15种常见食(药)用菌的3种总DNA提取方法比较研究

Study on the Comparison of three Genomic DNA Extraction Methods for 15 Familiar Edulis and Medicincal Fungi

本文研究了15种主要食(药)用菌共16个样品的3种总DNA提取方法之比较,它们分别是CTAB法、食品DNA提取试剂盒法和QIAGEN试剂盒法。提取结果分别经过核酸蛋白分析仪、电泳以及RAPD检测。紫外检测结果表明3种方法提取到的总DNA质量均不理想,电泳结果表明3种方法均能提取出小平菇、红菇、香菇、双孢蘑菇、竹荪、黑木耳、姬松茸等7种食(药)用菌总DNA,RAPD检测结果表明CTAB法适用于所有15种食(药)用菌提取用于PCR的总DNA。根据以上结果,我们认为可以选用CTAB法作为在检测这些食(药)用菌的转基因成分时的总DNA提取方法。

Edulis and medicinal fungus were sort of traditional healthy foods of China. Not only Chinese people liked them,but also many people in other countries liked them too. With the increasing of its output and export China, we found more andmore countries asked for the detection of genetically modified ingredients in the fungus. This required the study of transgenicmaterial detection techniques in fungus. The first problem to be resolved was the genomic DNA extraction method and the waysto analyze the quality of the extracted DNA. A high quality extracted genomic DNA was the basis of the following exact resultsof transgenic material detection. So this article set out a study on the genomic DNA extraction method appropriate for most ofthe common edulis and medicinal fungus by analyzing the quality of the extracted genomic DNA.Following 15 species of the common edulis and medicinal fungi(16 samples) were studied here, namely: Pleurotucomucopiae、Volvariella volvacea、Pleurotus sajur-caju、Agrocybe cylindraca maire、Ganoderma lucidum、Russulavinosa Lindbe、Pleurotus eryngii、Lentinus edodes、Agaricus bisporus、Dictyophora indusiata、spore of Ganodermalucidum、Tremella fuciformis Berk、Auricularia auricula、Flammulina velutipes、Pleurotus geesteyanus. Singer andAgaricus blazie. Three commonly used DNA extraction methods were selected, including CTAB method, Food DNA ExtractionKit and Qiagen DNA Extraction Kit. Of them the CTAB method refered for plant introduced in <Short Protocols in MolecularBiology> with a few improvings was adopted. The Food DNA Extraction Kit and Qiagen DNA Extraction Kit refered to theirmethod were provided with the kit. All extracted DNAs were assayed by UV spectrophotometer, agarose electrophoresis andRAPD(Random Amplified Polymorphic DNA) respectively. UV spectrophotometer detection results showed the quality of allextracted genomic DNA was poor as expected. Agarose electrophoresis results showed that all three methods could extract theDNA from the following 7 fungi, Pleurotu comucopiae, Russula vinosa Lindbe, Lentinus edodes, Agaricus bisporus, Dictyophoraindusiata, Auricularia auricula and Agaricus blazie, while CTAB method could extract the DNA of P.geesteyanus.Singer. TheFood DNA Extraction Kit could extract the DNA of V. volvacea and Qiagen DNA Extraction Kit could extract the DNA of T.fuciformis Berk additionally. The RAPD results showed that CTAB method could be used to extract all 15 fungi' s DNA for PCR,while the other two methods could be used to extract most of the 15 fungi' s DNA for PCR. According to the above results, wesuggested the choice of the CTAB method for DNA extraction when testing the transgenic ingredients for these edulis and medicinalfungi.All experiment results were confimed by another researcher in our programme.Due to all the values of UV spectrophotometer detection in this study were always abnormal, a comparison with the sameCTAB method on E. coli was made. The results showed that the values of DNA from E. coli were all good. While agaroseelectrophoresis could not be applied to all of the fungi, we had to select RAPD to analyze the DNA for PCR. How to improvethe extraction method to gain high quality DNA from edulis and medicinal fungus if there would be any better method speciallyfor analyzing them would be still needed for further studies.