沉香DNA提取方法及种质资源聚类分析研究

目的：分别建立适用于提取不同类型沉香样品（叶片、干燥枝条、药材）DNA的方法，通过ITS2序列对沉香药材进行种质资源聚类分析。方法：根据沉香叶片、干燥枝条、药材的组织差异，分别采用试剂盒法和优化的CTAB法提取DNA；利用紫外分光光度仪、琼脂糖凝胶电泳、PCR扩增及ITS2序列测定来检测DNA的纯度、浓度及可用性，以比较不同DNA提取方法的效率；将样品测序结果与从GenBank下载的沉香属（Aquilaria spp.）、拟沉香属（Gyrinops spp.），及沉香药材常见混伪品的ITS2序列，进行聚类分析。结果：优化的CTAB法提取得到的DNA纯度比试剂盒法稍低，但浓度更高。两种方法所提沉香总DNA进行ITS2序列的PCR扩增成功率和测序成功率均为100%。ITS2序列能准确鉴别出4个沉香混伪品和2个拟沉香物种，且15个沉香样品ITS2序列与Aquilaria subintegra（泰国）、Aquilaria sinensis（中国）、Aquilaria crassna（柬埔寨、泰国）三个沉香物种均100%相似。结论：本研究中的两种DNA提取方法均可用于沉香DNA条形码研究；优化CTAB法比试剂盒法更适用于药材DNA的提取，该方法为有关中药材DNA的提取提供了参考；ITS2序列可为沉香种质资源的鉴定和系统发育分析提供必要的实验依据。

A Study on the DNA Extraction Methods of Aquilaria and Cluster Analysis of Its Germplasm Resources

This study aimed at establishing DNA extraction methods suitable for Aquilaria samples of different types,such as leaves,dry branches and medicinal herbs for its authentication by DNA barcode,with cluster analysis of its germplasm resources with ITS2 sequences.As the differences between leaves,dry branches and medicinal materials of Aquilaria.,kit method and optimized CTAB method were adopted to extract DNAs.Ultraviolet spectrophotometry,agarose gel electrophoresis,PCR and ITS2 sequences were applied to detect the concentration,purity,availability of DNA,respectively.Then the extraction efficiency of the two methods were compared.The ITS2 sequences of Gyrinops spp.,Aquilaria spp.,and common adulterants of Aquilaria downloaded from GenBank and the sequenced results were clustered and analyzed.As a result,it was found that the purity of DNA extracted by the optimized CTAB method was lower than that by the kit method,while its concentration using the optimized CTAB method was much higher than that using the kit method.The total DNAs extracted by the two methods were amplified with PCR.Both the amplification success rate and the sequencing success rate for ITS2 were 100%.Four adulterants of Aquilaria and two species of Gyrinops were accurately identifyied using ITS2 sequences.The similarity between the fifteen samples and the three species of Aquilaria,including Aquilaria subintegra(Thailand),Aquilaria sinensis(China),and Aquilaria crassna(Cambodia,Thailand),was 100%.In conclusion,both the kit method and the optimized CTAB method can be applied to the studies of DNA barcode.However,the optimized CTAB method was more suitable for the DNA extraction of Chinese materia medica,which provided a reference for the DNA extraction of Aquilaria and other herbal medicines.The ITS2 sequence also provided scientific basis of germplasm identification and phylogenetic analysis.