4个地区独一味的ITS基因片段序列分析

Analysis of ITS gene sequence of Lamiophlomis rotata(Benth.) Kudo from the four locations in China

目的:检测独一味的ITS基因序列片段,并利用ITS序列作为遗传标记分析了来自4个不同地区(甘南玛曲、西藏林芝、四川若尔盖道班、四川若尔盖热当坝)的独一味的遗传变异情况。方法:采用Plant DNA Mini Kit法作为基因组DNA提取的最佳方法,提取植物独一味的核基因组DNA,并且建立了适合独一味的PCR反应体系,利用合成的特异性PCR引物对所提取的DNA中rRNA基因内转录间隔区序列进行套式扩增,扩增产物经琼脂糖凝胶电泳得到电泳图谱,并进行分析,测序。经Clusta 1X软件排序,MEGA3软件统计分析和分支分析,建立系统发育树,并计算各类群间的遗传距离。结果:琼脂糖电泳图谱及所测序列显示,来自于植物独一味的核DNA中rRNA基因内转录间隔区全长为670bp左右,甘南玛曲和西藏林芝的独一味遗传距离较小,四川与甘南玛曲和西藏林芝的独一味遗传距离较大。结论:基于ITS序列分析,作为品质突出的甘南玛曲独一味和其他地区的独一味相比,并未形成一个区别于其他地区独一味的一个独特的种。根据ITS序列特征所构建的系统树,与传统分类有所不同。

Objective： To discover the ITS sequence of Lamiophlomis rotata （Benth.） Kudo, and to analyze the genetic diversity situation of Lamiophlomis rotata（Benth.） Kudo from four different areas which locate at MaQu of Gansu province, RuoErGaiDaoBan of Sichuan province, Linzhi of Tibet Autonomous Region and RuErGaiReDangBa of Sichuan province by the ITS sequence which was regarded Genetic Markers. Methods： Plant DNA Mini Kit Method was considered to be an optimal technique. Based on the genomic DNA extracted by Plant DNA Mini Kit method, a reaction system suitable for Lamiophlomis rotata （Benth.） Kudo was established,with synthetic peculiar PCR primer, internal transcribed spaced sequences of rRNA gene were amplified in DNA extracted from wild Lamiophlomis rotata （Benth.） Kudo. And then the electrophoretic atlas of NPPCR amplified products on the agar sugar gel were analysed and measure its genic sequence. Statistical analyzing and branch analyzing were carried out by Clusta 1x software and genetic distance difference in every kinds can be calculated and the molecular phylogenetic tree was established by MEGA3. Result： The electrophoresis atlas and sequencing showed that the lengths of the internal transcribed spaced regions of the rRNA gene in DNA from Lamiophlomis rotata （Benth.） Kudo. had the same region about 670bp. The genetic distance of Lamiophlomis rotata （Benth.） Kudo from MaQu GanSu province, LinZhi Tibet Autonomous Region was closer than from RuErGai DaoBan Sichuan province and RuErGai ReDangBa Sichuan province. Conclusion： Based on the ITS sequence analysis the genetic diversity among the Lamiophlomis rotata （Benth.） Kudo. from MaQu,GanSu province which is high quality and other areas is not distinct. The phylogenic tree derived from the dendrograms based on the ITS sequence data was set up by the NJ contained some discrepancy from the traditional classification.