桑天牛遗传多样性研究中AFLP反应体系的建立

建立适合的AFLP反应体系研究桑树主要害虫桑天牛(Apriona germari)的遗传多样性,有助于从分子水平分析桑天牛不同地理种群的遗传多样性以及开辟害虫防治新途径。对提取的桑天牛成虫基因组DNA预扩增中的Mg2+浓度、dNTP浓度、引物浓度、TaqDNA聚合酶的用量以及选择性扩增中的预扩增产物稀释倍数、dNTP浓度、引物浓度、Mg2+浓度和Taq酶的用量等进行单因素试验,优化反应体系。确定预扩增反应体系中的Mg2+终浓度为1.50mmol/L,dNTP终浓度为0.15mmol/L,引物浓度为10.0×10-7mol/L,Taq酶用量为1.00U;确定选择性扩增反应体系中的预扩增产物稀释倍数为40倍,dNTP终浓度为0.15mmol/L,引物浓度为5.00×10-7mol/L,Mg2+终浓度为1.50mmol/L,Taq酶用量为1.00U。用优化后的反应体系扩增得到了条带清晰的桑天牛AFLP图谱。

Establishment of AFLP Reaction System for Genetic Diversity Analysis of Apriona germari

The establishment of proper AFLP reaction system for genetic diversity analysis of mulberry longicorn (Apriona germari) would facilitate analyses on genetic diversity of the pest populations from various geographic areas at the molecular level and open up a new way for pest control.In present study,single factor tests were conducted to optimize the reaction system for the pre-amplification of A.germari genomic DNA to determine concentrations of Mg2+,dNTP,and primers,and Taq DNA polymerase dosage.Single factor tests were also conducted to optimize the reaction system for the selective amplification of A.germari genomic DNA to determine the dilution times of pre-amplified product,concentrations of dNTP,primers,and Mg2+,and Taq DNA polymerase dosage.It was determined that the optimized pre-amplification system contained Mg2+ 1.50 mmol/L,dNTP 0.15 mmol/L,primers 10.0×10-7 mol/L each,and Taq DNA polymerase 1.00 U,and the optimized selective amplification system contained 40 times diluted pre-amplification product,dNTP 0.15 mmol/L,primers 5.00×10-7 mol/L each,Mg2+ 1.50 mmol/L,and Taq DNA polymerase 1.00 U.An AFLP graph of A.germari with clear DNA bands was obtained by using the optimized reaction system.