2.4 DNA extraction and real-time PCR
For each soil sample, total microbial genomic DNA was extracted from 0.5 g frozen soil using a MoBio PowerSoilTM DNA isolation kit (MoBio Laboratories, CA, USA) according to the manufacturer’s protocol. The extracted DNA was evaluated on 1% agarose gel. The quantity and quality of DNA extracts then was determined by NanoDrop ND-1000 (Thermo Scientific, USA), and stored at -20℃. The cbbL and nifHgene copy number were detected using gene-specific primers K2f / V2R (Yuan et al., 2012) and PolF / PolR (Poly et al.,2001; Simonet, Grosjean, Misra, Nazaret, & Normand,1991), respectively. The reactions were performed in 20 uL containing 10 uL of SYBR® Premix Ex Taq™ (Takara Bio Inc, Shiga, Japan), 0.2 uM of each primer, 1 uL of ROX Reference Dye, and 1 uL diluted DNA (1-10 ng) template in a final volume of 20 uL. Real-time PCR was conducted using an ABI7100 (Applied Biosystem, CA, USA) in the following conditions: 3 min at 95 ℃ for initial denaturing, followed by 40 cycles of 95 ℃ for 10 s, 62 ℃ (cbbL ) (Yuan et al., 2012) and 55 ℃ (nifH ) (Poly et al.,2001; Simonet, Grosjean, Misra, Nazaret, & Normand,1991) for 40 s, and 72 ℃ for 30 s, followed by a final extension for 5 min at 72 ℃. The concentration of plasmids was measured by a Nanodrop® NanoDrop ND-1000 and the standard copy numbers were calculated. The plasmids containing each target gene were diluted by 10 times successively with the spanning of 101–108and ten-fold serial dilutions were used as standard curves. The amplification efficiency ranged from 92% to 95% with the R2 values ranging between 0.998 and 0.999.