CAJ | 학술논문

[目的]以蕙兰(Cymbidium faberi Rolfe)基因组 DNA 为模板,对影响 ISSR-PCR扩增结果的因素进行筛选和优化,建立适合蕙兰的 ISSR-PCR 的最佳反应体系.[方法]利用改良的 CTAB 法提取蕙兰基因组 DNA,并对影响 ISSR-PCR 扩增结果的因素进行优化.[结果]获得了高质量的蕙兰基因组 DNA 并建立了最适的蕙兰 ISSR-PCR 体系(25 μl),即2.5 μl 10 × PCR buffer,2.0 mmol/L MgCl2,60 ng 模板 DNA,160 μmol/L dNTPs,1.25 U Taq DNA 聚合酶,0.4 μmol/L 引物,双蒸水15.85 μl;最佳扩增程序为:94 ℃预变性5 min;94 ℃变性30 s,复性温度比引物的 Tm 值低2～3 ℃,30 s,72 ℃ 延伸50 s,40 个循环;72 ℃延伸7 min.[结论]该优化体系的建立可为今后利用 ISSR 标记技术进行蕙兰遗传多样性研究提供依据.
[목적]이혜란(Cymbidium faberi Rolfe)기인조 DNA 위모판,대영향 ISSR-PCR확증결과적인소진행사선화우화,건립괄합혜란적 ISSR-PCR 적최가반응체계.[방법]이용개량적 CTAB 법제취혜란기인조 DNA,병대영향 ISSR-PCR 확증결과적인소진행우화.[결과]획득료고질량적혜란기인조 DNA 병건립료최괄적혜란 ISSR-PCR 체계(25 μl),즉2.5 μl 10 × PCR buffer,2.0 mmol/L MgCl2,60 ng 모판 DNA,160 μmol/L dNTPs,1.25 U Taq DNA 취합매,0.4 μmol/L 인물,쌍증수15.85 μl;최가확증정서위:94 ℃예변성5 min;94 ℃변성30 s,복성온도비인물적 Tm 치저2～3 ℃,30 s,72 ℃ 연신50 s,40 개순배;72 ℃연신7 min.[결론]해우화체계적건립가위금후이용 ISSR 표기기술진행혜란유전다양성연구제공의거.
[Objective] By using the genomic DNA of Cymbidium faberi Rolfe as template, the factors that affect the result of ISSR-PCR reaction system were researched and the optimal system was established.[Method] The genomic DNA was extracted from C.faberi Rolfe with method of modified CTAB.Different factors which affected ISSR amplification reaction were optimized.[Result] High-quality genomic DNA was obtained from C.faberi Rolfe.And the optimal reaction system was as follows: 25 μl amplification reactions system contained 2.5 μl 10 × PCR buffer, 2.0 mmol/L MgCl2, 60 ng template DNA, 160 μmol/L dNTPs, 1.25 U Taq DNA polymerase, 0.4 μmol/L ISSR primer and 15.85 μl ddH2O.The optimal amplification procedures were pre-denaturing for 5 min at 94 ℃, followed by 40 cycles of denaturing for 30 s at 94 ℃, annealing for 30 s at a temperature of 2-3 ℃ lower than melting temperature of each primer pair, extension for 50 s at 72 ℃.Then extension step of 7 min at 72 ℃ was performed.[Conclusion] The optimal system could provide a favorable basis for further study on genetic diversity of C.faberi Rolfe by using ISSR molecular marker technique.