摘要
为了能从分子水平探讨蒌蒿资源的遗传多样性、蒿属的系统分类和种质资源的鉴定,在此,报道采用小量法(SDS法)和大量法(CTAB法)提取蒌蒿DNA,建立优化的RAPD-PCR体系,并进行引物初步筛选。结果显示,两种抽提方法都可有效抽提出高质量的DNA,大量法得率要明显高于小量法,但小量法抽提的DNA也足够RAPD-PCR反应几十至上百次,不同的实验室可根据实验的需要进行选择。对两种方法抽提的DNA,都进行PCR扩增体系梯度摸索,最优的RAPD-PCR反应条件为:25μl反应体系中,含DNA模板约20ng,10×TaqDNA聚合酶缓冲液2.5μl,2μldNTPs(2.5mM),Mg2+2μl(25mM),TaqDNA聚合酶0.15μl(5U/μl),引物0.5μl(25μM),其余以双蒸水补充。在剔除了重复性差,条带模糊和单态的引物后,有九个引物表现稳定,扩增出来的条带清晰、多态性高。
To investigate the genetic diversity and authentication of this species in China and to probe into the phylogenetic taxonomy of the genus using molecular markers in the next step, two isolation methods of DNA （little extraction by SDS method and large amount of extraction by CTAB method）, optimization of RAPD-PCR condition and primers screening were firstly studied. The results showed that both isolation methods could effectively obtain high quality DNA, and the DNA amount harvested from little extraction method could be used in RAPD-PCR action for one hundred times at least in spite of much less than the production from large extraction. Different labs may select any of them according to the demand of their ex- periments. The subsequent RAPD-PCR condition was explored, using the DNA harvested from both methods, and the most suitable mixture was as follows： total 25μl reaction volume containing template DNA 20ng, 10× Taq buffer 2.5μl, 2μl dNTPs （2.5mM）, Mg^2＋ 2μl（25mM）, Taq DNA polymerase 0.15μl （5U/μl）, 0.5μl pimer （25μM） and ddH2O in the rest. Total nine stable and clear primers with high polymorphism were selected except for those with monomorphic and faint bands, using the DNA samples and the RAPD-PCR condition in our study.
出处
《中国农学通报》
CSCD
2006年第4期78-80,共3页
Chinese Agricultural Science Bulletin
