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中国农学通报

中国农学通报 ›› 2009, Vol. 25 ›› Issue (14): 27-31.

所属专题： 生物技术； 园艺

• 生物技术科学 • 上一篇    下一篇

树莓基因组DNA的提取及ISSR反应体系的正交优化

董晓莉,汤浩茹,陈 清,王小蓉,侯艳霞   

  • 收稿日期:2009-02-12 修回日期:2009-03-24 出版日期:2009-07-20 发布日期:2009-07-20

Extraction of Genomic DNA and Optimization of ISSR Reaction System with Orthogonal Design in Bramble

Dong Xiaoli, Tang Haoru, Chen Qing, Wang Xiaorong, Hou Yanxia   

  • Received:2009-02-12 Revised:2009-03-24 Online:2009-07-20 Published:2009-07-20

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摘要： 摘 要：以树莓优良野生种质插田泡（Rubus coreanus）为材料，比较了SDS法、CTAB法和改良CTAB法提取树莓基因组DNA的效果，结果发现三种方法均能提取到树莓DNA，但改良CTAB法优于CTAB法和SDS法，其所得的DNA质量最好，杂质最少。通过正交试验设计，对Taq酶，primer，dNTP和DNA用量进行了筛选，建立了树莓ISSR技术最优反应体系，即25 μl反应体系中含有1×PCR Buffer，2 mmol/L Mg2+，1.5 U Taq酶，0.2 μmol/L primer，0.4 mmol/L dNTP和20 ng DNA。采用引物UBC835和UBC873分别对10个树莓品种和8个树莓野生资源进行检验发现，该体系工作效果良好。

关键词: 转ScMV-CP基因甘蔗, 转ScMV-CP基因甘蔗, 土壤酶活性, 土壤微生物, BIOLOG 生态板, 土壤细菌多样性

Abstract: Abstract: Using one of the excellent germplasm resources of bramble Rubus coreanus as material, three different DNA-extracting methods (SDS method, CTAB method and modified CTAB method) were employed to be compared in order to isolate high-quality genomic DNA from young leaves. The results showed that the SDS method, the CTAB method and the modified CTAB method can extract DNA. But the modified CTAB method was better than the other two methods, because it could obtain high quality DNA with less impurity. By orthogonal design, a satisfactory ISSR reaction system was established, i.e. a total volume of 25 μl system, it contained 1×PCR Buffer, 2 mmol/L Mg2+, 1.5 U Taq DNA polymerase, 0.2 μmol/L primer, 0.4 mmol/L dNTP and 20 ng DNA template. We tested this reaction system and it worked well with primer UBC835 using in ten bramble cultivars and UBC873 using in eight bramble wild materials.