【作者】 孟庆文；

【导师】 曲延英； 王强；

【作者基本信息】 新疆农业大学 ， 作物遗传育种， 2007， 硕士

【摘要】 红景天,是一种珍稀的中药材,属于被子植物门(Angiospermae)景天科(Crassulaceae)红景天属(Rhodiola L.)多年生草本,药用有效成份红景天苷和苷元酪醇。在红景天生长与分布环境的差异性中,不同种或同一种的不同种群之间在遗传多样性水平呈现很大变化,这些变化和差异是红景天种间或种内不同群体对特定生态环境及其变化的长期适应和进化的结果。但是随着人们对红景天新的药理学作用认识的不断深入,需求的扩大,人为采挖、过度放牧以及大范围气候变化等因素都已影响到该种植物的生存,若不采取切实有效的保护措施,将导致红景天属植物生物资源的减少和遗传多样性的丧失。本研究通过AFLP分子标记技术对分布于中国新疆天山南部不同海拔地区12个种群的红景天(Rhodiola rosea L)进行种内遗传多样性分析,其目的为珍稀药用植物红景天的遗传多样性保护和植物资源的可持续利用提供理论依据。主要结果如下:1、建立一种用于药用植物红景天种内遗传多样性分析的AFLP分子标记方法。方法:红景天基因组DNA模板400 ng,25μL酶切体系中采用两步双酶切EcoR I(4 U,37℃)、Mse I(4 U,65℃),各3小时;在20μL连接体系中T4 DNA连接酶9 U,酶切DNA模板10μL,22℃连接过夜;50μL体系预扩增,Taq Plus酶2.5 U,dNTP 160μM,对应引物0.5μM,10×PCR Buffer(含Mg2+) 4.5μL,DNA模板量为连接体系稀释3倍取4μL;25μL体系选择性扩增,Taq Plus酶1.5 U,dNTP 80μM,对应引物0.25μM,10×PCR Buffer(含Mg2+) 2.5μL,DNA模板量为预扩增体系稀释3倍取3μL。2、9对引物分别对红景天三个地点(新农大实习林场、天池、一号冰川)共12个野生个体进行AFLP扩增。扩增得到214条清晰的谱带,其中多态带204条。这些种群特异带和种群间的共有带的不同揭示了各种群间的遗传差异及相似性,为遗传资源的保护和利用提供参考。3、在物种水平上,具有高的遗传多样性。物种水平多态百分率(PPL)为95.3%; Nei基因多样性H是0.266;shannon信息指数I是0.420。4、种群水平上,平均多态位点百分率(PPL)是63.7%;Nei基因多样性H是0.217;平均shannon信息指数I为0.330;观察等位基因数Na为1.637;有效等位基因数Ne是1.358。5、红景天野生种群间的遗传分化Gst为0.185,说明18.5%的遗传变异存在于种群之间,81.5%的遗传变异存在于种群内。红景天种群间基因流Nm为2.199。更多还原

【Abstract】 Rhodiola L., one of the important traditional Chinese herbal medicines, is a perennial herbaceous plant of the family Crassulaceae. Salidroside and tyrosol have been shown to possess the medical functions. In the different environment of growth of Rhodiola L., the level of genetic diversity is high. Intra- and extra-species genetic diversity of Rhodio L. reflects evolution and development to the special entironment. Due to changing consumer demands towards natural health products and the growing interest for unknown plant secondary metabolites and their applications in biotechnology, the natural resource and genetic diversity of Rhodiola L. is on the edge of exhaustion.In our study, Amplified fragment length polymorphism (AFLP) marker was used to analyze the genetic diversity and differentiation of Rhodiola L. in the various populations and attitudes in the south of Tianshan mountains (Xinjiang province). Our aim was to provide the theory of the artificial cultivation, the relationship within the active contents, zone and species. It is available for the conservation and sustainable development. The main results of this study are formulated as follows:1、To establish an AFLP method to analyze the level and change of intra-specific genetic diversity of Rhodiola L. Methods: Genome DNA as template was digested with EcoR I and Mse I by two steps in 25μL reaction system. Ligation was done with T4 DNA ligase at 22℃for overnight. The 50μL pre-amplification reaction mix contains 2.5U Taq plus DNA polymerase, 160μM dNTP, 0.5μM pre-amplification primers each, 4.5μL 10×PCR Buffer(Mg2+). Selective amplification is carried out using 1.5U Taq plus DNA polymerase, 80μM dNTP, 0.25μM pre-amplification primers each, 2.5μL 10×PCR Buffer(Mg2+) and added ddH2O to a 25μL volume.2、Genetic diversity in 12 individuals from three wild populations(the forest farm of Xinjiang Agricultural University, Tianchi Lake, No.1 glacier) were assessed by AFLP marker. The 9 primer combinations generated a total of 214 bands, of which 204 were polymorphic.3、The genetic diversity of Rhodiola L. at the species level is higher than that at the population level. As for wild population at species level, the percentage of polymorphic loci(PPL) was 95.3%, the Nei’s gene diversity(H) was 0.266, and the Shannon’s information index(I) was 0.420.4、At the population level, the estimates for the wild population PPL=63.7%, H=0.217, I=0.330, Na=1.637 and Ne=1.358.5、Genetic differentiation was high among populations, Gst was 0.185. This means 18.5% genetic variation that occurred between populations. The gene flow(Nm) was 2.199.更多还原