DNA for 183 animals, including 110 normal pigs and 73 PSL pigs from Tianzhong stock Corporation (Hubei, China), which came from the 185 animals described in our previous study . We collected the ear tissues of these pigs by using ear punches as our previous study described .
The porcine kidney cells (PK15) and the human kidney cells (293 T) were obtain from China Center for Type Culture Collection (CCTCC) and cultured using DMEM medium with high glucose (Hyclone, USA) supplemented with 10% FBS (PAN, Germany) in a humidified atmosphere of 5% CO2 at 37 °C.
Semi-quantitative test of the HOMER1–205 transcript
One primer was designed and the PK15 cDNA was used to amplify the sequence of exon 5 different from other transcripts in intron 4. The forward primer resided in intron 4 while the reverse primer resided in exon 6. The primer pairs used are as follows: HM-205 T former: 5′- GTCAAGTTTGAAAGTAAGTTTCCCT -3′; reverse: 5′- AGTATTTGCCCGGCTATCGG -3′; 18srRNA former: 5′- GAGACGGTGGGACAGCG -3′; reverse: 5′- GCCCTCGGTCGAGTTGTC -3′.
Promoter and transcription factor prediction
Bioinformatics website Neural Network Scan Service (http://www.fruitfly.org/seq_tools/promoter.html) was used to analyze the promoter regions in 3000 bp upstream the exon 1 and exon 5. JASPAR (http://jaspar.genereg.net/) were used to predict the transcription factor binding on the SNPs sites.
HOMER1 gene is located on the reverse strand. We used the reverse strand sequence as template. A total of 18 pairs of PCR primers were designed with Primer Premier 5.0 (Premier, Canada) based on the genomic sequence of the porcine HOMER1 gene (ENSSSCG00000014113) referring to Sscorfa11.1 assembly to amplify all exons and partial adjacent introns (Additional file 1). DNA pooling strategy was used to identify potential SNPs involved in the gene. Ten DNA samples were selected to construct a DNA pool. PCRs were performed in 25 μl volume containing, 22 μl mix (TSINGKE Gold-Green mix), 1 μl of genomic DNA (50 ng/μl), 1 μl of each primer (1 μM). The PCR reaction conditions were as follows: a pre-denaturation at 98 °C for 2 min, followed by 35 cycles of 10 s at 98 °C, annealing from 50 °C to 60 °C for 10 s, 10s/kb at 72 °C, and a final extension at 16 °C for 5 min. The PCR products were sequenced using the ABI DNA analyzer (Applied Biosystems, Foster City, CA, USA). The fragments were sequenced by Sangon (Shanghai, China).
Genotyping and association analysis
Among the identified SNPs within the region of HOMER1, 19 SNPs was selected according to their positions as the candidate marks to be genotyped. These SNPs were further genotyped for all experimental individuals using SNP capture on Hiseq4000 platform.
The Hardy-Weinberg test and Chisq.test in R were used to investigate the significant of those SNPs associated with PSL. p value < 0.05 was considered as statistically significant.
Linkage disequilibrium (LD) measure
Haploview 4.0 was applied to analysis the haplotypes of this ten significant SNPs accorded with the Hardy-Weinberg equilibrium .
Plasmid construction, transfection and dual-luciferase reporter assay
The samples with HP1 and HP2 were selected to clone the six fragments in different length. The ATG of HOMER1–205 was assigned as + 1. Six recombinant plasmids were constructed with primers shown in Additional file 2. Fragment H3W (GATC) or H3M (AGGT) covered from − 663~ + 154 bp contained wild or mutant allele of rs339135425, rs325197091, rs322755731 and rs343753765. Fragment H2W (TC) or H2M (GT) covered from − 276~ + 154 bp contained wild or mutant allele of rs322755731 and rs343753765. Fragment H1W (C) or H1M (T) covered from + 20~ + 154 bp contained wild or mutant allele of rs343753765. Those six fragments were amplified and inserted into the PGL3-Basic (Promega) between the restrictive enzyme cutting site KpnI and BgIII. Then those were transfected using lipofectamine 2000 (Invitrogen) into PK15 cells and 293 T cells. Plasmid DNA of each well used in the transfection containing 1 μg and 20 ng of the internal control vector pRL-TK Renilla/luciferase plasmid. The enzymatic activity of luciferase of the vector was measured by PerkinElmer 2030 Multilabel Reader (PerkinElmer).
The sequence of double-stranded small interfering RNAs targeting ARNT is 5′-CAGACAAGCUAACCAUCUUTT-3′ (GenePharma). 2 μl of siRNA, 0.2 μg of reconstructed plasmids were co-transfected into the cells using Lipofetamine 2000™ reagent for 4 h–6 h. Then the transfection mixtures were drew out, and the complete DMEM medium was added in each well. After 24 h, the enzymatic activity of luciferase was measured by PerkinElmer 2030 Multilabel Reader (PerkinElmer).
Western blotting was performed as our previously described . 10–12% SDS-PAGE gels were used to separate proteins and they were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). The membranes were blocked with 5% nonfat milk for 1 h and incubated with ARNT (Santa Cruz) primary antibody at 4 °C overnight. Membranes were washed with TBST (Tris - buffered saline containing 0.1% Tween 20) and incubated with HRP-conjugated secondary antibody (Proteintect) for 2 h. The relative expression normalized β-actin (Proteintect) was calculated in Image J software (NIH).
All experiments were performed independently three times, and data were presented as the means ± standard (SEM). Data were analyzed by two-tailed Student’s t-tests with SPSS version 16.0 (SPSS, Chicago, IL, USA). A p value < 0.05 was considered as statistically significant.