Cloning and Characterization of Molt-Inhibiting Hormone Gene in Black Tiger Prawn (Penaeus monodon)

• Main Authors: Lin Yu-Chung, 林禦崇
• Other Authors: Lu Jenn-Kan
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/66125274483847399616

碩士 === 國立海洋大學 === 水產養殖學系 === 89 === In the neurosecretory system of crustaceans, the X-organ sinus gland complex in the eyestalk has been shown to have a physiological and growth regulatory effects. The crustacean molt-inhibiting hormone (MIH) is released from the X-organ sinus gland complex and suppresses ecdysteroid synthesis by the Y-organ. In the present studies, degenerated primers were designed from the amino acid sequence of the neuropeptides of kuruma prawn (Penaeus japonicus) and other crustaceans. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) was performed using eyestalk complementary DNA of the tiger prawn (Penaeus monodon) as template. 3’ and 5’ Rapid Amplifications of cDNA Ends (RACE) methods were performed to clone entire cDNA encoding for molt-inhibiting hormone-like (MIH-like) neuropeptide. A partial DNA sequence (555bp) that coding for a protein homologous to the MIH gene was isolated, this cDNA fragment including an intron (244bp) and deduced amino acid of two exons consisting of 59 a.a. residues were identified. The sequence alignment analysis indicated that the deduced amino acid sequence of the tiger prawn is highly homologous to the MIH gene in other crustaceans, including kuruma prawn (Penaeus japonicus), sand shrimp (Metapenaeus ensis), Procambarus clarkii, Charybdis feriatus, Cancer magister, Cancer productus, Callinectes sapidus, Penaeus vannamei and Procambarus bouvieri, sharing 94.9%, 86.4%, 50.9%, 46.7%, 45.0%, 45.0%, 41.7%, 40%, 26.3% identity and the cDNA identity sharing 75.6%, 65.4%, 50.8%, 46.3%, 44.4%, 50.0%, 39.1%, respectively. In vitro transcription had been performed to produce a double-stranded RNA based on the MIH coding sequence of Penaeus monodon and these MIH dsRNA (220bp) were injected into the eyestalk of the tiger shrimp and resulting a slightly decrease of molting interval. The result of Southern Blot analysis indicated that at least one putative MIH gene is present in the Penaeus monodon genome. However, it is also possible that duplicated MIH genes sharing the same restriction enzyme cutting site may occur.