濒危植物珙桐AFLP反应体系的建立及引物筛选

Establishment and Primer Selection of AFLP Reaction System of Davidia involucrata

为研究不同生境下珙桐(Davidia involucrata Baill.)的遗传多样性,以珙桐叶片为材料优化建立珙桐的AFLP反应体系。对酶切与连接中DNA的浓度、预扩增的引物、选择扩增中Mg2+和dNTPs的浓度进行梯度比较分析。结果显示,酶切与连接过程中DNA的浓度变化对结果影响不大,预扩增的引物是否带有选择碱基对结果影响明显,选择扩增中dNTPs浓度的变化对最终谱带影响不大,而Mg2+浓度变化会对最终谱带产生影响,以1.75 mmol/L为宜。采用优化后的体系,从12对Mse I、EcoR I引物的144组AFLP选择扩增引物组合中筛选出能够得到清晰扩增谱带的7组引物组合,为今后的研究奠定了基础。

To study the genetic diversity of Davidia involucrata Baill.grow in different habitats,an AFLP-PCR system using the leaves of D.involucrata as material was established and optimized.The effects of main elements,including temple concentration for enzyme digestion and connection,primers for pre-amplification,dNTPs and Mg2＋ concentration,for selective amplification were tested.The results showed that the influence of DNA concentration and dNTPs concentration was slender;while the primers and concentration of Mg2＋ had significant effects on the reaction.The optimal Mg2＋ concentration in selective amplification was 1.75 mmol/L.7 combinations of primers with clear PCR bands were selected out of 144 combinations of 12 pairs of Mse I,EcoR I primers for further study.