中药黄草石斛rDNA ITS序列分析

目的　研究黄草石斛ITS片段遗传多样性,分析该片段在黄草药材DNA分子鉴别和石斛属植物系统学研究中的意义。方法　用一对引物进行PCR扩增,扩增产物纯化后用双脱氧终止法(Sangerdideoxy)测序。结果　获得核糖体DNA中ITS和5 8SrDNA完整序列,14个石斛类群的ITS 1与ITS 2序列的长度分别为228-233bp和242-247 bp。石斛种间ITS 1序列的差异百分率为11.79% - 31.58% ,ITS 2序列的差异百分率为10.29% - 25.30% ,金钗石斛种内ITS 1序列的差异百分率为0.87% ,ITS 2序列无差异。石斛各类群与外类群的差异百分率ITS 1序列为23.56% - 36.89% ,ITS 2序列为26.5.2 % - 33.31%。用NJ法根据ITS 1与ITS 2序列数据重建系统发生树。结论　两段序列在石斛种内保守,在种间有较大的差异,与外类群的差异最大,可作为中药黄草石斛分子鉴定的标记,而石斛属的系统发生关系尚须进一步研究

rDNA ITS SEQUENCING OF HERBA DENDROBII (HUANGCAO)

AIM To study the genetic diversity of ITS sequences of Herba Dendrobii (Huangcao) and analyze the utility of ITS sequences in molecular authentication of Herba Dendrobii (Huangcao)and phylogenetic of Dendrobium . METHODS The ITS gene fragment was amplified using a pair of primers. The PCR products were purified and sequenced by the methods of Sanger Dideoxy. RESULTS The DNA sequence of 228-233 bp ITS 1, 242-247 bp ITS 2 gene fragments and 5 8S rDNA were obtained from 14 samples of Dendrobium . The interspecific substitution varies from 11 79% to 31 58% at ITS 1 and 10 29% to 25 30% at ITS 2. The intraspecific substitution of D.nobile is 0 87% at ITS 1 and without difference at ITS 2. The substitution between Dendrobium and outgroup varies from 23 56% to 36 89% at ITS 1 and 26 52% to 33 31% at ITS 2. The phylogenetic tree based on ITS 1 and ITS 2 data was set up. CONCLUSION The ITS 1 and ITS 2 gene fragments were highly conservative at intraspecific level in Dendrebium , while they were less conservative at interspecific level in D.nobile . They were least conservative between Dendrobium and outgroup. Hence, the sequence of this fragment is a good molecular marker for authentication of the Huangcao. But, further study is necessary for the phylogenetic of Dendrobium .