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为了建立适合高良姜的扩增片段长度多态性(AFLP)技术体系,对AFLP技术中主要环节的反应条件进行了优化。研究结果表明,用改良CTAB法可提取高质量的高良姜基因组DNA,基因组DNA用限制性内切酶EcoRⅠ和MseⅠ各4U于37℃水浴2h可以被完全酶切,酶切片段与EcoRⅠ和MseⅠ接头在16℃连接过夜后,用优化的反应体系进行预扩增和选择性扩增,扩增产物经过6%变性聚丙烯酰胺凝胶电泳和银染后获得清晰的多态性指纹图谱,因此说明所建立的AFLP技术体系符合要求。
In order to establish an amplified fragment length polymorphism (AFLP) system suitable for Alpinia officinalis, the reaction conditions of the main components of AFLP technology were optimized. The results showed that high quality Alpinia officinarum genomic DNA could be extracted by modified CTAB method. Genomic DNA was completely digested with 4U of restriction endonucleases EcoRⅠand MseⅠ in water bath at 37 ℃ for 2h. The digested fragment was ligated with EcoRI and MseⅠ After ligation overnight at 16 ° C, preamplification and selective amplification were performed using an optimized reaction system. The amplified product was subjected to 6% denaturing polyacrylamide gel electrophoresis and silver staining to obtain a clear polymorphic fingerprinting, thus indicating The established AFLP technology system meets the requirements.