| Heat stress is the most important stressful pathogenic factors of pig industry in southern China,to elucidate the function of key molecular that play important role in the process of heat stress,will facilitate the breeding process of a heat resistance breed(lines)because of the supply of a gene material,as well helped to the control and prevention of heat stress.In this study,we cloned the porcine SOCS3 and SOCS4 gene and predicted the potential molecular function.The real time quantitation polymerization chain reaction(q RT-PCR)methods were used to detect the expression of SOCS3/4 m RNA in the differental tissues of heat stressed pig.A cell model of HSP70 overexpressing were also established for the exploration of SOCS3/4 regulation machinism to nuclear factor-?B where 293 T cell line and IPEC-J2 cell line were used.The results show that:(1)The porcine SOCS3 gene consists of 690 bp of ORF,encodes a protein precursor with 229 amino acids,which shares high amino acid sequence identity with that in Ba Mei pig(98.2%),Equus caballus(97.4%),Bos Taurus(96.5%),Homo sapiens(97.8%)and Mus musculus(96.4%).The molecular weight of SOCS3 protein is 25.1k Da,and PI is 8.84.It also has the classic structural trait of SOCS family,in which a central SH2 domain,a C-terminal 40-amino-acid SOCS box domain and a 12-amino-acid KIR domain are consisted.We also investigated the dynamic of SOCS3 m RNA in thymus gland,spleen,mesenteric lymph node and intestine lamina propria of pigs during 10 days consecutive heat stress,and found that the expression of SOCS3 in mesenteric lymph node were decreased on day 3,day 6 and day 9 after heat stress begin significantly(P?0.05).But to the spleen and intestine lamina propria,there were a drastic up-regulation on day 6 and day 9,although a reduced tendency was displayed on day 3(P?0.05).Porcine SOCS3 negatively regulates the activity of NF-?B which induced by overexpressing of HSP70 in both of 293 T cell line and IPEC-J2 cell line.On the other hand,TNF-? expose amplicated the induce effect of HSP70 to NF-?B activity.We also showed that SOCS3 inhibited the phosphorylation of I?B-? and activated NF-?B via the degradation of Mal protein,a upstream molecular of NF-?B signal pathway.(2)The porcine SOCS4 gene consists of 1326 bp of ORF,encodes a protein with 441 amino acids,285 amino acids of them form the N-terminal,which shares high amino acid sequence identity with that in Changbai pigs(100%),Bos Taurus(96.1%),Capra Hircus(96.1%),Felis catus(95.2%),Equus caballus(94.6%),Canis lupus familiaris(94.3%),Homo sapiens(93.0%)and Mus musculus(88.1%).The molecular weight of SOCS4 protein is 50.5 k Da,and p I is 6.60.q RT-PCR results showed that the expression of SOCS4 in thymus gland after heat stress was significantly upregulated on day 3 and 9(P?0.05).As well in mesenteric lymph node,where it significantly upregulated on day 1 and 6(P?0.01).Difference to above mentioned tissues,it were significantly decreased in small intestine and spleen(P?0.05).Porcine SOCS4 negatively regulates the activity of NF-?B which induced by overexpressing of HSP70 in both of 293 T cell line and IPEC-J2 cell line.On the other hand,TNF-? expose amplicated the induce effect of HSP70 to NF-?B.We also showed that SOCS4 inhibited the phosphorylation of I?B–? and activated NF-?B,but this function does not via Mal protein.Our studies cloned the porcine SOCS3/4 c DNA successfully,and found a significant changes of SOCS3/4 m RNA expression in heat stressed pigs with a tissue and time dependence manner.Porcine SOCS3/4 negatively regulates the activity of NF-?B which induced by overexpressing of HSP70 in both of 293 T cell line and IPEC-J2 cell line.TNF-? expose amplicated the induce effect of HSP70 to NF-?B.We also showed that SOCS3/4 inhibited the phosphorylation of I?B–? and activated NF-?B,in witch,SOCS3 work via the degradation of Mal protein,a upstream molecular of NF-?B signal pathway,but not SOCS4. |
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