【作者】 王雅丽；

【导师】 贺学礼；

【作者基本信息】 河北大学 ， 植物学， 2012， 硕士

【摘要】 为探明药用植物根围AM真菌遗传多样性，于2010年8月在河北省安国市3个样地(中药材种植基地、霍庄、大户村)用随机抽样法采集7种中药材根围土壤样品，分离筛选双网无梗囊霉(Acaulospora bireticulata)为试验菌种，进行孢子DNA提取、PCR扩增、序列测定及聚类分析，研究了双网无梗囊霉遗传多样性与土壤因子、宿主植物之间的关系，并选取两条代表菌株所测序列，连同从NCBI数据库中下载的4属28种AM真菌相应序列构建系统发育树，并从土壤中提取AM真菌DNA，纯化，PCR扩增后进行变形梯度凝胶电泳（DGGE）分析。取得的主要试验结果如下：1.自然条件下，7种中药材植物根系均能与AM真菌形成良好共生关系。双网无梗囊霉基因序列保持了高度稳定性，具有广谱性。rDNA ITS区测序及Bioedit序列分析表明，土壤条件和宿主植物对AM真菌DNA序列有一定影响，在真菌ITS区出现0~8个碱基差异，但3个样地7种植物双网无梗囊霉DNA相似性在99.2%以上，基因序列保持了高度稳定性。说明双网无梗囊霉对样地和宿主植物没有严格依赖性和选择性，在样地和植物间基因序列保持了高度稳定性，其分布具有广谱性。2. AM真菌遗传多样性与土壤因子密切相关。同一样地不同中药材双网无梗囊霉DNA相似性系数和土壤因子相似性很高，不同样地同一中药材AM真菌DNA碱基序列相似系数低于前者，可见遗传多样性与土壤因子密切相关。3.聚类分析结果显示，环境和土壤条件对双网无梗囊霉基因序列的影响大于宿主植物的影响。土壤因子比宿主植物对AM真菌群落影响更大。不同样地双网无梗囊霉DNA序列比较，大户村与霍庄相似性系数较高，AM真菌在分子水平上无显著差异，这两个样地先聚在一起，而种植基地与其他样地相似性系数较低，后聚在一起，样地间土壤因子比较，大户村与霍庄相似，与基地差异大。进一步说明序列差异受土壤因子影响大于宿主植物。可能是由于试验样地为农田土，采用人工施肥，同一样地土壤因子基本相同，且植物为1年生，故双网无梗囊霉DNA碱基差异受宿主植物影响不明显。4本试验建立了检测土壤AM真菌群落的PCR-DGGE体系。研究结果表明，巢式PCR反应的第一轮反应体系中土壤DNA模板量约2-5ng。第二轮反应以第一轮扩增产物原液为模板，在退火温度为67℃时扩增的条带最为明亮。通过对DGGE体系的优化得知，选用变性梯度为40%-60%的聚丙烯酰胺凝胶在90V电压下电泳7h分离下的条带效果最佳，DGGE图谱的条带分离结果清晰、丰富。DGGE分析结果表明，受样地和宿主植物共同影响，AM真菌种群结构表现出多样化。更多还原

【Abstract】 To elucidate the genetic diversity of AM fungi associated with the medicinal plant, wecollected soil samples (0~30cm depth) from the rhizosphere of seven medicinal plants inPlanting Site, Huozhuang and Dahu Village in Anguo of Hebei Province in August2010.We choose Acaulospora bireticulata for representative and carried on the spore DNAextraction, PCR increases, sequence determination and cluster analysis, to find out therelationship between the genetic diversity of A. bireticulata and soil factors, and thenchoose two fungus’ DNA sequence from Scutellaria baicalensis in Planting Site andDendranthema morifolium in Huozhuang for representative to make the phyletic evolutiontree derived from phylogenetic inference analysis of18S rRNA gene (partial) to28S rRNAgene (partial) nuclear ribosomal sequences. The DNA region we measured is18SrRNA(partial), ITS1,5.8S rRNA, ITS2and28S rRNA(partial). We extracted AM fungus’DNA from soil, and then carried on the purification,PCR amplification and the deformationgradient gel electrophoresis (DGGE) analysis.The main results are as follows:1.AM fungi could infect all host plants, the similarity of DNA sequences arrived to99.2%, which maintained a high stability in generation, and showed A. bireticulata’s broadspectrum. In the region of18S,5.8S and28S, A. bireticulata’s DNA were highlyconservative,10strains had no difference in any gene position spot. In the region of ITS1and ITS2there were several gene variations, in ITS1region A. bireticulata’s DNA sequencehad1~4different bp, in ITS2region10strains’ DNA sequence had0~5different bp.2. The genetic diversity of AM fungus is closely related to soil factors.Cluster analysisresults showed that the similarity of DNA sequence in the same soil sample were higherthan that in different soil sample, so the DNA sequence in the same soil sample gathers inthe same place first, then with different soil sample last.3. The effect that environment and soil conditions on the gene sequence of A.bireticulata outweigh the effects of host plants.The similarity of DNA sequences of A.bireticulata between different Chinese medical plants in the same soil samples were veryhigh, some of which arrive to100%. On the contrary, The similarity of DNA sequences ofA. bireticulata in the same Chinese medical plants but in different soil samples were lowerthan the former, obviously came to the result that DNA sequences of A. bireticulata were more closely related with soil factors than with host plants. Because of the joint influencesfrom soil texture and the host plant, the DNA sequences of A. bireticulata were remarkablydifferent, the biggest difference of DNA sequence came from S. baicalensis in Huozhuangand Bupleurum chinense in Planting Site, which arrives to7different bp in1700bp.4.PCR-DGGE detection system was built up using nested PCR for detecting AM fungiin the different tillage farming soils.In the first round,2-5ng of DNA templatewas suitablefor the PCR amplification.In the second round,the first round PCR productwas used astemplate with annealing temperature at67℃.In DGGE detecting, the polyacrylamide gelwith40%-60%denaturing gradient was used,the voltage of90V andelectrophoretic time of7h showed better effect on PCR product separation. By the common effects from thesample plot and host plant, AM fungal population structure shows diversity.更多还原