| Hypophthalmichthys molitrix is one of the major freshwater fishes in China,it‘s aquaculture model and cultivation techniques, have been completely mature, but H.molitrix are sensitive to hypoxia and low degree of tolerance,it easily lead to a large area of death phenomenon.In this study, we described the c DNA cloning, m RNA tissue distribution and gene expression patterns in the liver, brain and gill under hypoxia of the cell cycle protein gene(Cyclin) named Cyclin A, Cyclin B, Cyclin D, Cyclin E, checkpoint kinase 1 and cell division cycle 25 A in H. molitrix. Simultaneously, we detected hepatocyte and brain cells apoptosis under hypoxic stress by annexin V/PI with flow cytometry and TUNEL. The results are as follows:1.Cloning and gene expression patterns of cell cycle genes under hypoxia in H. molitrix.We Designed the conserved primer according to the gene sequence of Danio rerio in Gen Bank, the complete c DNAs sequence of Cyclin A, Cyclin B and Cyclin E genes and the core fragment of Cyclin D, Chk1 and Cdc25 A genes in H. molitrix were obtained using the homology cloning and RACE method. Cyclin A c DNA sequence full-length was 1654 bp with a 1185 bp open reading frame(ORF) encoding 394 amino acid residues, a 73 nucleotides of 5’-UTR and a 396 nucleotides of 3’-UTR; Cyclin B c DNA sequence full-length was 1523 bp with a 1194 bp open reading frame(ORF) encoding 397 amino acid residues, a 77 nucleotides of 5’-UTR and a 252 nucleotides of 3’-UTR; Cyclin E c DNA sequence full-length was 1881 bp with a 1239 bp open reading frame(ORF) encoding 412 amino acid residues, a 212 nucleotides of 5’-UTR and a 430 nucleotides of 3’-UTR. Multiple polypeptide sequence alignment showed high identity of Cyclin A and Cyclin B within Cyprinus carpio(99% and 100%, respectively) and high identity of Cyclin E and Cyclin B within the zebra fish(99%, respectively).RT-PCR revealed that the cell cycle genes m RNA was expressed in some of the tissues in H.molitrix. There was a high expression in liver and brain, and a low expression in gill in Cyclin A; there was a high expression in heart, a low expression in other tissues in Cyclin B; there was a high expression in liver, brain, kidney and heart, and a lowexpression in other tissues in Cyclin D; there was a high expression in muscle, brain and liver, and a low expression in other tissues in Cyclin E; there was a high expression in liver and brain, no expression in spleen and a low expression in other tissues in Chk1; there was a high expression in heart, brain and liver, a low expression in gill and no expression in other tissues in Cdc25 A. At the same time, Real-time PCR suggested that control of silver carp from normal to floating and death under hypoxia stress, in the liver, Cyclin A and Cyclin E gene expression decreased gradually(P<0.05); Cyclin B and Cyclin D expression had no significant change(P>0.05); the expression levels of Chk1 gene increased and then decreased(P<0.05); the expression levels of Cdc25 A gene decreased and then increased(P<0.05); in the brain, the expression levels of Cyclin A and Cdc25 A gene decreased and then increased(P<0.05); Cyclin B and Cyclin D expression had no significant change(P>0.05); the expression level of Cyclin E gene decreased(P<0.05); the expression levels of Chk1 gene increased and then decreased(P<0.05); in the gill, Cyclin A and Cyclin E gene expression decreased gradually(P<0.05); Cyclin B and Cyclin D expression had no significant change(P>0.05); the expression levels of Chk1 gene increased and then decreased(P<0.05); the expression levels of Cdc25 A gene decreased and then increased(P<0.05). In the study, the result showed that the expression of the four cell cycle protein genes(Cyclin A,Cyclin E), Chk1 and Cdc25 A were regulated by hypoxia stress.2. The apoptosis of hepatocyte and brain cells I n H.molitrix(Hypophthalmichthys molitrix) under hypoxic stress were detected by annexin V/PI with flow cytometry and TUNELThe apoptosis of hepatocyte and brain cells in H.molitrix under hypoxic stress were detected by annexin V/PI with flow cytometry and TUNEL. The result of annexin V/PI with flow cytometry show that the rate of hepatocyte apoptosis are 8.85±0.24, 22.06±0.57 and 22.91±0.49 at control, floating and death stage, respectively; the rate of brain cells apoptosis are 8.18±0.45, 24.80±0.63 and 24.86±0.21 at control, floating and death stage, respectively. The result of TUNEL show that the rate of hepatocyte apoptosis are 3.80±0.16, 36.78±0.34 and 37.27±0.35 at control, floating and death stage,respectively; the rate of brain cells apoptosis are 6.74±0.30, 35.04±0.40 and 35.71±0.41 at control,floating and death stage,respectively. The results showed that compared with the normal control group, the apoptosis of hepatocyte and brain cells in silver carp significantly increased under hypoxic stress, with the hypoxia time lengthened, the number of the apoptosis cells were not significant changed. |
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