松毛虫AFLP反应体系的建立及引物筛选

Establishment of AFLP reaction system and screening of primer pairs for pine caterpillars

为研究不同环境条件下松毛虫的遗传多样性,以油松毛虫为材料研究松毛虫的AFLP反应体系。对选择性扩增中的预扩增产物稀释倍数、Mg2+浓度、dNTP浓度、引物浓度和Taq酶浓度进行了比较分析。结果显示,在选择性扩增体系中,预扩增产物稀释40倍,Mg2+浓度1.5 mmol/L、dNTP浓度0.2 mmol/L,引物浓度0.500pmol/L,Taq酶用量为1U,扩增产物经5%变性聚丙烯酰胺凝胶电泳可以得到稳定的结果。用优化的AFLP反应体系,以油松毛虫、赤松毛虫和马尾松毛虫为材料筛选引物,从81对引物组合中筛选出10对多态性高的引物组合。

To examine the genetic diversity of Dendrolimus tabulaeformis in different environment conditions, AFLP reaction system was optimized. In the selective amplification reaction, some important factors affecting the selective amplification were studied, including the dilute multiple of the pre-amplification production, Mg^2＋ concentration, dNTP concentration, primer concentration and the different dosage of Taq DNA polymerase. It was proved that the optimized system of AFLP for D. tabulaeformis was built： 40 dilute multiple for pre-amplification production, Mg^2＋ 1.5 mmol/ L, dNTP 0.2 mmol/L, prime 0. 500 pmol/L, Taq DNA polymerase 1U in 20 μL selective amplification reaction system. The steady results could be achieved in 5 % PAGE electrophoresis. Applying the optimization of AFLP reaction system ten pairs of informative primers were identified from eighty-one pair primers with the materials as D tus tabulaeformis, D. spectabilis and D. punctatus.