小叶锦鸡儿基因组DNA的提取及AFLP反应体系的建立

以小叶锦鸡儿幼嫩叶片为材料，采用改良的SDS法提取其基因组DNA，通过优化AFLP技术体系中的几个主要因素，建立了适合小叶锦鸡儿的AFLP银染反应体系。改良的SDS法能通过在提取液中加入β-巯基乙醇防止氧化、加入PVP去除酚类等物质，获得满足AFLP分析要求的纯度高、完整性好的基因组DNA；用EcoRⅠ和MseⅠ37 ℃双酶切4 h后可以将500 ng的基因组DNA完全切开。酶切产物和接头经16℃连接过夜后，用带有1个选择性碱基的引物和带有3个选择性碱基的引物分别进行预扩增和选择性扩增，扩增产物经变性聚丙烯酰胺凝胶电泳分离，用AgNO₃染色，得到了清晰的指纹式样。小叶锦鸡儿AFLP反应体系的建立为利用该技术研究小叶锦鸡儿的遗传多样性奠定了实验基础。

Genomic DNA Extraction and AFLP Analysis System Establishment of Caragana microphylla

The genomic DNA in the fresh leaves of Caragana microphylla was extracted using a modified SDS method, and then an AFLP silver-stained analysis system suitable for C.microphylla was established by optimizing several main factors. We found that the genomic DNA obtained had good purity and integrity for AFLP analysis with mercapto-ethanol in the extracting solution to prevent formation of oxides and PVP to eliminate phenol from genomic DNA. 500 ng genomic DNA was digested for 4 h with EcoRⅠand MseⅠrestriction enzymes, ligated to adaptors for 12 h at 16℃, pre-amplified using EcoRⅠ+1 and MseⅠ+1 primers and then amplified using EcoRⅠ+3 and MseⅠ+3 primers. The amplified products were resolved in denaturing ployacrylamide gels and stained with silver. With this system, clear ploymerphic fingerprints were obtained, which can be taken as a base for future study of AFLP for C.microphylla populations.