硅胶膜离心柱法能高效、可靠地提取黄秋葵的DNA

The Method of Silica Gel Membrane Centrifugal Column Can be Used to Extract DNA from Okra with Efficiency and Reliability

黄秋葵(Abelmoschus esculentus)组织中富含影响其DNA提取质量的多糖、多酚。为探究这一特性,本研究以黄秋葵幼叶为材料,探索了一种新的基因组DNA提取方法——硅胶膜离心柱法,利用硅胶膜离心柱与自配的溶液提取了高质量的DNA。此法先用不含十六烷基三甲基溴化铵(cetyl-trimethyl-ammonium bromide, CTAB)的缓冲液去除一部分次生代谢物;然后用3%CTAB缓冲液分离DNA;再用氯仿/异戊醇(24:1)抽提,之后用硅胶膜离心柱纯化DNA。运用琼脂糖凝胶电泳、ISSR-PCR扩增和限制性酶切等方法检测DNA的质量,并与传统CTAB法、十二烷基硫酸钠(sodium dodecyl sulfate, SDS)法和高盐低pH法进行比较分析。结果表明,本法提取的DNA完整性好,纯度高;能被Eco RⅠ完全消化;ISSR扩增得到的条带最清晰、多态性好,明显优于其它3种传统方法。此外,本方法也成功用于石斛(Dendrobium)、葡萄(Vitis spp.)、无花果(Ficus carica)、芦荟(Aloe vera)等植物基因组DNA的提取,因此具有广泛的适用性,可为富含多糖植物基因组DNA的提取提供参考。

High contents of polysaccharides and polyphenols in okra(Abelmoschus esculentus)tissues influence the quality of isolated DNA.According to this character,we developed a new genomic DNA isolation method named silica gel membrane centrifugal column method,whi ch combines commercially available silica columns with home-made solutions,to extract high quality DNA from okra leaves in this study.This method involves removing most of secondary metabolites with CTAB-free buffer from homogenate,then isolating DNA by modifiedCTAB buffer containing 3%CTAB and 1%%β-mercaptoethanol,wiping off protein contaminations with chloroform/isopentanol(24:1),and purifying DNA by spin-column.The DNA samples isolated by silica gel membrane centrifugal column method,CTAB method,SDS method,high salt and low pH method,were assessed by agarose gel electrophoresis,ISSR-PCR amplification,and restriction enzyme digestion,respectively.The results showed that the extracted DNA from young leaves by silica gel membrane centrifugal column method was integral and pure,and completely digested by Eco RⅠ.ISSR amplification displayed clear polymorphic patterns.Therefore,our newly-developed protocol produced higher quality of DNA than the other methods in okra.Moreover,we showed that this method could also be successfully applied to other species such as Dendrobium,Vitis spp.,Ficus carica L.and Aloe vera.Thus,our method has a wide applicability and provides a solution for the genomic DNA extraction from plant tissues accumulating polysaccharides.