【作者】 李忠虎；

【导师】 蔺海明； 陈垣；

【作者基本信息】 甘肃农业大学 ， 作物栽培学与耕作学， 2007， 硕士

【摘要】 我国党参种质资源丰富,但资源调查研究工作相对薄弱,加之不同地区之间相互引种,致使至今党参属植物种质及其亲缘关系不清,给党参资源研究、保存、评价、利用以及新种质的创新等带来很大困难。本研究采用AFLP(扩增片断长度多态性)技术,在建立较稳定的党参AFLP优化体系的基础上,筛选出2对扩增谱带清晰、多态性好的引物组合,构建了12份党参材料的指纹图谱,分析了其遗传多样性,以期为党参品种鉴定、亲缘关系分析、高密度遗传图谱构建、核心种质构建等奠定基础。主要研究结果如下:1.针对党参组织中多糖、蛋白质等含量较高的特点,采用改良的CTAB法,并除去糖类等杂质,筛选出适于AFLP分析的党参基因组DNA最有效的提取方法。结果表明:DNA提取液中加入2%CTAB,175mmolL-1的山梨醇和6.25gL-1的亚硫酸钠等物质,所提取党参的基因组DNA纯度高、基本无降解,适于AFLP分析要求。2.通过对党参AFLP反应体系进行系统研究,建立了适于党参AFLP分析的最佳体系。酶切体系总体积15uL中含纯化DNA300ng,EcoRI 15U,EcoRI Buffer 2uL,先用EcoRI 37℃酶切7h,然后加入MseI 5U,65℃酶切5h,再80℃保温20min,灭活限制性内切酶的活性,然后将离心管置于冰上,降温后稍离心,收集酶切产物。连接体系为:5uL酶切完成的DNA样品,MseI adaptor(10pmol/uL) 5uL,EcoRI adaptor(10pmol/uL)1uL,T4DNAligase 0.5uL,T4DNAligase buffer 2.0uL,ddH2O6.5uL,混匀,稍离心,在低温水浴锅中22℃连接7h,简短离心收集连接产物。取连接产物1uL进行预扩增。预扩产物稀释50倍进行选择性扩增。选择性扩增完成后,加入5uL Loading buffer,95℃变性5min,变性后立即冷却,置于-20℃保存备用。取4uL变性的选扩产物在6%的变性聚丙烯酰胺凝胶上进行电泳,然后进行银染程序。3.在党参AFLP分析过程中,采用银染检测技术代替放射性同位素,避免了同位素的危害,使实验操作更加方便安全,而且缩短了实验周期,探讨了银染操作方法和试剂纯度等影响银染效果的因素。4.筛选出的两对引物在12个不同来源地的党参样品中共扩增出188个条带,平均每对引物扩增出94个;其中多态性条带173个,占总扩增条带数的92.02%。用遗传一致性系数和遗传距离对党参部分种质资源进行综合UPGMA聚类,结果表明:在遗传距离为0.63的水平上将供试党参材料分为3个类群。第一个类群包括7个不同来源地的党参样品,其中来自甘肃省陇西县城关镇和甘肃省陇西县首阳镇的白条党参遗传距离最近,为0.2392,首先聚在一起,然后和甘肃省陇西县渭河乡的白条党参、甘肃省华亭县西华乡的党参、甘肃省渭源县七圣乡的白条党参、甘肃省渭源县莲峰乡的白条党参以及甘肃省文县中寨乡的素花党参聚为一类。第二个类群有4个党参样品,分别为甘肃省甘谷县的白条党参、重庆市兹狄种植研究所的川党参、甘肃省甘南山区的野生党参和吉林省梅河口市进化镇的轮叶党参。第三个类群只有一个党参样品,即为采自云南省红河市的鸡蛋参。更多还原

【Abstract】 China is abundant in Codonopsis germplasm resources. But there’s little investigation work of Codonopsis resources had been done and due to a great many of introduction planting among different areas, it caused the code of germplasm resources and genetic relationship confused that makes the researching、conserving、appraising、utilizing and innovating of new Codonopsis ermplasm resources much difficult. The research adopted the technology of AFLP (Amplified Fragment Length Polymorphism), and to construct stable optimized structure, and then based on it selected 2 pairs of clear amplified band、high polymorphic primer combinations, established 12 fingerprinting pattern of Codonopsis samples, analyzed the genetic polymorphism of them, then the work of identifying、analyzing genetic relationship、establishing high density fingerprinting pattern and constructing of core germplasm of Codonopsis should be based on it. The main result shown as follows:1. In accordance with the characteristic of high content of polysaccharide and protein in organize of Codonopsis, improved CTAB method was adopted, and eliminate polysaccharide and other impurities, then selected the best efficient method which fitted for AFLP analysis of extracting genomic DNA of Codonopsis. The result showed that to add 2%CTAB、175mmolL-1 sorbierite and 6.25gL-1 sodium sulfite in DNA extracting solution, then the extracted genomic DNA of Codonopsis are with high purity、basically no degradation and fitted for AFLP analysis.2. Through the system study on AFLP reaction system of Codonopsis, established optimized system fitted for AFLP analysis. The overall volume of 15uL digestion system contained purified DNA300ng,EcoRI 15U,EcoRI Buffer 2uL,first digested 7h by EcoRI in 37℃, then added MseI 5U, digested 5h in 65℃, kept constant temperature 20 minutes in 80℃, inactivated the activity of restriction enzyme, then put the eppendorf on the ice, centrifuge briefly and collected the products.of digestion after cooling down.The linked system contained: 5uL DNA of digestion, MseIadaptor(10pmol/uL) 5uL,EcoRI adaptor (10pmol/uL)1uL,T4DNAligase0.5uL,T4DNAligasebuffer2.0uL,ddH2O6.5uL,bed blending, centrifuge briefly,then linked 7h in low temperature water bath in 22℃, centrifuge briefly and collect products of link.Take 1uL linked products used for pre-amplification.The pre-amplified produce was diluted 50 times with TE buffer and used for selective amplification.After the selective amplification,the Codonopsis samples were denatured by adding 5 uL Loading buffer and heated up in 95℃for 5min,then cooled down on ice immediately.Then conserved in -20℃for standby.Took 4uL denatured PCR reaction products to electrophoresis on 6% denatured polyacrylamide gels and then stained by silver.3. In the process of the AFLP analysis, the technology of silver staining was used instead of radioactive isotope, because it avoided the harm of the radioactive isotope, so experiments operation are more convenient and safe ,and it shortened the periods of the experiments. The factors of affecting the effects of silver staining,such as operation methods and reagent of silver staining also have been discussed.4. In the experiment, two primers been selected can amplify 188 bands, each primers can amplify 94 bands and 173 of 188 amplified bands were polymorphic bands, which acccouts for 92.02%.To cluster part of Codonopsis germplasm resources by UPGMA in genetic identity and genetic distance,the results showed that: Codonopsis samples could be classified into three clusters at 0.63 level of genetic distance. There are seven Codonopsis samples from different places in the first cluster, among which the Codonopsis pilosula (Franch.)Nannf from Chengguan town and Shouyang town, Longxi county in Gansu province have the closest genetic distance that is 0.2392.There are four Codonopsis samples in the second cluster and only one Codonopsis sample in the third cluster, namely, C.convolvulacea from Honghe town in Yunnan province.更多还原