建立胡杨抗逆研究的cDNA-AFLP反应体系

Constructing cDNA-AFLP reaction system of abiotic stress study for Populus euphratica

以胡杨为研究材料,建立适用于胡杨抗逆研究的cDNA-AFLP反应体系,对影响反应体系的关键因素(RNA提取、内切酶组合、预扩增体系、选择性扩增体系)进行了探索和优化。结果表明:通过改进后的CTAB法能成功获得高质量的RNA;EcoRⅠ/MseⅠ的组合4.5h内将ds-cDNA酶切完全,与相应接头连接过夜后产物直接用于预扩增;稀释10倍的预扩增产物作为模板用于选择性扩增;通过优化体系得到的选择性扩增产物,银染检测后条带清晰稳定,经RT-PCR检测,证明优化后的cDNA-AFLP技术差异显示的结果可以准确反映基因在植物体中差异表达的真实情况,并利用该技术成功筛选出42对可以获得带型丰富且重复性好的引物组合。

The cDNA amplified fragment length polymorphism（cDNA-AFLP） system for Populus euphratica was established after optimizing several key factors,which may affect cDNA-AFLP analysis.The results indicated that optimized CTAB method could be suit for extracting ideal RNA from P.euphratica;the dscDNA could be digested completely by the combination of EcoRⅠ and Mse Ⅰ for 4.5 hrs;the reducible result can be obtained when the pre-amplification products were diluted to 10 times for selective amplification template;the amplified products resulted from this reaction system were determined by silver-staining,and the fragments were visualized clearly and stably;detected by RT-PCR,the optimized differential display cDNA-AFLP technique can accurately reflect the true differences of gene expressions among plants.In addition,42 pairs of primer arrays,producing repeatable and prolific bands,were successfully selected by this improved method.This investigation establishes a cDNA-AFLP system for P.euphratica,which builds a foundation for further study on stress-response genes in P.euphratica.