Studies On Cloning And MRNA Expression Of Porcine CRHR1 And CRHR2 Genes

In the modern swine production, the stress syndrome, including weaning stress, the slaughter stress, heat stress and so on, brings great loss in the pig industry economically. As a result, a great deal of efforts has been made to control porcine stress, especially on the sides of feed and environment management. But pig stress is controlled by not only environmental but also genetic factors. Thus, it is indispensable to uncover the molecular genetic mechanism of the pig stress.As stress arises, the excitement of hypothalamus-pituitary-adrenal (HPA) axis causes the release of hypothalamic corticotropin-releasing hormone (CRH), as well as neuropeptide hormone to the hypothalamic portal system, which stimulates adrenocorticotropic hormone (ACTH) secretion by binding the corticotropin-releasing hormone receptor (CRHR) in the cell in the front of pituitary. With the effect of ACTH in blood on the adrenal cortex in the peripheral organs, glucocorticoid (GC) is released. Finally, GC is a key hormone to the stress which can affect the whole body.Therefore, in order to uncover the molecular mechanism of pig stress, this study focused on pig CRH receptor genes CRHR1 and CRHR2 basing on the physiological process of pig stress described above. Gene cloning, sequence analysis, protein structure and function prediction of these two genes were finished as well as their mRNA expression analysis in the brain of weaning piglets.The main research works and conclusions are as follows:(1) The first complete coding sequence (CDS) of CRHR1 and CRHR2 are obtained using the approach of combining homology and electronic cloning. CRHR1 gene contains a 1,248 bp open reading frame (ORF) encoding 415 amino acids while CRHR2 gene contains a 1,236 bp ORF encoding 411 amino acids. Analysing the two genes and database, we found six single nucleotide polymorphism sites.(2) The alignment analysis of CDS sequences of CRHR1 and CRHR2 among rats, mice, cows, dogs and pigs shows that the consistency of CRHR1 gene in pig and other mammals is more than 91%, while the comparability of CRHR2 is more than 86%. The comparability of the protein sequences of CRHR1 and CRHR2 of pig with other mammals are more than 97% and 88% respectively. It verified that CRHR1 and CRHR2 gene are highly conserved in mammals.(3) We predicted the basic features, protein structure and molecular function of porcine CRHR1 and CRHR2 proteins with bioinformatics methods. The results indicate that porcine CRHR1 and CRHR2 belong to G-protein-coupled receptor family, which consist of a GPCR domain, including four extracellular domains, seven transmembrane domains and four intracellular domains. CRHR1 and CRHR2 proteins structure also contains a hormone receptor domain that suggests those two genes have the basic characteristics and functions of hormone receptor. Bioinformatics results show that porcine CRHR1 and CRHR2 proteins are very conservative in the key structural and functional region. This suggests that porcine CRHR1 and CRHR2 proteins should have the similar function with some known proteins.(4) CRHR1 and CRHR2 mRNA expression was detected in the brains of weaning and non-weaning piglets using real-time quantitative PCR method. The result shows the effects of weaning stress on the mRNA expression level of CRHR1 and CRHR2 in the piglet brain exist though not significant (p>0.05) statistically.Obviously, our research built an important foundation for finding the effective genetic anti-stress approach and developing the anti-disease breeding of pig.