摘要
将cDNA扩增片段长度多态性(cDNA-AFLP)分析方法在中国对虾(Fenneropenaeus chinensis)中进行了应用,旨在建立1套适于中国对虾研究的cDNA-AFLP反应体系。对总RNA提取、cDNA双链合成、双酶切反应、连接反应、预扩增反应、选择性扩增反应和银染等步骤进行了优化和改进。结果表明,用EcoRⅠ/MseⅠ双酶切、核心序列加3个选择性碱基的选择性扩增引物、1∶1摩尔比的EcoRⅠ端与MseⅠ端选择性扩增引物比和12μL选择性扩增体积,可得到十分稳定的结果,所得产物经电泳和银染后可获得条带清晰的图像。该方法的建立为cDNA-AFLP技术在中国对虾功能基因组研究中的应用奠定了基础。
The aim of this study is to establish an appropriate cDNA-AFLP （cDNA-Amplified Fragment Length Polymorphism） analysis system for Fenneropenaeus chinensis. The processes of cDNA-AFLP, includ- ing total RNA extraction, synthesis of double-stranded cDNA, double enzyme digestion reaction, adapter ligation reaction, pre-amplification and selective amplification reactions and argent dyeing, were optimized and improved. It was proved that quite steady results could be achieved by using EcoR I /Mse I double enzyme digestion, three selective base primers 1：1 （w： w） EcoR I/Mse I selective primers, and 12 μL volume of selective amplification. The pictures of the production by way of electrophoresis and argent dying were very good, the band in focus and background less disturbed. The establishment of the system provided a foundation for the application of cDNA-AFLP techniques to the functional genomics research of Fenneropenaeus chinensis.
出处
《中国海洋大学学报（自然科学版）》
CAS
CSCD
北大核心
2009年第6期1208-1212,共5页
Periodical of Ocean University of China
基金
国家自然科学基金项目(40706052)
国家科技支撑计划(2006BADO1A13)
公益性农业行业专项(nyhyzx07-042)
国家虾产业技术体系(nycytx-46)资助
关键词
AFLP
中国对虾
反应体系
AFLP
Fenneropenaeus chinensis
reaction system
