Molecular Cloning, Characterization, Expression Analyses Expression Of Megalobrama Amblycephala GRHR And GR

Corticotropin-releasing hormone receptor(CRHR) and Glucocorticoid receptor(GR) is the key hormone receptor of hypothalamic-pituitary-adrenal(HPA) axis. They play an important role in the ion balance, endocrine, immunity and stress. As a common phenomenon, Stress is good for animals to adapt to the environment. But it is harmful under the excessive stress events. M. amblycephala is an economic breeding species in China which shows inferior ability to resist stress compared with other Four Major Chinese Carps. However, this fish species is an ideal object for stress research. At present, there are few researches based on these receptors of M. amblycephala. Therefore, we intends to obtain more complete information of the pathway by monitoring the key receptor gene expression on HPI axis under stress state in this study, that will be great to prevente the stress related diseases of aquatic animals and promote the production of fishery.The M. amblycephala CRHR and GR mRNA was cloned by RACE PCR. The sequences were analyzed with biology information technology. The expression profiling of M. amblycephala CRHR and GR in different tissues was analyzed by quantitive real-time PCR, and its expression variation to stress was detected after exogenous cortisol injection.The results are as follows:1. Obtained the full length of M. amblycephala CRHR1 mRNA, containing a 1290 bp open reading frame(ORF), and encoding 429 amino acids. Amino acid sequence alignment showed that M. amblycephala CRHR1 had the highest sequence identity with C. auratus CRHR1, 99.07%. Tissue expression analysis showed that the highest expression of CRHR1 was observed in Hypothalamus, followed in Pituitary and the lower expression was observed in Liver, Kidney, Muscle, Skin, Heart. After cortisol injection, serum glucose come to a high plateau from 2 h to 24 h, and the serum cortisol reach a peak at 12 h. The variation tendency of the CRGR1 mRNA is different in different tissues with cortisol injection. In Pituitary, there was a remarkable decrease immediately after cortisol injection, but in Head kidney and Heart, there was a striking increasing. In Hypothalamus, the change of CRHR1 seems like a unimodal pattern, increasing slowly at the early stage and then slowly decreasing at the end.2. Obtained the full length of M. amblycephala GR1 a, GR1 b and GR2 mRNA, containing a 2199, 2226, and 2241 bp open reading frame(ORF), and encoding 732, 741 and 746 amino acids, respectively. Sequence homology analysis and phylogenetic analysis showed that M. amblycephala GR1 was clustered with cyprinidae fish GR1, and M. amblycephala GR2 was clustered with cyprinidae fish GR2. Tissue expression analysis showed that the higher expression of GR1 was observed in Heart, Spleen, Ovarian and Skin. So does GR1 b.While GR2 showed a higher expression in Liver, Hypothalamus, Pituitary, Gut, Testis and Muscle. After cortisol injection, the variation tendency of the GRs m RNA is different in different tissues. In Hypothalamus, the changes of GR1 put up no clear variation tendency; while the changes of GR1 b and GR2 increasing slowly at the early stage and then gradually stabilized. In Pituitary, GR1 increasing at the early stage then decreasing at the later; And GR1 b show some volatility; The variation tendency of GR2 seems like a unimodal pattern. In Head kindey, all the receptors expression was decrease at the early stage. In Gill, all the receptors expression was striking increasing at 2h. In Skin, the variation tendency of GR1, GR1 b and OCC all seems like a unimodal pattern, but the location of the peak is different. In Liver, the variation tendency of GR1 and GR1 b all seems like a unimodal pattern, while the expression of HSP70 and PEPCK was striking increasing during a short period of time.