CAJ | 학술논문

[目的]试图通过ISSR指纹图谱的构建,为真藓科（Bryacae）植物的种类鉴定提供分子分析数据。[方法]为获得标准试验程序,首先利用正交试验设计的方法对真藓科植物的ISSR-PCR反应的5因素（Mg2＋、dNTPs、引物、DNA模板、TaqDNA聚合酶）4水平进行试验。[结果]最适扩增条件是：在20μl PCR反应体系中,5ng模板DNA,0.2μmol/L引物,2.25mmol/LMgCl2,0.6U Taq DNA聚合酶,0.4mmol/LdNTPs。最适退火温度为48～50℃。利用此结论使用6条ISSR引物对14种真藓科及相关的提灯藓科（Mniaceae）植物分别进行PCR扩增,共扩增出86条带,多态性带为86条,多态率达100%。根据扩增结果进行NJ聚类分析,得到的支序图呈星状。[结论]ISSR指纹能够在种级分类水平提供适度的多态性,利用引物UBC-808、811以及826构建的ISSR指纹图谱能够区分所有供试植物,为利用ISSR指纹技术解决真藓科植物种级水平分类关系问题时提供了分子辅助证据的可行性。
[목적]시도통과ISSR지문도보적구건,위진선과（Bryacae）식물적충류감정제공분자분석수거。[방법]위획득표준시험정서,수선이용정교시험설계적방법대진선과식물적ISSR-PCR반응적5인소（Mg2＋、dNTPs、인물、DNA모판、TaqDNA취합매）4수평진행시험。[결과]최괄확증조건시：재20μl PCR반응체계중,5ng모판DNA,0.2μmol/L인물,2.25mmol/LMgCl2,0.6U Taq DNA취합매,0.4mmol/LdNTPs。최괄퇴화온도위48～50℃。이용차결론사용6조ISSR인물대14충진선과급상관적제등선과（Mniaceae）식물분별진행PCR확증,공확증출86조대,다태성대위86조,다태솔체100%。근거확증결과진행NJ취류분석,득도적지서도정성상。[결론]ISSR지문능구재충급분류수평제공괄도적다태성,이용인물UBC-808、811이급826구건적ISSR지문도보능구구분소유공시식물,위이용ISSR지문기술해결진선과식물충급수평분류관계문제시제공료분자보조증거적가행성。
[Objective] The aim was to provide molecular basis for the identification of species in the moss family Bryaceae by the construction of inter-simple sequence repeats （ISSR） fingerprinting. [Method] In order to seek standardizing PCR reaction set-up, an orthogonal design was used to optimize ISSR-PCR amplification system of Bryaceae in five factors （Mg2＋, dNTPs, primer, DNA template, Taq DNA polymerase） at four levels respectively. [Result] A suitable ISSR reaction system was obtained, namely： 20 μl reaction system containing 5 ng of DNA template, 0.2 μmol/L primer, 2.25 mmol/L MgCl2, 0.6 U of Taq DNA polymerase, 0.4 mmol/L dNTPs. Proper annealing temperature was found at 48-50 ℃.The above system and six ISSR-PCR primers were used for the PCR amplification of 14 samples from Bryaceae and the related species in Mniaceae. A total of 86 bands were amplified, all showed polymorphism. NJ cluster analysis showed a star-shaped cladogram. [Conclusion] The results manifested that ISSR fingerprinting could provide the appropriate degree of polymorphism at low taxonomic level, so it would be a useful tool to provide additional evidence for resolving taxonomic relationships at the species level of Bryaceae.