丹参及鼠尾草属植物的rDNA ITS序列分析

Analysis of rDNA ITS sequences of Radix et Rhizoma Salviae Miltiorrhizae and plants of Salvia L.

目的研究丹参ITS片段遗传多样性,分析该片段在丹参药材DNA分子鉴别和鼠尾草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测序。结果获得核糖体DNA中ITS和5.8SrDNA完整序列,13个鼠尾草属植物ITS1与ITS2序列的长度分别为222～224bp、220～223bp。鼠尾草属植物种间ITS1序列差异百分率为0～12.16%,ITS2序列的差异百分率为0.5%～13.51%;丹参种内ITS1序列的差异百分率为0～0.9%,ITS2序列的差异百分率为0～0.5%;鼠尾草属各类群与外类群的差异百分率ITS1序列为16.07%～20.27%,ITS2序列为15.91%～20.27%。用邻接法(NJ)根据ITS1与ITS2序列数据建立系统发生树。结论两段序列在丹参种内保守,在属间有较大的差异,与外类群的差异最大,可作为中药丹参分子鉴定的标记,而鼠尾草属植物的系统发生关系尚须进一步研究。

Objective To study the genetic diversity of ITS sequences of Radix et Rhizoma Salviae Miltiorrhizae （RRSM） and plants of Salvia L. and analyze the utility of ITS sequences in molecular authentication of RRSM and phylogenetic of plants of Salvia L. 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 222-224 bp ITS1, 220-223 bp ITS2 gene fragments and 5.8S rDNA were obtained from 13 samples of Salvia L. The interspecific substitution varied from 0% to 12.16% at ITS1 and 0. 50% to 13.51% at ITS2. The intraspecific substitution of RRSM is 0% to 0. 9% at ITS1 and 0% to 0. 5% at ITS2. The substitution between group of Salvia L. and outgroup varied from 16.07% to 20. 27% at ITS1 and 15.91% to 20. 27% at ITS2. The phylogenetic tree based on ITS1 and ITS2 data was set up. Conclusion The ITS1 and ITS2 gene fragments are highly conservative at intraspe cific level in RRSM, while they are less conservative at interspecific level in Salvia L. They are least conservative between group of Salvia L. and outgroup. Hence, the sequence of this fragment is a good molecular marker for authentication of RRSM and can be used for further study on the phylogenesis for plants of Salvia L.