| In this study, we described the cDNA cloning, mRNA tissue distribution and gene expression patterns in the heart, brain, liver, spleen, kidney and muscle under hypoxia of the tubulin gene named TUB84, TUBA2and the inducible heat shock protein70(HSP70) in H. molitrix. Simultaneously, we detected the protein expression of the inducible heat shock protein70(HSP70) and constitutive heat shock protein70(HSC70) in the organizations above under hypoxia. In addition, the protein expression of HSP70in brain during the rescue process after asphyxia also is determined. The results are as follows:1. Cloning and gene expression patterns of TUB84and TUBA2under hypoxiaThe complete cDNAs sequence of TUB84and TUBA2gene in H. molitrix were obtained using the homology cloning and RACE method. TUB84cDNA sequence full-length was1490bp with a894bp open reading frame (ORF) encoding297amino acid residues, a453nucleotides of5’-UTR and a143nucleotides of3’-UTR. The full-length cDNA of TUBA2is1815bp and the open reading frame is1007nucleotides flanked by a105nucleotides of5’-UTR and a703nucleotides of3’-UTR. Multiple polypeptide sequence alignment showed high identity of TUB84and TUBA2within the zebra fish (98%and97%, respectively).RT-PCR showed the TUB84and TUBA2mRNA in H. molitrix were highly expressed in heart, brain, muscle, spleen, liver, kidney and gill, and there was no significant expression differences among these tissues. At the same time, Real-time PCR suggested that The TUBA2gene expression rose slightly only in the heart at2hah (P>0.05), while TUB84gene had no significant changes in all tissues. At4hah, there were a significant down-regulated expression in heart (P<0.05) and a slight rise in brain (P>0.05) for TUB84gene, while TUBA2gene expression rose slightly in brain, heart and muscle (P>0.05). At6hah, there were a significant rise in liver, kidney and muscle for TUB84gene (P<0.05), and a significant decrease in heart (P<0.05). At the same time, TUBA2gene expression increased sharply in liver, spleen and kidney (P<0.05), but in heart an opposite result was observed. At8hah, both TUB84gene expression and TUBA2gene expression had obvious rise in kidney (P<0.05), besides, in the liver the TUBA2gene expression also rose significantly (P<0.05). At10hah, the TUB84gene expression decreased to the control level in kidney but increased significantly (P<0.05) in spleen, while the TUBA2gene expression decreased significantly in both liver and kidney (P<0.05), but it was still higher than the control group in liver and lower in kidney (P<0.05). Therefore, the expression of tubulin gene TUB84and TUBA2were regulated by hypoxia stress, but the exact mechanism remains to be further studied.2. Cloning and expression analysis of an inducible heat shock protein70gene in silver carpThe homology cloning and RACE PCR were employed to clone the cDNA of an inducible HSP70and PCR amplification of translated region with DNA template was used to check its introns. The tissue expression and expression patterns of HSP70mRNA under the condition of acute hypoxia were analyzed by RT-PCR and real-time PCR, respectively. The full-length cDNA of HSP70is2339bp and the open reading frame is1932nucleotides flanked by a67nucleotides of5’-UTR and a340nucleotides of3’-UTR. The ORF encoded a predicted polypeptide of643amino acids. Intron absent in the translated region accorded with the characteristic of inducible type HSP70.RT-PCR revealed that the HSP70mRNA was expressed in all the seven tissues and there was a high expression in heart and brain, a low expression in liver, muscle, spleen and kidney. Real-time PCR indicated that in all tissues, the expression level of HSP70mRNA changed variously under hypoxia. in heart, it increased radically until up to the highest at4hah, then dropped, but it was still higher than the control group obviously at10hah (P<0.05). In brain, it had a upward tendency on the whole which was markedly higher than the control level as well (P<0.05). In spleen and kidney, it rose until10hah achieving its highest level, while there was no significant change at the early time in the muscle, but it increased suddenly at10hah (P<0.05). In liver, HSP70mRNA increased at4hah (P<0.05) but decreased to the control level at6hah, then it increased again to reach its highest expression level at10hah. Thus, it could be inferred that the expression of inducible heat shock protein70gene was regulated by hypoxia stress, and the up-regulation of HSP70mRNA in the various organizations may be a mechanism of silver carp that was response to hypoxia stress, which may provide a theoretical basis for us to study silver carp under hypoxia.3. Expression patterns of two heat shock protein under hypoxiaELISA showed there was no significant change in all tissues above for HSC70protein expression, but some different expression patterns for HSP70in sliver carp. In liver, the HSP70had a fluctuant expression whose expression level was higher than the control level except at6hah (P<0.05). HSP70protein contained in heart increased at first, then dropped under hypoxia, which reached the highest level at4hah. In brain, the expression level increased until8hah, then decreased at10hah, but its expression was higher than the control level all the time (P<0.05). In spleen and muscle, the expression level increased significantly at8hah. In kidney, there was a fluctuant expression, and its expression level was lower than the control level at4hah (P<0.05), then it increased to the highest expression level at8hah, but decreased again at10hah.Some rescue experiments was tested on the sliver carp which suggested it was about15-25minutes for sliver carp recovering to swim normally after asphyxia. Real-time PCR showed HSP70protein expression was higher than the control level at15min and30min after asphyxia (P<0.05), then it decreased to the control level until180min after asphyxia, which suggested HSP70had played a significant role from asphyxia to recovering. |
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