Table 4. Oligonucleotide primers of qRT-PCR for validation of DEGs.
| Gene name | Putative function | GO category | Pathway name | Primer name | Nucleotide sequence (5′-3′) | Expected product |
|---|---|---|---|---|---|---|
| ACTIN | - | - | - | ACTIN-F | TGTGTAGCGGTGAAATGCG | 140bp |
| ACTIN-R | CATCGTTTACGGCGTGGAC | |||||
| METL | Aspartokinase II | Aspartate family amino acid biosynthetic process (Biological process) | Metabolic pathways | METL-F | AAGGTGTAGTTGCTGGAGAGGT | 130bp |
| METL-R | GCGTGTGAAGAGACATCAAGGA | |||||
| METE | 5-methyltetrahydropteroyltriglutamate | Methylation (Biological process) | Metabolic pathways | METE-F | CTTACGAGGCGGGCATTCAG | 151bp |
| METE-R | AAGCGGGTGATGGCAAAGC | |||||
| air-2 | alanine racemase | regulation of cell shape, peptidoglycan biosynthetic process (Biological process) | Metabolic pathways | air-2-F | AACGCTTTCTCTGGCTCCCTA | 125bp |
| air-2-R | CGACATCAGCACGGCATTCA | |||||
| air | alanine racemase | peptidoglycan biosynthetic process, alanine metabolic process (Biological process) | Metabolic pathways | air-F | ACCGCACCTTCACCCTCAA | 209bp |
| air-R | GAACAGCACCACCTCGTCAC | |||||
| AHA2142 | Acetyl-CoA acetyltransferase | signal transduction, metabolic process, cholesterol metabolic process (Biological process) | Metabolic pathways | AHA2142-F | GGAGACATTGCCGAAGTGACC | 118bp |
| AHA2142-R | CTACCTCATAGTGCCGCTCAAC | |||||
| gyrB1 | DNA gyrase subunit B | DNA topoisomerase type II (ATP-hydrolyzing) activity, ATP binding, metal ion binding, DNA replication origin binding, GTPase activity (Molecular process) | Metabolic pathways | gyrB1-F | GCGGAATGTTGTTGGTGAAGC | 173bp |
| gyrB1-R | CTACGAAGGCGGCATCAAGG | |||||
| gyrA | DNA gyrase subunit A | DNA topoisomerase type II (ATP-hydrolyzing) activity, magnesium ion binding, protein heterodimerization activity (Molecular process) | Metabolic pathways | gyrA-F | GTCTTCTCGTCCACCTCCACT | 222bp |
| gyrA-R | CAACATTCCGCCTCACAACCT |
The data revealed that the upregulation or downregulation of these six genes was consistent with the RNA-Seq results. Together, these results indicate that the qRT-PCR and RNA-Seq results were reliable overall; however, further studies to determine the molecular mechanisms of resistance to enrofloxacin are required (Fig 7).