2.6 Cloning, sequencing, and phylogenetic analyses
In order to further identify the main T-RFs appearing in the profiles,
clone libraries were established both the cbbL and nifHgenes.The DNA extracted from the three replicates of each IM treatment
were mixed and served as the template for PCR. The PCR amplification was
performed with the same primers as before but without the fluorescent
labels. Subsequently, the PCR products were ligated into the plasmid
vector pGEM-T, and transformed into Escherichia coliJM109 (Takara Bio
Inc, Shiga, Japan) by the manufacturer’s
instructions.
Randomly selected clones (approximately 200 clones for each clone
library) were screened through the reamplification with the
vector-specific primers M13F and M13R and sequenced by the Sangon
Biotech (Shanghai) Co., Ltd. Chimeras were detected using Decipher
(Wright, Yilmaz, & Noguera, 2012) , and the BLASTn program was used to
identify the most similar sequences from GenBank (NCBI). Based on 97%
sequence similarity, 45 and 49 operational taxonomic unit (OTU) were
obtained from 95 to 246 clones in the cbbL and nifH clone
library, respectively.
Only
those OTUs containing at least two sequences were selected for
phylogenetic analysis and T-RF assignment, and one representative
sequence of these OTUs were defined for T-RFLP analysis as described
above. Phylogenetic trees were constructed by the neighbor-joining
method using MEGA 5.0 (Tamura et al., 2012). Bootstrapping (1000
replicate reconstructions) was used to test the reliability of
phylogenetic reconstructions. All of the sequences generated in this
study have been deposited in the GenBank database under accession
numbers MF430858 to MF430936 for cbbL and MF6633454 to MF6633523
for nifH .