基于ITS序列的中国药用石斛及其混伪品的分子鉴定

DNA molecular identification of Herba Dendrobii and its adulterant species based on ITS sequence analysis

目的：通过测定17种药用石斛的rDNA ITS全序列,构建分子系统树,在分子水平对待检种进行鉴别,为石斛的分子鉴定提供依据。方法：采用改良的CTAB法提取石斛叶片DNA,PCR扩增rDNAITS区全序列,产物回收纯化后直接测序,运用Bioedit,MEGA4.0等软件分析石斛属植物的rDNA ITS序列的特征。结果：建立了17种药用石斛rDNA ITS区碱基全序列数据库,其中,ITS1的长度为228-234 bp,GC量为45.7%-53.0%,变异位点167个,占总位点67.34%,信息位点106个,占总位点42.74%;ITS2长度为241-247 bp,GC量为44.8%-55.7%,变异位点165个,占总位点66.27%,信息位点115个,占总位点46.18%。属间的遗传距离为0.295,石斛种间的平均遗传距离为0.142,种内各居群间的平均遗传距离为0.002。结论：利用17种石斛的全序列数据库及遗传分析软件,通过对待检种rDNA ITS区进行序列测定,可以在分子水平对石斛不同种质进行鉴别,为石斛的分子鉴定提供依据。

To identify Herba Dendrobii and its adulterant species on molecular level, the rDNA ITS sequences of 17 species of Herba Dendrobii were studied. Genomic DNA of Dendrobium was extracted using the modified cetyhrimethyl ammonium bromide （CTAB） method. The PCR products of the rDNA ITS sequences of Dendrobium （32 materials） were purified and then sequenced. The characteristic of the sequences and the genetic distance were compared between Bulbophyllum odoratissimum and Dendrobium, Dendrobium interspecies and different populations. Phylogenetic trees were constructed using the UPGMA method by the biology softwares including BioEdit, MEGA4.0 etc. The PCR products were purified and then sequenced. It was built up that the database of rDNA ITS sequences of 17 species of Herba Dendrobii （32 materials）. The ITS1 was 228-234 bp, the GC content accounting for 45.7%- 53.0%. Its variable sites were 167, accounting for 67.34%. The Parsim-Informative positions were 106, accounting for 42.74%. The ITS2 was 241-247 bp, the GC accounting for 44.8% -55.7%. The variable sites were 165, accounting for 66.27%. The Parsim- Informative positions were 115, accounting for 46.18%. The genetic distance between B. odoratissimum and Dendrobium was 0.295. The average genetic distance was 0. 142 between Dendrobium species, and there were 2-156 variable nucleotides. The average genetic distance between different populations was 0.002, and there were 2-156 variable nucleotides. The genetic distance between B. odoratissimum and Dendrobium was greater than that of Denrobium interspecies. Meanwhile, the genetic distance between Denrobium species was also greater than that of different populations （varieties）The molecular phylogeny tree was constructed on the database of rDNA ITS the sequences of 17 species of Herba Dendrobii using the biology softwares. Then 10 materials on molecular level were authenticated. It is concluded that using of the whole sequences database of 17 species of Herba Dendrobii and heredity analysis softwares, and measuring the sequences of rDNA ITS of the inspected species, can authenticate the Dendrobium on molecular level, and provide basis for molecular authentication.