| 黑斑原鮡AFLP分析体系的建立 |
| The Construction of AFLP Analysis System in Glyptosternum maculatum |
| 投稿时间：2010-07-28 修订日期：2010-12-24 |
| DOI： |
| 中文关键词:AFLP 黑斑原鮡 反应体系 |
| 英文关键词:AFLP Glyptosternum maculatum, reaction system |
| 基金项目:国家海洋公益性行业科研专项（２０１００５０１３）；海洋渔业科学与技术浙江省重中之重学科开放课题（２０１００１０５）；浙江海洋学院人才引进启动项目（２１１３５０１１５０９）。 |
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| 中文摘要: |
| 建立黑斑原鮡(Glyptosternum maculatum)AFLP反应体系,对DNA提取、双酶切、连接、预扩增、选择性扩增和银染效果进行了探索。结果表明,40μLTaqI酶切体系中65℃先酶切2h,EcoRI酶切和连接同时进行,EcoRI酶和T4DNA连接酶的用量均以2U为宜,温度与时间分别为37℃和3h,连接产物稀释10倍作为预扩增的模板。利用这种优化的反应体系,5对选择性扩增引物组合表现出较高的稳定性、清晰度和多态性,它们分别是:E-AAC/T-AAT、E-AAC/T-AAG、E-AAG/T-ACA、E-AAG/T-AAT和E-AAT/T-AGA。5对引物组合对96个样本扩增,共得到332条清晰可辨、可重复的条带,平均每对引物组合扩增出66.4条带,其中51条带是多态的。将AFLP技术用于黑斑原鮡的研究,通过优化体系的建立,为其种质资源的研究奠定基础。 |
| 英文摘要: |
| In this study，amplified fragment length polymorphism(AFLP) reaction system was established for Glyptosternum maculatum，mainly optimized DNA extraction，two enzyme digestion，ligation，pre-amplification，selective amplification and the effect of silver-staining. As a result，template DNA(100-400 ng) was double digested in 40μL of reaction mixture using 5 U Taq I and 10 U EcoR I enzymes at 65℃ for 2 h and then 37℃ for 3 h. Using this suitable AFLP reaction system，five primer combination which were E-AAC/T-AAT, E-AAC/T-AAG, E-AAG/T-ACA，E-AAG/T-AAT and E-AAT/T-AGA can amplify clear polymorphic and stable bands. Five primercombinations detected 332 products，51 of them(15.4%)were polymorphic in at least one population. AFLP technique was applied in Glyptosternum maculatum research; utilization of this improved technique may lay the groundwork for further research of germplasm resource for Glyptosternum maculatum. |
| 郭宝英,谢从新,祁鹏志,吴常文.2011.黑斑原鮡AFLP分析体系的建立[J].水生态学杂志,32(3):127-131. |
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