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。
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 40 μL 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 primer combinations 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.
出处
《水生态学杂志》
北大核心
2011年第3期127-131,共5页
Journal of Hydroecology
基金
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)
关键词
AFLP
黑斑原鮡
反应体系
AFLP
Glyptosternum maculatum
reaction system
作者简介
郭宝英，1980年生，女，讲师，主要从事水生生物分子遗传学与育种学研究。E—mail：guobaobao1981@yahoo．com．cn
通讯作者：谢从新。E-mail：xiecongxin@mail．hzau．edu．cn
