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【目的】基于聚合酶链式反应—变性梯度凝胶电泳(PCR-DGGE)对桑树根、茎、叶组织内生菌多样性的分析,结合浓度、纯度、PCR扩增性等指标,比较4种DNA提取方法之优劣。【方法】采用常见的DNA提取方法十六烷基三甲基溴化铵(CTAB)法、缓冲液振荡(SPBS)法、液氮研磨(LNG)法和试剂盒(KIT)法提取桑树各组织DNA,从PCR-DGGE多样性等多方面对4种方法进行比较。【结果】对于桑树根和茎,DNA提取浓度最高的方法是LNG法,最低的是SPBS法;桑树叶情况则完全相反。桑树各组织,KIT法获得的DNA纯度均最高。桑树各组织通过16S r DNA PCR-DGGE比较内生细菌多样性,适宜的DNA提取方法是LNG法或CTAB法,不宜采用KIT法提取DNA。内生真菌ITS PCR-DGGE分析结果完全不同,最佳提取方法是KIT法,最不适宜的DNA提取方法因组织不同而异。【结论】对于桑树内生菌研究,最佳的DNA提取方法因组织不同而异,还与研究的内生菌种类有关系。
【Objective】 The objective of this study was to analyze the endophytic fungi diversity in roots, stems and leaves of mulberry by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) The advantages and disadvantages of DNA extraction methods. 【Method】 CTAB method, buffer oscillation (SPBS) method, liquid nitrogen grinding (LNG) method and KIT method were used to extract the tissues of mulberry DNA, PCR-DGGE diversity and other aspects of the four methods were compared. 【Result】 For mulberry roots and stems, the highest concentration of DNA was extracted by LNG method and the lowest was SPBS method. The situation of mulberry leaf was the opposite. Mulberry organizations, KIT DNA purity obtained the highest. The diversity of endophytic bacteria was compared by 16S rDNA PCR-DGGE in mulberry tissues. The suitable method for DNA extraction was LNG method or CTAB method, and DNA should not be extracted by KIT method. Endophytic fungi ITS PCR-DGGE analysis results are completely different, the best extraction method is KIT method, the most inappropriate DNA extraction methods vary by tissue. 【Conclusion】 For the study of mulberry endophytes, the best DNA extraction method varies from tissue to tissue, and is also related to the type of endophyte studied.