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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。
OBJECTIVE: To establish a molecular phylogenetic tree based on the rDNA ITS sequences of 17 species of medicinal Dendrobium and to identify the species at the molecular level to provide evidence for the molecular identification of Dendrobium. Methods: The DNA of Dendrobium was extracted by modified CTAB method. The full sequence of rDNA ITS was amplified by PCR. The products were recovered and sequenced directly. The characteristics of rDNA ITS sequences of Dendrobium were analyzed by software such as Bioedit and MEGA4.0. Results: The full-length rDNA ITS region database of 17 medicinal Dendrobium species was established. Among them, the length of ITS1 was 228-234 bp, the GC content was 45.7% -53.0%, the number of variation sites was 167, accounting for 67.34% , 106 loci, accounting for 42.74% of the total loci; ITS2 length was 241 ~ 247 bp, GC content was 44.8% -55.7%, 165 loci were polymorphic, accounting for 66.27% of the total loci and 115 loci, 46.18% of the total site. The genetic distance between genera was 0.295, the average genetic distance between Dendrobium species was 0.142, and the average genetic distance among all populations was 0.002. Conclusion: Based on the full sequence database of 17 species of Dendrobium and genetic analysis software, the sequence of rDNA ITS region of the tested species can be used to identify different germplasms of Dendrobium at the molecular level, which provides the basis for the molecular identification of Dendrobium.