Cloning And Expression Of CRH And CRH-R1 From Ctenopharyngodon Idellus

Corticotropin releasing hormone (CRH) is the key factor of the hypothalamic-pituitary-adrenal axis (HPA), which not only acts on the pituitary gland, but also widely in the central nervous system and the hypothalamus. It plays a vital role in vertebrates to regulate stress response. In this study, These genes of full-length cDNA of CRH and CRH-Rlwas cloned from grass carp, Ctenopharyngodon idellus, and the expression of CRH was analyzed in the heart, spleen, kidney, liver, brain, skin, head kidney and gill in healthy grass fish respectively. The main results of this thesis are as follows:1. Degenerate primer was designed according to sequences of zebrafish, human and carp CRH gene in GenBank. The mid-sequence of CRH gene was amplified from grass carp, Ctenopharyngodon idellus by PCR. And then we obtain the sequence of full-length CRH cDNA of grass carp, Ctenopharyngodon idellus using 5'-RACE and 3'-RACE. CRH cDNA sequence was analyzed using bioinformatics, The full length of CRH gene cDNA is composed of 907bp, including a 489bp open reading frame, which encodes 163 amino acids, flanked by a 126 bp of 5' untranslated region(UTR) and a 292 bp of 3'-UTR, its molecular weight is about 18KD.2. The special primers of CRH and ?-actin gene of grass carp, Ctenopharyngodon idellus were designed to analyze CRH gene express in different tissues by Real time PCR. The expression profile of CRH was detected in heart, kidney, head kidney, brain, skin, gills, liver, spleen of grass carp, Ctenopharyngodon idellus. The results showed that CRH was the highest expression in the brain. The Immunohistochemical technology was used to further to analyze the express of CRH in the sames tissues of grass carp, Ctenopharyngodon idellus. The results reveals that there are expressed in heart, liver, spleen, kidney, head kidney, brain, gill tissue, respectively.3. Degenerate primer was designed according to sequences of zebrafish, human and carp CRH-R1 gene in GenBank. The mid-sequence of CRH-R1 gene was amplified from grass carp, Ctenopharyngodon idellus by PCR. And then we obtain the sequence of full-length CRH-R1 cDNA of grass carp, Ctenopharyngodon idellus using 5'-RACE and 3'-RACE. CRH-R1 cDNA sequence was analyzed using bioinformatics, The full length of CRH-R1 gene cDNA is composed of 2174bp, including a 1831bp open reading frame, which encodes 429 amino acids, flanked by a 540 bp of 5'untranslated region(UTR) and a 334 bp of 3'-UTR, its molecular weight is about 49KD.