哲罗鱼AFLP技术体系建立的研究

Study on Technology System Built for AFLP of Hucho taimen(pallas)

以哲罗鱼为材料提取DNA,采用两组限制性内切酶组合酶切基因组,并对酶量、酶切时间、扩增时稀释倍数及镁离子的浓度进行优化,结果表明200 ngDNA用4 U的EcoRI和PstI在37℃酶切4 h后,再分别用4 U的Tru9I和TaqI在65℃酶切4 h后用3 U的T4连接酶连接3 h,进行预扩增,预扩增产物稀释100倍后选择扩增效果最佳,得到了清晰稳定的扩增图谱;并对两组酶切组合的扩增结果进行了比较,结果显示EcoRI、Tru9I酶切组合扩增的条带数要多于PstI、TaqI酶切组合扩增的条带数,但后者的多态性要高于前者。本研究为采用该技术对哲罗鱼基因组的研究提供了参考。

Nowadays,amplified fragment length polymorphism（AFLP） is one of the most regularly used molecular markers.In this study,the aim DNA was extracted from Hucho taimen（pallas）.The genomic DNA was digested with two groups of restriction enzymes.The quantity of the restricting enzymes, the time of the digested reaction,times of pre-amplification product dilution and concentration of Mg^2＋ were optimized.200 ng genomic DNA was digested for 4 h at 37 ℃ with 4 U EcoRⅠ,PstⅠ and then 65 ℃ for 4 h with 4 U Tru9Ⅰ and TaqⅠ,respectively;the product was ligated for 3 h with 3 U ligase and was pre-amplificated;the products of pre-ampification was diluted 100 times for selective amp lification;the amplification patterns were obtained clearly and stably.Comparison the obtained results with two pairs of restriction enzymes showed that the group of EcoRⅠ and Tru9Ⅰ obtained more amplification bands than the group of PstⅠ and TaqⅠ,but the polymorphic bands of the later exceed the former.An useful tool was provided to check the Hucho taimen（pallas） genetic variations and was helpful for molecular assisted selection of breeding.