A functional mutation associated with piglet diarrhea partially by regulating the transcription of porcine STAT3.

The present study aimed to search for functional mutations within the promoter of porcine STAT3 and to provide causative genetic variants associated with piglet diarrhea. We firstly confirmed that STAT3 expressed higher in the small intestine than in the spleen, stomach and large intestine of SPF piglets, respectively (P < 0.05). Then, 10 genetic variations in the porcine STAT3 promoter region was identified by direct sequencing. Among them, three mutations SNP1: g.-870 G>A, SNP2: g.-584 A>C and a 6-bp Indel in the promoter region that displayed significant differential transcriptional activities were identified. Association analyses showed that SNP1: g.-870 G>A was significantly associated with piglet diarrhea (P < 0.05) and the GG animals had lower diarrhea score than AA piglets (P < 0.01) in both Min and Landrace population. Further functional analysis revealed that E2F6 repressed the transcriptional efficiency of STAT3 in vitro, by binding the G allele of SNP1. The present study suggested that SNP1: g.-870 G>A was a piglet diarrhea-associated variant that directly affected binding with E2F6, leading to changes in STAT3 transcription which might partially contribute to piglet diarrhea susceptibility or resistance.

A 12-bp indel in the 3'UTR of porcine CISH gene associated with Landrace piglet diarrhea score.

Variations in Cytokine inducible SH2-containing protein (CISH) gene influence human susceptibility to common infectious diseases, but little is known about CISH in swine. The objectives of this study were to 1) determine porcine CISH (pCISH) mRNA expression level in different tissues of piglets, 2) predict putative functional genetic variations within pCISH, 3) investigate the association between a identified variation in the 3'UTR and piglets phenotype traits in Min (n = 226) and Landrace (n = 186) population, and explore the function of this variation. Results of quantitative PCR showed pCISH mRNA expressed in all the collected tissues with higher level in lung and ileum than colon (p < 0.05). In-silico analysis indicated none of the functional ns-SNPs existed in pCISH coding region. Results from the characterizing of 3'UTR presented a novel 12-bp insertion/deletion (indel) mutation. Statistical analysis demonstrated that this 12-bp indel associated with piglets diarrhea score in the Landrace population, and animals with AA genotype (12-bp insertion) presented lower diarrhea score when compared with BB (p < 0.05) or AB (p < 0.01) carriers. The in vitro study indicated that the luciferase activity of reconstruct plasmid psiCHECK-2-CISH-AA or psiCHECK-2-CISH-BB was significantly lower than the negative control (p < 0.05), and luciferase activity of psiCHECK-2-CISH-AA was higher than that of the psiCHECK-2-CISH-BB (p < 0.05). Although results herein suggested the 12-bp indel might affect Landrace piglet susceptibility to diarrhea, further association studies in more populations are needed before this preliminary finding could be used for pig breeding.

Identification of mutations in porcine STAT5A that contributes to the transcription of CISH.

Identification of causative genes or genetic variants associated with phenotype traits benefits the genetic improvement of animals. CISH plays a role in immunity and growth, however, the upstream transcriptional factors of porcine CISH and the genetic variations in these factors remain unclear. In this study, we firstly identified the minimal core promoter of porcine CISH and confirmed the existence of STATx binding sites. Overexpression and RT-qPCR demonstrated STAT5A increased CISH transcriptional activity (P < 0.01) and mRNA expression (P < 0.01), while GATA1 inhibited CISH transcriptional activity (P < 0.01) and the following mRNA expression (P < 0.05 or P < 0.01). Then, the putative functional genetic variations of porcine STAT5A were screened and a PCR-SSCP was established for genotype g.508A>C and g.566C>T. Population genetic analysis showed the A allele frequency of g.508A>C and C allele frequency of g.566C>T was 0.61 and 0.94 in Min pigs, respectively, while these two alleles were fixed in the Landrace population. Statistical analysis showed that Min piglets with CC genotype at g.566C>T or Hap1: AC had higher 28-day body weight, 35-day body weight, and ADG than TC or Hap3: CT animals (P < 0.05, P < 0.05). Further luciferase activity assay demonstrated that the activity of g.508A>C in the C allele was lower than the A allele (P < 0.05). Collectively, the present study demonstrated that STAT5A positively regulated porcine CISH transcription, and SNP g.566C>T in the STAT5A was associated with the Min piglet growth trait.