Liu Jing, He Tao, Chun Ze
Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China.
Zhongguo Zhong Yao Za Zhi. 2009 Nov;34(22):2853-6.
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 cetyltrimethyl 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.
为在分子水平上鉴别石斛及其伪品，对17种石斛的rDNA ITS序列进行了研究。采用改良十六烷基三甲基溴化铵（CTAB）法提取石斛的基因组DNA。对32份材料的石斛rDNA ITS序列的PCR产物进行纯化后测序。比较了密花石豆兰与石斛、石斛种间及不同居群之间序列的特征及遗传距离。利用包括BioEdit、MEGA4.0等生物学软件，采用UPGMA法构建系统发育树。PCR产物经纯化后测序。建立了17种石斛（32份材料）的rDNA ITS序列数据库。ITS1为228 - 234 bp，GC含量占45.7% - 53.0%。其可变位点有167个，占67.34%。简约信息位点有106个，占42.74%。ITS2为241 - 247 bp，GC含量占44.8% - 55.7%。可变位点有165个，占66.27%。简约信息位点有115个，占46.18%。密花石豆兰与石斛之间的遗传距离为0.295。石斛种间平均遗传距离为0.142，有2 - 156个可变核苷酸。不同居群间平均遗传距离为0.002，有2 - 156个可变核苷酸。密花石豆兰与石斛之间的遗传距离大于石斛种间的遗传距离。同时，石斛种间的遗传距离也大于不同居群（品种）间的遗传距离。利用生物学软件在17种石斛rDNA ITS序列数据库上构建了分子系统发育树。然后对10份材料进行了分子水平的鉴定。结果表明，利用17种石斛全序列数据库及遗传分析软件，测定待检物种的rDNA ITS序列，可在分子水平上对石斛进行鉴定，为分子鉴定提供依据。
