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、Mg2+、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、Mg2+、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20 ?L的反应体系中，模板、Mg2+、dNTPs、引物、Taq DNA聚合酶的最佳浓度分别为15ng、2.7 mmol/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。 本实验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, Mg2+, 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 20?L ISSR-PCR reaction system of K. alvarezii, the most suitable concentration of template, Mg2+、dNTPs、primer、Taq DNA polymerase were 15ng, 2.7 mmol/L, 0.2mmol/L, 0.1mmol/L and 1.5U, respectively. And the PCR program for ISSR was 5 min initial denaturation at 94℃, then followed by 32 cycles of 50 seconds at 94℃ (denaturation), 60 seconds at 48℃ (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
赵素芬,何培民. 长心卡帕藻ISSR - PCR反应体系的正交优化研究[J]. 中国生物工程杂志, 2008, 28(专刊): 163-167.
. Study on Optimization of ISSR-PCR Reaction System for Kappaphycus alvarezii by Orthogonal Design. China Biotechnology, 2008, 28(专刊): 163-167.
https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2008/V28/I专刊/163
Cited
