摘要
以油松毛虫为材料,对T4DNA连接酶不同用量、预扩增中的Mg2+浓度、dNTP浓度、引物浓度以及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.最终建立了适合松毛虫的AFLP反应体系.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.
In order to establish AFLP technique system by using the genomic DNA of Dendrolimus tabulaeformis Tsai et Liu as test material,different dosages of T4 DNA ligase,concentration of Mg2＋,dNTP and primers were optimized in preamplification reaction system,and the dilute multiple of preamplif ication production and the concentration of Mg2＋,dNTP,primers and different dosages of Taq DNA polymerase were also optimized in selective amplification reaction systerm. Very steady results were achieved. In the linking reaction, T4 DNA ligase 2 U was added to the ligation system. The preamplification system included Mg2＋ 1.5 mmol/L, dNTP 0.2 mmol/L, Pstl primer and Taql primer 5.0×10.7 μmol/L, and 40 dilute multiple of preamplification production. The selective amplification systerm included Mg2＋ 1.5 mmol/L, dNTP 0.2 mmol/L, PstI primer and Taql primer 5.0× 10^-7μmol/L, Taq DNA polymerase 1 U. By using D. tabulaeformis Tsai et Liu, D. spectabilis Butler and D. punctatus （Walker） as test materials, ten pairs of informative primers were identified from eighty-one pairs of primers by optimizing the AFLP reaction system. The result established a strong foundation for the AFLP analysis of pine caterpillar. Fig 9, Ref 17
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2009年第6期875-879,共5页
Chinese Journal of Applied and Environmental Biology
基金
国家自然科学基金项目(No.30771739)资助~~
关键词
松毛虫
AFLP
反应体系
引物筛选
遗传多样性
pine caterpillar
AFLP
reaction system
primer screening
genetic diversity
