扇脉杓兰AFLP反应体系的建立

Establishment of AFLP Reaction System for Cypripedium japonicum Thunb.

通过SDS法、高盐低pH法、常规CTAB法、高盐CTAB法和改良CTAB法提取扇脉杓兰(Cypripedium japonicum Thunb.)幼叶基因组DNA,并对其AFLP反应体系进行了优化。结果表明:改良CTAB法所提取的DNA电泳条带清晰无污染,A260和A280比值在1.8～2.0,用限制性内切酶酶切后条带均一,酶切时间以4 h为宜。最终确定50μL PCR反应体系的最佳条件为:预扩增产物稀释20倍4μL,dNTP(2.5 mmol/L)3μL,Mg2(+25 mmol/L)4μL,MseⅠ(10μmol/L)和EcoRⅠ(10μmol/L)各1.5μL,Taq DNA聚合酶(5 U/μL)0.3μL。

Genomic DNA was extracted from young leaves of Cypripedium japonicttm Thunb. by SDS method, high salt and low pH method, routine CTAB method, CTAB with high salt method and modified CTAB method, respectively, and an optimized AFLP reaction system was established for Cypripedium japonicum Thunb.. The resnhs showed that DNA electrophoresis strips, which were extracted by the modified CTAB method, were clear and without contamination, and the proportion of A260 to A280 was from 1.8 to 2.0. After double digestion by restriction enzyme, the strips were uniform. 4 h is appropriate for enzyme restriction. The optimum PCR reaction system （50 μL） consisted of 4 μL 20 fold dilution of the pre-amplification product, 3 μL dNTPs （2.5 mmol/L）, 4 μL Mg2＋ （25 mmol/L）, 1.5 μL Mse I （10 μmol/L）, 1.5 μL EcoR I （10 μmol/L） and 0.3 μL Taq DNA polymerase （5 U/μL）.