长蛸ISSR-PCR优化反应体系的建立及优化研究 |
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引用本文: | 汤永磊,周超,祁鹏志,吴常文,郭宝英.长蛸ISSR-PCR优化反应体系的建立及优化研究[J].浙江水产学院学报,2012(1):28-32. |
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作者姓名: | 汤永磊 周超 祁鹏志 吴常文 郭宝英 |
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作者单位: | [1]浙江海洋学院海洋科学学院,浙江省海洋养殖装备与工程技术重点实验室,浙江舟山316004 [2]华中农业大学水产学院,湖北武汉430072 |
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基金项目: | 国家海洋公益性行业科研专项(201005013);浙江省大学生科技创新活动计划(新苗人才计划,2011R4111012);农业部海洋与河口渔业资源及生态重点开放实验室开放课题(开-10-01) |
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摘 要: | 以长蛸DNA为模板,利用单因子试验分别对影响长蛸ISSR-PCR反应的DNA浓度、Taq DNA聚合酶、Mg2+、dNTPs和引物的浓度进行了优化,并通过梯度PCR确定最适退火温度,最终确定长蛸最佳反应体系和PCR扩增程序为:25μL体系,其中包括Taq DNA聚合酶1.5 U,Mg2+2 mmol/L,dNTPs 0.15 mmol/L,引物0.4μmol/L,DNA模板量约50 ng确立退火温度为52℃。利用所建立的ISSR-PCR反应体系,通过对24份野生长蛸样本的检验,获得了清晰、重复性好、多态性高的条带,研究结果对于把ISSR标记技术引入研究长蛸不同地理群体遗传多样性和遗传结构具有重要意义。
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关 键 词: | 长蛸 ISSR 体系优化 |
Establishment and Optimization of ISSR-PCR System in Octopus variabilis |
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Authors: | GUO Bao-ying |
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Institution: | GUO Bao-ying, ZHOU Chao, QI Peng-zhi, et al (1. Marine Science School of Zhejiang Ocean University, Key Laboratory of Mariculture Equipments and Engineering Technology of Zhejiang Province, Zhoushan 2. College of Fisheries, Huazhong Agricultural University, Wuhan 316004; 430070, China) |
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Abstract: | The influence of Mg2+, dNTPs, Taq DNA polymerase, ISSR primer and template DNA concen- tration on the result of ISSR-PCR amplification was analyzed using the genomic DNA of Octopus variabilis. The reaction system was as follows: 2 mmol/L Mg2+, 0.15 mmol/L dNTPs, 1.5 U Tat/ DNA polymerase, 0.4 Ixmol/L ISSR primer, 50 ng template DNA. The suitable annealing temperature was 52 ℃. The established system was further confirmed in 24 wild O. variabilis samples. This reaction system would provide the basis for the diversity analysis of O. variabilis with ISSR markers. |
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Keywords: | Octopus variabilis ISSR system optimization |
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