基于实时荧光定量PCR技术对不同盐碱程度土壤甲烷氧化菌比活性的研究

Specific Activity of Methanotrophs in Saline-Alkaline Soils Retrieved from A Fluorescent Quantitative Real-time PCR Technique

为了探究不同盐碱程度土壤甲烷氧化菌比活性与甲烷吸收速率的关系,通过室内CH4培养实验,采用实时荧光定量PCR技术,研究3种不同盐碱程度土壤,即:轻度盐化土壤(SA)、强度盐化土壤(SB)、盐土(SC)CH4吸收速率和甲烷氧化菌比活性。以含有甲烷氧化菌功能基因pmo A片段的重组质粒为标准品,测得标准曲线的相关系数r2为0.9977,扩增效率为86%,溶解曲线峰值均一。结果表明:轻度盐化土壤(SA)、强度盐化土壤(SB)、盐土(SC)CH4吸收速率分别为28.4、20.6、17.7 ng·kg-1·h-1,表现为轻度盐化土壤(SA)>强度盐化土壤(SB)>盐土(SC)。土壤CH4吸收速率与土壤甲烷氧化菌比活性显著正相关,Person相关系数r=0.788(P=0.012,n=9)。由Monte Carlo法检验后表明:p H、土壤浸提液电导率EC与土壤甲烷氧化菌比活性显著负相关,相关系数分别为-0.943(P=0.000 1,n=9)和-0.895(P=0.001,n=9),而容重ρb、总磷TP、总氮TN、有机碳OC与土壤甲烷氧化菌比活性无显著相关性(P>0.05)。较高p H和EC的盐碱土壤,土壤甲烷氧化菌比活性低,CH4吸收速率低。

In order to explore the relationship between Specific Activity of methanotrophs and methane uptake rate in different levels of saline-alkaline soils, most studies on CH4 uptake potential in saline-alkaline soils are limited to confined field trials. In methanotrophs culture experiments, we used fluorescent quantitative real-time PCR technique to investigate CH4 uptake rate and methanotrophs specific activity in three saline-alkaline soils:slightly saline soil (SA), severely saline soil (SB), saline soil (SC). The recombinant plasmid containing pmoA gene was used as standards to optimize the experimental conditions and generate a standard curve. The correlation coefficient (r2) of the standard curve was 0.997 7 and the amplification efficiency was 86%. The dissocition curves of the real time qPCR demonstrated uniform peaks. The results showed that CH4 uptake rates of SA, SB and SC were negatively related to soil salinity, i.e., SA>SB>SC. Soil CH4 uptake rate increased with the growth of specific activity, showing a significant positive correlation (r=0.788, P=0.012, n=9). Monte Carlo test showed that methanotrophs specific activity was significantly negatively correlated with pH (r=-0.943, P=0.000 1) and EC (r=-0.895, P=0.001), while correlations between methanotrophs specific activity and bulk density(ρb), total phosphorus(TP), total nitrogen(TN) and organic carbon(OC) were insignificant (P>0.05). Saline-alkaline soils of high pH and EC appeared to have lower methanotrophs specific activity and CH4 uptake rates.