cDNA-AFLP比较当归早薹基因转录差异反应体系的建立

Optimization of cDNA-AFLP Reaction System for Early Bolting Angelica sinensis(Oliv.)Diels

建立了利用cDNA-AFLP技术对当归早薹相关的差异表达基因进行筛选的方法,优化了影响cDNA-AFLP试验结果的关键因子,包括选择性扩增模板量、酶离子浓度、dNTP浓度。结果表明,以1μL稀释10倍的预扩增产物作为模板、1.5 mmol/LMgCl2、0.6 mmol/L dNTP,进行的选择性扩增反应获得DNA片段分子量范围较广,在100-1 000 bp长度范围中均有扩增片段,是较为理想的选择性扩增的结果。聚丙烯酰胺凝胶电泳验证了试验结果,在优化条件下利用部分引物组合可以较好地比较早薹当归在基因转录水平发生的差异。

In this study,the reaction system of cDNA-AFLP for Angelica sinensis was established,template,dNTP,Mg2＋ of selective amplification was optimized.The stable reaction system was established：20 μL selective amplification reaction system containing 1 μL diluted pre amplification products,1.5 mmol/L MgCl2,0.6 mmol/L dNTP.In this reaction system,amplification products in the length range about 100-1 000 bp.cDNA-AFLP products were resolved in a 6% denaturing polyacrylamide sequencing gel run with 1×TBE electrophoresis buffer.