摘要
为了建立适合白桦的cDNA-AFLP体系,对反应体系中的酶切、预扩增、选择性扩增的引物筛选等几个重要的影响因素进行优化,得到了适合白桦的cDNA-AFLP反应体系。选择白桦雄花为研究材料,用改良的CTAB法提取总RNA,采用低频酶EcoRⅠ和高频酶MseⅠ酶切双链cDNA。通过研究发现,预扩增中Mg2+浓度为2mmol/L,引物浓度为0.3μmol/L,dNTP浓度为0.25mmol/L,Taq酶浓度为1U,退火温度控制在60℃时,预扩增结果较好。另外,从16对引物组合中选出11对选择性较好的引物组合,扩增的产物经过6%的聚丙烯酰胺凝胶(PAGE)电泳和银染检测,得到清晰、分辨率高、信号强度明显且重复性好的结果,表明该cDNA-AFLP分析体系适用于白桦相关的功能基因的研究。
Several significant influencing factors,including enzyme digestion system,pre-amplification and primer screening in selective amplification,were optimized,and a suitable cDNA-AFLP reaction system for birch（Betula platyphylla Suk.）was set up.The plant material used was the male flower of wild birch.Total RNA was isolated by improved CTAB-LiCl method,and double-stranded cDNA was cut with low-frequency enzyme EcoRⅠ and high-frequency enzyme MseⅠ.According to these experiments,the optimal conditions for pre-amplification were obtained as the concentration of Mg^2＋ 2mmol/L,primer 0.3μmol/L,dNTP concentration 0.25mmol/L,Taq enzyme 1U,and the annealing temperature of 60 degrees C.Moreover,11 out of 16 primer pairs were selected in selective amplification.The finally amplified fragments were separated on 6% polyacrylamide gel,and clear,abundant and repeatable electrophoresis bands with high resolution were obtained.It indicates that the established cDNA-AFLP system is suitable for the analysis of functional-related genes of birch.
出处
《东北林业大学学报》
CAS
CSCD
北大核心
2010年第10期15-18,共4页
Journal of Northeast Forestry University
基金
教育部科学技术研究重点项目(109053)
中央高校基本科研业务费专项基金(DL09CA06)资助
