应用PCR-DGGE研究铜冶炼厂附近根际土壤微生物生态变化

Analysis of Rhizosphere Microbial Community Structures in Heavy Metal-contaminated Soils Using PCR and Denaturation Gradient Gel Electrophoresis(DGGE)

应用DGGE指纹图谱技术对重金属污染土壤植物修复过程中根际微生物的生态变化进行了研究。在植物的生长旺季采集根际土壤样品,共采集香根草、油菜、芥菜和对照11个样品,并进行土壤重金属分析和微生物群落结构分析。结果表明,在植物种植后,根际土壤重金属的NH4NO3和EDAT提取态均比对照(未种植植物)低。PCR-DGGE图谱分析不同植物根际土壤微生物DGGE图谱有着明显的差异性。DGGE图谱分析结果显示植物根际微生物的Shannon指数都有所增加,香根草根际微生物多样性程度最高为0.9347,其次为油菜和芥菜,其多样性指数分别为0.7655、0.6940。主成分分析(PCA)结果可以看出,主成分分析能够对不同植物根际和对照分开,并且显示出不同样品之间有着差异性。通过对土壤不同提取态的重金属含量与Shannon指数的相关性分析可以看出,各种不同提取态的重金属都跟Shannon植物有着负相关,但NH4NO3提取态的Cu和Zn与Shannon指数没有显著性的相关性,而EDTA提取态的Cu和Zn则与Shannon指数有显著性的负相关性,其相关系数为0.9539,EDTA提取态的Cu还与Shan-non指数呈极显著性的负相关,其相关系数为0.9931。

Heavy metals contaminated rhizosphere was investigated with respect to microbial populations and functions by applying cultivation-independent molecular methods. Several species （Vetiveria, rape, mustard） were selected in the field study. The results showed that the availability of heavy metals in rhizosphere soils was decreased compared to the control （no plant）. Among these plants, Vetiveria had the best effect on decreasing the availability of heavy metals in rhizosphere. The genomic DNA was also extracted and purified from different rhizosphere soils. The 16S rDNA genes （V3 region） were amplified by using the specific primers （F357GC and R518）. Soil bacteria community analysis was carried out using activation-independent methods. Partial 16S rDNA genes were amplified from soil bacterial community DNA by PCR, using primers bound to evolutionarily conserved regions within these genes in the eubacteria. The diversity of PCR-amplified products was transformed to genetic fingerprints using denaturing gradient gel electrophoresis （DGGE）. Two standard methods dimensioning techniques （PCA） and Shannon diversity index analysis were used to analysis the DGGE result. PCA analyzed the data directly by a binary bandmatching table. The two principal components （PC） explained a low of the total variation （PC1=25.819%; PC2= 16.545%）. The PCA could separate the different groups completely, all the rhizosphere samples of one plant type were localised together in the same area. Shannon diversity index of rhizosphere soil microorganism was higher than that of the control （0.653 6）. Among the three plants, Vetiveria had the highest Shannon diversity index （0.934 7）. The concentrations of chemical forms of Cu and Zn were negatively correlated with the Shannon diversity index. There existed significantly negative correlations between chemical forms of Cu and Zn extracted by ethylenediaminetetraacetic acid （EDTA） （EDTA-Cu and EDTA-Zn） and the Shannon diversity index （r=0.953 9, P〈0.05）, and the correlation was very significant between EDTA-Cu and the Shannon diversity index （r=0.993 1, P〈0.01）. The change of chemical forms of heavy metals would result in the change of microorganism diversity in soil. These results may help in the further research on the soil environment chemical behavior of heavy metals, the synthesis toxicity of heavy metals to soil ecosystem and the uptake of heavy metals by different plants.