龙眼AFLP银染体系的建立与优化

目的]优化龙眼AFLP银染体系中的影响因素。方法]以23份龙眼品种和2份龙眼近缘植物龙荔为试材,通过对扩增片段长度多态性(AFLP)反应体系中的几个影响因素(DNA提取、酶切与连接、电泳与银染)进行研究,建立起龙眼AFLP银染分析体系。结果]用紫外分光光度计检测样品DNA质量和浓度,发现提取的DNA吸光度A260/A280比值在1.8左右,A260/A230的比值大于2.0,说明所提取的DNA样品纯度较高,无蛋白质或盐类的污染。酶切与连接中采用37℃水浴5 h,然后转入65℃水浴2 h的方法能保证酶切充分,又能防止酶切过度。电泳和银染时用4℃预冷的显影液进行显影,可以降低显影速度。结论]采用该技术体系得到的AFLP指纹式样清晰,重复性强,适宜用作龙眼品种鉴别和遗传多样性分析。

Establishment and Optimization of Suitable Silver-stained AFLP Reaction System in Longan

Objective] The research aimed to optimize some factors affecting silver-stained AFLP reaction system of longan.Method] With 23 longan cultivars and 2 longli genetic-related with the longan plant as the tested materials,the amplified fragment length polymorphism(AFLP) analysis system was established through studying some factors affecting the AFLP analysis system such as quality of DNA extraction,the restriction enzyme digestion-ligation system,gel electrophoresis and silver-staining.Result]When the DNA quality and concentration in samples were determined by ultraviolet spectrophotometer,it was found that the ratio of DNA absorbency at A260/A280nm was about 1.8 and that at A260/A230nm was over 2,showing that the purity of the extracted DNA sample was higher without protein or salts pollution.In the restriction enzyme digestion-ligation system,using water bath first at 37 ℃ for 5h then at 65℃ for 2 h could not only make full restriction enzyme digestion and but also control excess restriction enzyme digestion.During gel electrophoresis and silver-staining,using the developer precooled at 4℃ for development could reduced the developing rate.Conclusion] Using this AFLP reaction system could get a repeatable AFLP fingerprints with high definition,so it is suitable for cultivar identification and genetic diversity analysis in longan.