Molecular Cloning And Expression Patterns Of Hypoxic Stress Related Genes In Hypophthalmichthys Molitrix

Silver carp (Hypophthalmichthys molitrix) is an important proportion in Chinese aquaculture. It is hypoxia-intolerance and easily affected by environmental stress. The characteristic of silver carp caused great loss in the long-term breeding. Considering the mechanisms under hypoxia plays an important role in breeding of silver carp. By the homology gene cloning and RACE methods, the cDNA full length of insulin-like growth factor binding protein-1(IGFBP-1) and insulin-like growth factor-Ⅰ(IGF-Ⅰ) were cloned from liver of silver carp. The cDNA full length sequences were analysed in bioinformatics. At the same time, the full length cDNA sequences of α-tubulin and α-actin were cloned from muscle of silver carp. The cDNA sequences of the above genes were analysed in bioinformatics and the protein structures and functions were forcasted. IGFBP-1mRNA was detected in different tissues of silver carp by RT-PCR. The IGF-Ⅰ mature protein was subcloned into pET-32(a)+. IPTG was used to induce the expression of IGF-Ⅰ. The characterization of the expression patterns of the genes under hypoxia of0h,2h,4h,6h,8h,10h were analysed by Real-time PCR. The expression of IGFBP-1and IGF-I protein under hypoxia in liver were analysed by Elisa. The main results were:(1) IGFBP-1cDNA is1081bp in length which including73bp5’-untranslated region,219bp3’-untranslated region and789bp open reading frame which encodes262amino acids. The homology of nucleotide sequence of IGFBP-1was89%,86%,80%,75%,73%,73%and73%compared with Cyprinus carpio, Danio rerio, Oncorhynchus tshawytscha, Scophthalmus maximus, Micropogonias undulatus, Pelteobagrus fulvidraco and Lateolabrax japonicus, respectively. Two IREs and one HAS were existed in3’-untranslated region, indicating that IGFBP-1is regulated by insulin and hypoxia induced factor.RT-PCR results showed that IGFBP-1mRNA were expressed in muscle, heart, brain, kidney, liver, gill and spleen. It showed high expression in liver, heart and muscle, but a low expression in brain. IGFBP-1was induced significantly under hypoxia. Real-time PCR revealed that in liver, the expression level increased significantly at6hours under hypoxia, and decreased a little after that but still at a high level compared to the control group (P<0.05).(2) The whole open reading frame (ORF) of IGF-I is486bp, and the homology of nucleotide sequence in open reading frame is99%,98%and95%compared with Hypophthalmichthys nobilis, Ctenopharyngodon idella and Cyprinus carpio respectively. The homology of nucleotide sequence in ORF of silver carp was76%compared with Homo sapiens.The precursor peptide of IGF-I contains S, B C, A, D and E regions, when it turns into the mature peptide, S and E regions were removed. Multiple polypeptide sequence alignment showed that silver carp IGF-I contains B, C, A, D and E regions. The analysis of E region indicated that the cloned silver carp IGF-I belongs to IGF-IEa-2subtype.The IGF-I mature protein were subcloned into pET-32(a)+and the recombinant protein was induced by IPTG SDS-PAGE revealed that pET-32a (+)-IGF-I m could express the recombinant protein. The expression of the mature IGF-I protein was induced highly by the optimization of different times and different IPTG concentrations on the expression of pET-32a (+)-IGF-I m. The results showed that when IPTG concentration was0.7mM and the inducing time at2h were the best condition.(3) a-tubulin and a-actin were cloned from muscle of silver carp, a-tubulin cDNA is1565bp in length with an open reading frame of1365bp which encodes451amino acids. RT-PCR results showed that a-tubulin mRNA were expressed in muscle, heart, brain, kidney, liver, gill and spleen. It showed high expression in brain and liver, but a low expression in spleen. Real-time PCR was used to explore the expression level in muscle of silver carp. The results showed that a-tubulin was increased significantly at4h under hypoxia, but decreased after that till12h under hypoxia (P<0.05). a-actin cDNA is1399bp in length with an open reading frame of1134bp which encodes377amino acids. RT-PCR results demonstrated that a-actin mRNA was detected in muscle, heart, brain, kidney, liver, gill and spleen. Real-time PCR revealed that in muscle, brain, heart and kidney, the expression level of a-actin was decreased under hypoxia compared to the control group, it was increased a little in liver, while in spleen, no significant change was detected (P<0.05).