河北安国7种中药材AM真菌遗传多样性研究

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 bi-reticulata)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。结果表明,双网无梗囊霉可侵染不同样地不同宿主植物,3个样地7种中药材双网无梗囊霉DNA相似性达99.2%以上,其遗传特征保持了高度稳定性,双网无梗囊霉在样地和植物间具有广谱性。同一样地不同中药材根围土壤因子相似性很高,AM真菌DNA碱基序列相似系数也很高,而不同样地同一中药材根围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。土壤因子对双网无梗囊霉基因序列的影响大于宿主植物的影响。

AM fungal genetic diversity in seven medicinal plant rhizospheres in Anguo City of Hebei Province

To shed more light on the genetic diversity of AM fungi associated with medicinal plants, soil samples were collected in August 2010 in the 0~30 cm depth soil rhizosphere of 7 medicinal plants in Planting Site, Huozhuang Village and Dahu Village of Anguo City, Hebei Province. caulospora bireticulata was used for spore DNA extraction, PCR amplification, sequence determination and cluster analysis to determine the relationship between genetic diversity of A. bireticulata and soil factors. The 2 fungus DNA sequences from Scutellaria baicalensis and Dendranthema morifolium were used for tree phyletic evolution derived by phylogenetic inference analysis of 18S rRNA gene (partial) to 28S rRNA gene (partial) nuclear ribosomal sequences. The measured DNA regions were 18S rRNA (partial), ITS1, 5.8S rRNA, ITS2 and 28S rRNA (partial). The results showed that AM fungi likely infected all host plants, with 99.2% similarity in A. bireticulata DNA sequences in 3 sampling plots. A high generation stability was noted, indicating a broad spectrum of A. bireticulata. In the 18S, 5.8S and 28S regions,A. bireticulata DNA was highly conservative, with 10 strains having no difference in gene position spot. A significant gene variation was noted in the ITS1 and ITS2 regions. When ITS1 region position spot was located in 291~379 bp, A. bireticulata DNA sequence exhibited 1~4 bp difference. Also when ITS2 region position spot was located in 538~742 bp, DNA sequences for 10 strains exhibited 0~5 bp difference. Cluster analysis showed that the similarities of A. bireticulata DNA sequences among different medicinal plants in the same sampling plot were higher than those among different sampling plots of the same medicinal plant. The similarities in A. bireticulata DNA sequences among different medical plants in the same sampling plot were very high; some as high as 100%. On the contrary, the similarities in A. bireticulata DNA sequences of the same medical plants among different sampling plots were low. This suggested that A. bireticulata DNA sequences were closely related with soil factors. Because of the combined effect of soil texture and host plant, A. bireticulata DNA sequences were remarkably different. The highest difference in DNA sequence was in the Huozhuang-based S. baicalensis and Plant Site-based Bupleurum chinense; with difference up to 7 bp in 1 700 bp.