| The increasing industrial and agricultural activities have caused rising water deterioration, with the capacity of carrying oxygen decreased. Causing oxidative stress reaction of the aquatic animals, the yield of the aquaculture declined consequently. Silver carp(Hypophthalmichthys molitrix) accounts for an important proportion in Chinese aquaculture. Easily affected by the environmental stress, silver carp is known as hypoxia-intolerance. The mechanisms under hypoxic stress of this hypoxia-intolerance species are still unknown. Considering the significance of the mechanisms applying to the anti-stress breedings, it will be very exciting to work on it. The gynogenesis technology can provide experimental animals with better consistent genetic background. With the homology gene cloning methods, three functional genes HIF-la, SODs and Transferrin, which play important role during the hypoxic stress, were cloned the first time. Analyzing the cDNA full length sequences of the genes, the protein structures and functions were forcasted. The characterization of the expression patterns of the genes analyzed by real-time PCR (SYBR green I), including tissue-specific expression patterns, embryogenetic expression patterns. In addition, acute hypoxia induced expression patterns of the two genes were also analyzed.1. HIF-1 alpha:alpha subunit of a transcription factor which respond to changes in available oxygen in the cellular environment.HIF-la cDNA is 3828 bp in length with an 2325 bp open reading frame(ORF) encoding a 774 amino acids (aa). Multiple polypeptide sequence alignment showed high identity of HIF-la within the cyprinid fish (88-95%). Analyzing the conserved domain database, a bHLH structure, two PAS structures, a C-TAD domain and a pVHL recognized sequence were found on silver carp HIF-1αprotein.Silver carp HIF-la mRNA were highly expressed in heart, brain, muscle and gill with slightly expressed in liver, kidney and spleen. HIF-1αwas detected throughout the embryogenesis in a highly dynamic pattern with highest level at cell cleavage stage (2-4 cells) and decreasing levels during post-cell cleavage stages. Acute hypoxia differently affected the expression of HIF-1αin liver and gill. In liver, the expression level increased at 9 hah (P<0.05) and kept increasing until 18 hah (50 bold compared to the control group). In gill, HIF-la increased at 3 hah and reached highest expression level at 12 hah (15 bold compared to the control group), and decreased after that until to the control level (P<0.05).2. SODs:a class of enzymes that play important antioxidant defense role in nearly all cells exposed to reactive oxygen species.Full-length Cu/Zn-SOD cDNA consisted of 753 bp with a 465 bp ORF encoding a 154 aa. A 1008 bp long cDNA of Mn-SOD was obtained, relatively, which encoded a 224 aa. Both Kozak sequence and polyadenylation signal locating on the sequence revealed the integrity of the sequences we cloned. The conservative sequence and motif also proved the silver carp SODs represented all the essential characteristics of known SOD orthologues.Both Cu/Zn-SOD and Mn-SOD were detected in heart, brain, liver, kidney, spleen, muscle, gill and blood. Cu/Zn-SOD and Mn-SOD were expressed throughout the embryogenesis, indicating their important roles during embryonic development especially at the cleavage stage. Acute hypoxia suppressed expression of Cu/Zn-SOD and Mn-SOD in liver significantly, up-regulated them in gill relatively, indicating that tissue-specific expression of Cu/Zn-SOD and Mn-SOD is an important molecular mechanism of adaptation to hypoxia.3. Transferrin:a liver-derived iron transport protein whose plasma concentration increase following exposure to hypoxia, which is mediated by hypoxia-inducible factor-1The entire Transferrin cDNA sequence is 2365 bp long and the open reading frame is 2025 bp and encodes a protein with 674 amino acids. Forecasting molecular characteristics of the gene by online software, the result showed that it was composed of two domains, and had a signal peptide of 15 amino acids which located the 21 st amino acid of N-terminal.Transferrin gene had the highest expressional level in liver, then the spleen. There was no expression in other tissues. During embryogenesis, the expression of transferring mRNAs was first detected at the gastrula stage and the expression level increased steadily until the tail bud embryo. Transferrin mRNA showed marked reduction after the stage of tail bud embryo until larvae. Results from the present study indicate that Transferrin played an important role in organogenesis during embryogenesis.This study cloned 3 functional gene related to hypoxic stress, the full length of the cDNAs will benefit the following research related. The expression patterns during embryogenesis and under hypoxic stress illustrate the important function of the genes under hypoxic stress in transcriptional level, wich will contributed to further research on the hypoxic stress reaction in fish. |
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