小麦叶片cDNA-AFLP扩增反应体系的优化

Optimization of cDNA-AFLP Amplification Reaction System in Wheat Leaves

【目的】建立并优化小麦叶片cDNA-AFLP扩增反应体系,进行cDNA-AFLP分析,挖掘新春6号相关抗旱基因。【方法】在盆栽小麦苗期水分胁迫下叶片cDNA-AFLP的试验中,分别对20μL预扩增和选择性扩增反应体系中的4种反应条件影响因子进行不同梯度优化的研究,包括引物、dNTP、Mg2+及TaqDNA聚合酶的用量。【结果】PCR预扩增20μL反应体系中,引物(40 mM)1.0μLd、NTP(2.5 mM)1.8μL、Mg2+(25 mM)2.2μL、TaqDNA聚合酶(5U)0.5μL时预扩增效果较好;PCR选择性扩增20μL反应体系中,引物(40 mM)0.6μL、dNTP(2.5 mM)2.0μL、Mg2+(25 mM)1.6μL、TaqDNA聚合酶(5U)0.4μL时,可得到更多清晰可辨的TDFs(transcriptderived fragments)。【结论】试验通过对引物、dNTP、Mg2+、TapDNA聚合酶用量等因子进行筛选,获得较为理想的预扩增和选择性扩增体系,得到了更多有效的条带,为利用cDNA-AFLP研究小麦抗旱相关基因的分离及其克隆奠定了良好的基础。

[ Objective] To establish and optimize cDNA - AFLP amplification reaction system for wheat leaves, cDNA- AFLP were analyzed, drought resistant gene were found in Xinchun 6. [ Method ] In the pot experiment of wheat at seedling stage under water stress, the optimization of cDNA- AFLP reaction system was studied by using wheat leaves as samples. Different levels of concentration of primer, dNTP mixture, Mg^2＋ and Taq DNA polymerase were studied in 20 μL amplification reaction system. [ Result] Results showed that the quite unmber of clear and distinguishable TDFs bands could be found when the volume of primer was 1.0 μL （40 mM）, dNTP was 1.8 μL （2. 5mM）, Mg^2＋ was 2.2 μL （25 mM）, TaqDNA was 0.5μL （5U） in the preliminary amplification reaction system and the volume of primer was 0.6 μL （ 40mM ）, dNTP was 2.0 μL （ 2.5 mM ）, Mg^2＋ was 1.6 μL （25 mM ）, TaqDNA was 0.4 μL （5U） in the selective amplificaion reaction system. [Condusion] The study will set good base for segregating and cloning wheat gene to use cDNA - AFLP.