| 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. |
