Computational protocol: Cry1Ac Transgenic Sugarcane Does Not Affect the Diversity of Microbial Communities and Has No Significant Effect on Enzyme Activities in Rhizosphere Soil within One Crop Season

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Protocol publication

[…] Soil total microbial DNA was extracted from soil samples using the MOBIO Ultraclean Soil DNA Isolation Kit according to the manufacturer's instructions manufacturer. For the analysis of soil bacterial diversity and fugal diversity, a 196 bp fragment of the bacterial 16S rDNA gene and a 390 bp fragment of the fungal 18S rDNA gene was amplified using primers F338-GC and R534, and FR1-GC and FF390, respectively. PCR was carried out in a volume of 50.0 μL as above, using the following program: an initial denaturation at 95°C for 8 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 55/50°C for 16S rDNA/18S rDNA for 30/45 s, extension at 72°C for 30 s/2 min, and a final extension at 72°C for 10 min. Then the PCR products (2 μL) were detected by electrophoresis on 1.5% (w/v) agarose gels stained with ethidium bromide for 1 h at 100 V to verify that similar concentrations of PCR products had been amplified from each soil sample. The remaining PCR products (45 μL) were then analyzed by DGGE (Bio-Rad D-Code™ Universal Mutation Detection System, Bio-Rad, Shanghai, Co., Ltd.) using a 40–60% denaturing gradient (100% denaturant contained 7 M urea and 40% formamide) on a 6.5% (w/v) polyacrylamide gel for bacterial samples, or a 45–60% denaturing gradient for fungal samples (Vainio and Hantula, ). DGGE gels were prepared in advance and were allowed to polymerize for at least 5 h. Gels were run at 80 V and 60°C for 14 h in 1 × TAE (Tris acetate-EDTA buffer) re-circulating buffer for bacterial 16S rDNA or at 50 V and 60°C for 18 h in 1 × TAE for fungal gels. DGGE gels were stained by silver staining according to the method of Radojkovic and Kušic ().Band quantitative analysis of DGGE gel used the Quantity One band analysis package (Bio-Rad, Shanghai, Co., Ltd.) and statistical analysis used the method described by Fromin et al. ().Bands of interest were excised from the DGGE gel and eluted into a PCR tubes with the sterile distilled water (20 μL). After extraction at 4°C overnight, 2 μL of the solution was used to re-amplify the excised fragment using the same primer pair and PCR conditions as previously described. PCR products were purified from a 1.5% (w/v) agarose gel using the gel extraction kit (Promega), sub-cloned into the pMD19-T vector (TaKaRa) and transformed into competent E. coli DH5α cells (Tiangen). Sequencing was carried out by Invitrogen Co., Ltd., Shanghai, China. Then the sequences were identified by blast search alignment on the NCBI (National Center for Biotechnology Information). Uncultured/environmental sample sequences were excluded from both fungal and bacterial search parameters. For identification based on blast search homology, the criteria used were consistent similarity at ≥98% to the same species or genus. Sequences identified were submitted to the GenBank database using the submission tool sequin (http://www.ncbi.nlm.nih.gov/Sequin/index.html). All sequences that were sequenced successfully were submitted to GenBank (Accession numbers: KP693619–KP693681). Multiple alignments were made automatically using Clustal X software with minor manual adjustments (Barriuso et al., ). Phylogenetic analysis of the aligned sequences was performed using MEGA 5.02. In the neighbor-joining tree generated, the statistical robustness of the tree and the reliability of the branching patterns were confirmed by 1000 bootstrapping replicates (Saitou and Nei, ). The other parameters settings were describe as follows: nucleotide sequence evolution model using “maximum composite likelihood,” substitutions to include using “d: transitions+ transversions,” rates among sites using “uniform rates” and pattern among lineages using “same (homogeneous)” (Barriuso et al., ). The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, ). […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics