热带果树基因组DNA提取方法的改良及Southern blot分析

摘要

Tissues of most tropical fruit trees contain polysaccharide and polyphenolics that restrict the extraction of high-quality genomie DNA for further molecular biology analysis. This experiment was conducted to develop a simple, universal and effective modified CTAB method for genomie DNA extraction from tissues of tropical fruit trees for southern blot analysis. This optimized procedure includes two steps. First, before the DNA was extrac- ted, the polysaccharide and polyphenolics were pre-removed through the STE lysis buffer by centrifugation. In addition, possible contamination by secondary metaboliates could be avoided by picking up the cotton-shaped ge- nomie DNA floccules resuspended in liquid at the DNA precipitation step with ethanol. With the modified CTAB method, high-quality DNAs were extracted from young leaves of the three representative tropical fruit trees, ba- nana (Musa spp. ) , papaya (Carica papaya L. ) and longan (Dimocarpus longan Lour. ). After digested with different restriction enzymes, fragments of banana ethylene receptor gene (GenBank accession No. AF113748), papaya phytoene desaturase gene (DQ779922) and longan flowering-related LEAFY homologous gene (DQ160214) were used as probes for further Southern blot analysis, respectively. The hybridization bands were clear with high background. The results indicate that the modified CTAB method, with pre-treated conjugation by DNA pick-up, is a simple and effective protocol for genomic DNA isolation from tropical fruit trees, which pro- duces high-quality genomic DNA available for further Southern blot analysis and other tests in molecular biology.%以香蕉、番木瓜、龙眼为代表材料，建立一套适合热带果树基因组DNA提取的方法——改良CTAB法。将提取各基因组DNA经限制性内切酶完全消化后，分别以香蕉内源乙烯受体基因（AF113748）、番木瓜八氢番茄红素脱氢酶基因PDS（DQ779922）、龙眼开花相关基因LEAFY（ADQ160214）为探针进行Southern blot杂交，3种植物的基因组DNA适宜上样量的60μg，杂交条带清晰，背景弱，说明采用改良CTAB法，能保证基因组DNA提取的浓度和纯度，符合Southern blot的要求。