大麻基因组DNA提取及AFLP体系的建立

Extraction of DNA and Construction of AFLP Reaction System in Hemp

本研究以大麻嫩叶为材料,以改进的SDS法,提取了高质量的大麻基因组DNA,经过酶切、连接、预扩增、选择性扩增、银染等试验条件的优化,建立了AFLP反应体系。研究结果表明:在常规的SDS提取液里加入8μL/mLβ-巯基乙醇(V/V)和3%PVP(M/V)能够提取到高质量的适用于AFLP的基因组DNA;选取150ng大麻基因组DNA来进行酶切,EcoRⅠ和MseⅠ各0.5U,在37℃酶切4h,即可完全酶切。最优的选择性扩增体系为20μL反应体系中含有1.0U Taq DNA polymerase、1.0μL25mmol/L Mg2+、0.4μL10mmol/LdNTPs、50ng/μL引物各1.0μL、4.0μL稀释50倍的预扩增产物及2μL10×PCR Buffer;使用该方法,获得了清晰、稳定的图谱并筛选到了18对多态性较好的AFLP引物组合。

In this study,we used the leaves of hemp as test material,isolated high quality genomic DNA from them by using modified method of SDS,and established the optimized AFLP（amplified fragment length polymorphism） reaction system through optimization of several important factors in DNA restriction reaction,ligation reaction,pre-amplification,selective amplification and silver-staining. The results indicated that high quality genomic DNA extracted by routine SDS extracting solution added 8 μL/mL β-mercaptoethanol（V/V） and 3% PVP-40（M/V） was suitable for AFLP analysis in hemp. In enzyme digestion,150 ng genomic DNA could be fully digested with only 0.5 U EcoRⅠor MseⅠat 37℃ for 4 h. The optical selection amplification system was 20 μL reaction mix containing 1.0 U Taq DNA polymerase,1.0 μL 25 mmol/L Mg2＋,0.4 μL 10 mmol/L dNTPs,50 ng/μL primers each 1.0 μL,4.0 μL 50×diluted product of pre-amplification and 2 μL 10×PCR Buffer. Clear and stable amplification patterns can be obtained and 18 pairs AFLP primer combinations with good genetic diversity were selected based on this technological system. This AFLP system will provide a useful tool for genetic diversity analysis,gene mapping,molecular assisted selection and identification of variety.