Animals, housing and sample collection
This study was approved by the Animal Care and Use Committee of Nanjing Agricultural University (SYXK Su 2017-0007). A total of 500 piglets from 65 litters were selected in the Huaiyin pig breeding farm (Huaian, Jiangsu, China). Piglets were weaned at 35 d of age and moved into new empty pens with their original littermates in a nursery room at two days before mixing. Then, 9 or 10 weaned pigs with the same sex and similar body weight from different litters were mixed in pens of dimension 2.5 m × 2.2 m. The pens were equipped with slatted floors, stainless-steel feeders and nipple drinkers to allow ad libitum access to feed and water. Ear tissues of weaned piglet were collected and genomic DNA was extracted by a standard phenol/chloroform method.
Behavioral assessment
A digital video recording system (Hikvision DS-2CE56C2P-IT3 3.6 mm; Hikvision network hard disk video recorder DS-7808HW-E1/M; Hikvision Digital Technology Co. Ltd., Hangzhou, Zhejiang, China) was used to continuously record the behavior of piglets for 72 h after mixing. A video camera was installed over each pen. To individually identify pigs in the video recording, all pigs in each pen were marked different numbers on the back of pigs using a spray paint (7CF, Shenzhen Zhaoxin Energy Co., Ltd, Shenzhen, Guangdong, China) before mixing. The definitions of aggressive behavioral traits used were described in our previous studies [16, 17] with some additional new traits. Specifically, 9 indicators were used to quantify aggression, and their description and definition are shown in Table 1. A fighting behavior was recorded when it lasted for more than 3 s. For the same pair of pigs, the intervening period between each fight event was at least 8 s [18].
SNP identification and genotyping
To identify the functional SNPs of ARHGAP24 gene regulating the aggressive behavior of weaned pigs after mixing, specific primers (Table S1) were used to amplify ARHGAP24 gene, including the coding regions, partially adjacent introns and the 5′- and 3′ flanking regions according to the reference genome sequence of pigs (GenBank accession number: NC_010450.4). The DNA sequences contained potential SNPs of ARHGAP24 gene from 224 aggressive and docile pigs were amplified by polymerase chain reactions (PCR). PCR was performed using 1.1×T3 Super PCR Mix (TsingKe, Nanjing, Jiangsu, China) and the amplified PCR products were sequenced using the Sanger method. The DNA sequences of porcine ARHGAP24 gene were aligned by the DNAMAN software (Lynnon BioSoft, Quebec, QC, CA) and SnapGene Viewer software (BSL Biotech LLC, Chicago, IL, USA).
Linkage disequilibrium (LD) estimation and association analyses
The extent of LD between the identified SNPs were estimated using Haploview 4.2 (Broad Institute of MIT and Harvard, Cambridge, MA, USA). The association analyses for aggressive behavioral traits were performed using the GLIMMIX procedure of SAS 9.4 software with the following the generalized linear mixed model: =µ+ + + + + +c× + , where, is the phenotypic value of aggressive behavioral trait for each pig; μ is the overall mean; is the fixed effect of the genotype or haplotype combinations; is the fixed effect of the parity; is the fixed effect of the gender; is the individual random additive genetic effect, distributed as N (0; A ), with the additive genetic variance ; is the random effect of the pen; c is the regression coefficient of covariate ; is the body weight before mixing as a covariate; is the random residual, distributed as N (0; I ), with the additive genetic variance .
Promoter prediction of the porcine ARHGAP24 gene
The promoter region of the porcine ARHGAP24 gene was predicted by Promoter 2.0 (http://www.cbs.dtu.dk/services/Promoter/) [19] and Neural Network Promoter Prediction (https://www.fruitfly.org/seq_tools/promoter.html) [20]. Putative transcriptional binding start sites caused by the SNPs mutation in the 5′ flanking or UTR region of the ARHGAP24 gene were predicted by JASPAR 2020 (http://jaspar.genereg.net/) [21], AnimalTFDB 3.0 (http://bioinfo.life.hust.edu.cn/AnimalTFDB/#!/) [22].
Plasmid construction
The promoter region of the porcine ARHGAP24 gene was amplified by PCR using Vazyme LAmp Master Mix (Vazyme Biotech, Nanjing, Jiangsu, China). Subsequently, plasmids containing variable lengths of truncated the porcine ARHGAP24 promoter were individually amplified using different forward primers and a common reverse primer (ARHGAP24-P1: −33/+352, ARHGAP24-P2: −308/+352; ARHGAP24-P3: −670/+352; ARHGAP24-P4: −1113/+352; ARHGAP24-P5: −1572/+352; ARHGAP24-P6: −1976/+352), and the primers contained MluI and XhoI (Takara Bio Inc., Dalian, Liaoning, China) recognition sequences, respectively (Table S2). Subsequently, the amplified fragments were inserted into the multiple cloning sites of the pGL3-basic vector to generate luciferase reporter plasmids. Moreover, specific regions containing rs335052970, rs344700648, and rs339198696 were amplified using ARHGAP24-Haplotype primers contained recognition sequences of MluI and XhoI (Table S2). The DNA samples of piglets were amplified using primers (Table S2) targeting the promoter region of porcine ARHGAP24 gene containing p53 transcription factor binding element (PBE) motif and then cloned into pGL3-basic vector by MluI and XhoI. The plasmid with PBE was used as DNA template, amplified by point mutation primer-ARHGAP24-PBE-MUT (Table S2). The cDNA fragments of p53 were amplified using a forward primer containing an EcoRI (Takara Bio Inc., Dalian, Liaoning, China) site and a reverse primer containing a XhoI site (Table S2) and connected to the eukaryotic expression vector pcDNA3.1 (+) (pcDNA3.1-p53). The plasmid structures were sequenced to confirm the integrity of the constructed fragments.
Cell culture, cell transfection, and luciferase assays
Human embryonic kidney 293T (HEK 293T) cells were used for promoter activity analysis. Firstly, HEK 293T cells were cultured in an incubator at 37 °C and 5% CO2. Cells were plated in 12-well plates and 3 wells for each group. On the following day, the plasmids contained the variable length of ARHGAP24 promoter fragments, the rs335052970 A or G allele and haplotypes were individually co-transfected into the HEK 293T cells with pRL-TK Renilla luciferase reporter vector (Promega, Madison, WI, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Controls were the pGL3-basic and pGL3-control luciferase reporter gene vector. After 24 h, cells were harvested with passive lysis buffer (Promega, Madison, WI, USA). The cell lysates were assayed for reporter gene activity using a dual-luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
Primary neural cells were prepared from cerebral cortices of weaned piglets, as previously described [23]. In brief, cerebral cortices were removed from the piglet’s brains. Then, the meninges and microvessels were carefully removed in ice precooled D-Hanks' Balanced Salt Solution (HBSS, Gibco, Grand Island, NY, USA), and the brain tissue were minced into small pieces of about 1 mm3. After papain (Biofroxx, Einhausen, Germany) and DNase 1 (BioFroxx, Einhausen, Germany) were added respectively, they were digested in an incubator at 37 °C for 30 min. After the digestion was terminated with Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Grand Island, NY, USA), they were sub packed into 15 mL centrifuge tubes for centrifugation for 10 min. The porcine neural cells were cultured in DMEM supplemented with 20% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) at 37 °C in a humidified atmosphere with 5% CO2 for 48 h. Endotoxin-free plasmids of pcDNA3.1-p53 and pcDNA3.1 were transfected into primary neural cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), respectively. The siRNA of p53 and ARHGAP24 were designed and chemically synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd. The primer sequence is shown in Table S2. Either scrambled siRNA or p53 siRNA plasmids were transfected into primary neural cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was performed using the ChIP assay Kit (ab156907, Abcam, Cambridge, UK) according to the manufacturer’s instructions. Briefly, a total of 2 × 106 neural cells were fixed with 1% formaldehyde (Aladdin, Shanghai, China) via gentle swirling at room temperature for 10 min. Then, the fixing reaction was stopped by the addition of 1 mL of 2.5 M glycine solution (Beyotime, Shanghai, China). Nuclei were digested into 300-500 bp fragments. Inputs and immunoprecipitation (IP) samples were incubated with 2 μL of p53 antibody (AF0879, Affinity, Suzhou, Jiangsu, China) or 0.8 μL non-immune IgG overnight at 4°C on a rocking platform, and precipitated DNA fragments containing PBE motifs were detected using PCR with the specific primers listed in Table S2.
Immunofluorescence staining
After 10 days of culture, the porcine neural cells were identified by immunofluorescence staining. Briefly, the cells were washed three times with phosphate-buffered saline (PBS, HyClone, Logan, UT, USA) and fixed for 30 min in 4% paraformaldehyde (Beyotime, Shanghai, China). The cells were subsequently incubated in ice-cold 0.5% Triton X-100 (Beyotime, Shanghai, China) for 20 min and washed a further three times with PBS. Next, the cells were incubated with QuickBlock blocking buffer (Beyotime, Shanghai, China) at room temperature for 1 h. The cells were incubated with anti-beta III Tubulin (Tuj1) antibody (dilution 1:100; Abcam, Cambridge, UK) at room temperature for 1 h. The cells were then washed three times and incubated with fluorescein conjugated goat anti-rabbit IgG (H+L) antibody (dilution 1:500; Proteintech, Wuhan, Hubei, China) in the dark. After 1 h of incubation at room temperature, the cells were washed three times. The cell nuclei were stained with 4ʹ,6-Diamidino-2-Phenylindole (DAPI, Beyotime, Shanghai, China) in the dark. After washing three times, the glass slides were sealed with fluorescence anti-quenching agents (Beyotime, Shanghai, China). Images were captured using confocal microscopy (Zeiss, LSM 700; Oberkochen, Germany).
RNA extraction and quantitative reverse transcription-PCR (RT-qPCR)
The cells were harvested at day 1 post-transfection. Total RNA of porcine neural cells was extracted by using the Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The purity of RNA was detected by the NanoDrop 2000 (Thermo Fisher Scientific, Fremont, CA, USA). To quantify the mRNA expression level of ARHGAP24 and p53, total RNA was reverse-transcribed into cDNA using the HiScript III RT SuperMix (Vazyme Biotech, Nanjing, Jiangsu, China). The RT-qPCR was performed on Quantum Studio 5 quantitative PCR instrument (Applied Biosystems, Foster, CA, USA) using SYBR Green master mix (Vazyme Biotech, Nanjing, Jiangsu, China) and the specific primers (Table S2). Relative expression levels were calculated by using the 2−ΔΔCt method [24]. Coding gene expression levels were normalized to the expression of GAPDH. For the RT-qPCR reaction, each treatment had at least 3 biological replicates.
Western blotting
Cell protein lysates were harvested using 200 µL ice-cold radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) with 1% phenylmethyl sulfonyl fluoride (PMSF, Beyotime, Shanghai, China). Total protein extracts were separated on 4–20% SDS–PAGE gels (Genscript Biotech, Nanjing, Jiangsu, China) and then blotted onto polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). After blocking with QuickBlock™ blocking buffer (Beyotime, Shanghai, China) for 30 min, the PVDF membranes were incubated overnight with the following primary antibodies: immunoreactive proteins were detected with a rabbit polyclonal antibody for anti-p53 (1:1,000; AF0879, Affinity, China), anti-ARHGAP24 (1:1,000; DF9858, Affinity, China) and a rabbit polyclonal antibody for anti-GAPDH (1:5,000; AF7021, Affinity, China). The appropriate anti-rabbit secondary antibody (1:8,000; S0001, Affinity, China) was used to incubate the membranes. ECL Peroxidase Color Development Kit (Vazyme, Nanjing, Jiangsu, China) was used in chromogenic reaction according to the manufacturer’s instructions. The protein bands visualization was performed by the ChemiDoc XRS + system (Bio-Rad, Hercules, CA, USA). The band density was analyzed using ImageJ software.
Statistical analyses
Data were analyzed using SAS 9.4 (SAS Institute Inc, Cary, NC, USA). Chi-square tests were used to analyze the difference of allele frequency between the most aggressive and the least aggressive pigs. The behavioral data were analyzed using GLIMMIX procedure with a model option DIST = EXPO. The relative fluorescence activity value was normalized by negative control pGL3-basic. The significance of luciferase activity statistics was analyzed by unpaired two-sided student’s t-test and one-way ANOVA analysis. The results are presented as mean ± standard error of the mean (SEM), and P < 0.05 was considered significant.