长心卡帕藻ISSR-PCR反应体系的正交优化研究

Study on Optimization of ISSR-PCR Reaction System for Kappaphycus alvarezii by Orthogonal Design

采用CTAB法提取了大型经济海藻长心卡帕藻(Kappaphycus alvarezii)的基因组DNA。通过单因子梯度试验,确定了影响简单重复序列间区(ISSR)扩增结果的模板、Mg^(2+)、dNTPs、引物、Taq DNA聚合酶的适宜浓度、退火温度和反应的最佳循环次数;利用正交试验优化了模板、Mg^(2+)、dNTPs、引物、Taq DNA聚合酶的配比浓度。结果表明:在进行长心卡帕藻的ISSR扩增时,在总体积为20μl的反应体系中,模板、Mg^(2+)、dNTPs、引物、Taq DNA聚合酶的最佳浓度分别为15ng、2.7mmol/L、0.2 mmol/L、0.1 mmol/L、1.5 U;退火温度为48℃。反应程序为94℃预变性5 min,然后经94℃变性50 s、48℃退火1 min,72℃延伸2 min,进行32次循环,最后在72℃再延伸8 min。结果为卡帕藻属和麒麟菜属类海藻的遗传多样性、种质鉴定、分子标记及辅助育种等研究提供了有用的手段。

The genomic DNA of the seaweed Kappaphycus alvarezii was extracted by CTAB method.The template,Mg^(2+),dNTPs,primer,Taq DNA polymerase concentrations,and annealing temperature and the best cycle times were optimized for ISSR- PCR reaction system in K.alvarezii with single-factor and orthogonal design experiment.In 20μl ISSR-PCR reaction system of K.alvarezii,the most suitable concentration of template,Mg^(2+),dNTPs,primer,Taq DNA polymerase were 15ng,2.7 mmol/L,0.2mmol/L,0.1mmol/L and1.5U,respectively.And the PCR program for ISSR was 5 min initial denaturation at 94℃,then followed by 32cycles of 50 seconds at 94℃(denaturation),60 seconds at 481(annealing),120 seconds at 72℃(extension),and a final 8 minutes extension at 72℃.These results consequently provide a useful means for the research of genetic diversity,molecular marker,germplasm identification and auxiliary breeding in Kappaphycus and Eucheuma.