 2.50μL,Mg2+浓度为2.00mmol/L,dNTPs浓度为0.15mmol/L,引物浓度为0.60μmol/L,Taq DNA聚合酶用量为1.25U;在此基础上,对最佳反应体系的退火温度进行了筛选确定;在确定退火温度后,采用最佳反应体系,用ISSR引物UBC808和RAPD引物OPE07进行稳定性和通用性验证,结果表明该优化反应体系具有较好的稳定性和通用性。

Using 13 Alternariasmall-spored species as materials,the orthogonal design were used to optimize ISSR and RAPD reaction system for Alternariasmall-spored species,four influding factors on ISSR and RAPD,including Mg2＋,dNTPs,TaqDNA polymerase and primer at four levels.The results showed that the optimal ISSR and RAPD reaction system for Alternariasmall-spored species were established as follows：in 25μL ISSR-PCR system were 10×PCR Buffer2.50μL,2.50mmol/L Mg^2＋,0.10mmol/L dNTPs,0.30μmol/L primer,1.25 UTaq DNA polymerase;in ISSR-PCR system were 10×PCR Buffer 2.50μL,2.00mmol/L Mg^2＋,0.15mmol/L dNTPs,0.60μmol/L primer,1.25 UTaq DNA polymerase;decided the annealing temperature of optimal reaction system,then ISSR primer UBC808 and RAPD primer OPE07 were used to carry out stable and adaption in the optimal ISSR and RAPD reaction system at the optimal annealing temperature,indicating that the optimal ISSR and RAPD reaction system were stable and adapted.