鲤鱼cDNA-AFLP技术反应体系的建立

Establishment of cDNA-AFLP System for Cyprinus carpio

将cDNA扩增片段长度多态性(cDNA-amplified fragment length polymorphism)技术应用在鲤鱼基因的转录表达研究中,本文通过对cDNA-AFLP主要步骤的优化和改进,建立起鲤鱼cDNA-AFLP反应体系。结果表明,采用MseⅠ和BstYⅠ酶切鲤鱼cDNA100ng,分别处理2h和3h后,以接头浓度分别为2pmol/μL和0.2pmol/μL、T4连接酶浓度为0.12U/μL的反应体系进行连接。以稀释50倍的预扩增产物为模板、BstYⅠ和MseⅠ比例为1:4的引物配比进行选择扩增可得到稳定的扩增结果。本研究建立的cDNA-AFLP技术将为鲤鱼转录组研究建立基础和技术支持。

cDNA-amplified fragment length polymorphism technology was used for gene transcript expression in Cyprinus carpio, in this study we established an appropriate cDNA-AFLP reaction system by optimization and improvement the main steps of cDNA-AFLP. The result proved that the digestion 100 ng cDNA with MseⅠ and BstYⅠ after two and three hours separately, the ligation reaction system consisted mainly of 0.2 pmol/μL adapterⅠ, 2 pmol/μL adaperⅡ, and 0.12 U/μL T4 ligate enzyme. And we could achieved the stable amplification result by taking the preamplification 50 times dilution as the template, the BstYⅠ and MseⅠ selective primer ratio of 1：4. The establishment of cDNA-AFLP reaction system in this investigation would provid a foundation and technical support for the transcriptomic research of Cyprinus carpio.