Tissue Distribution, Developmental Expression Pattern Of Zebrafish P8, P8-1, P8-L2 Gene And The Response To Stress

Transcription factors play an important role in both an animals'growth and development and in response to the enviromental stress. Transcription factors with a basic helix-loop-helix (bHLH) domain are one of the largest transcription factor families. p8, also named com1 or Nupr1, p8 encodes a transcription cofactor with a basic helix-loop-helix (bHLH) domain. Recently, p8 has been investigated with reference to regulation of stress in the mammals, but its role in the fish is little known. Fish lived in a highly variable environment and it is easy for them to be influenced or stressed by changing environment. This directly influences the fish immune responses greatly and could lead to disease outbreaks. In this study, Zebrafish, was chosen to clone the p8, p8-L1 (p8-like 1) and p8-L2 (p8-like 2) genes both belong to p8 super family, to study the tissue distribution, expression pattern in embryonic developmental stages of p8 and its regulation in response to some environmental stressors. Further, an attempt has been made to illustrate the biological mechanism of p8, which is necessary to instruct the fish farming in theory.Bioinformatics analysis showed, the full-length zebrafish p8 cDNA consists of 730bp, is in 3 chromosome, and encodes 75 amino acids with a molecular weight of 8.961KDa and isolectric point of 8.433. Zebrafish p8 protein was found to encode bHLH, a well-conserved domain during the 39-65 amino acids and a well-conserved NLS sequence during the 60-74 amino acids. Zebrafish p8-L1 and p8-L2 cDNA consists of 742bp and 813bp, both are in 5 chromosome, have the same ORF and encodes 76 amino acids with a molecular weight of 8.801KDa and isolectric point of 8.86. Zebrafish p8 genome contains two introns and three exons, which is 225bp, 138bp, 353bp respectively. p8 sequence comparison and phylogenetic analysis show that the amino acid sequences of zebrafish p8 have a closer similarity to its vertebrate counterparts than the invertebrate. Zebrafish p8-L1 genome also contains two introns and three exons, which is 291bp, 208bp, 147bp respectively, whereas zebrafish p8-L2 genome only contains one intron, which is 813bp. The same to zebrafish p8, they are both contain Phospho_p8 domain.The tissue distribution and developmental expression of zebrafish p8 were detected by Quantitive real-time PCR. It was found that zebrafish p8 mRNAs had a wide distribution in all tested tissues and developmental stages of embryos. However, zebrafish p8 was expressed strongly in liver, brain and its expression was the highest in backbone, whereas expressed at a lower intensity in the gills, intestine and muscles. The expression of zebrafish p8 mRNA varied much from cleavage stage to hatching stage. Zebrafish p8 mRNA was expressed maternally at high levels in cleavage stage, reduced sequentially from blastula to segmentation stage to reach the lowest point, but elevated sharply at hatching stage. It is possible that zebrafish p8 is expressed widely as a housekeeping gene and performs an important function in the early embryogenesis. The tissue distribution of zebrafish p8-L1 was different from that of zebrafish p8 evidently, whereas zebrafish p8-L2's was similar with zebrafish p8's. Besides, The developmental expression of zebrafish p8, p8-L1 and p8-L2 were further detected by in situ hybridization to whole-mount zebrafish embryos, which was nearly consistency with Quantitive real-time PCR analysis. The results revealed that the anti-sense probes gave intense ubiquitous staining from the cleavage stage to blastula stage, and was up to the gastrula stage. At the segmentation stage, the expression of zebrafish p8 as a whole decreased and was concentrated in the head and the ventral side of the notochord, which almost can not examined in the dorsal side. With the full development of the somites, the expression of zebrafish p8 in the whole was decreased sequentially, which was also strong in the head and weak in the trunk and tail. In the hatching stage, zebrafish p8 mRNAs were only expressed in the head and ventral fins slightly. The expression pattern of zebrafish p8-L1 and p8-L2 were also similar with that of zebrafish p8. This specific pattern of expression indicates that zebrafish p8, p8-L1 and p8-L2 might all play an essential role in the development of central nervous system and play a part in most tissues eyes and internal organs such as heart, blood in zebrafish.Zebrafish were exposed to several stressors such as starvation challenge, temperature challenge, osmotic pressure and pH changes. The response to different enviromental stressors was detected by Quantitive real-time PCR using backbone mRNA. Quantitative real-time PCR assay suggested that zebrafish p8 mRNA expression was significantly up-regulated with exposure to the stressors. We provide evidence that zebrafish p8 could respond to a more wide range of cellular stressors, further supporting that zebrafish p8 acts as a stress-related gene. Zebrafish p8-L1 and p8-L2 transcripts are both up-regulated in response to osmotic pressure and pH changes.Finally, an expression vector including the cDNA encoding for zebrafish p8 and 5'additional tags of pET28a was constructed and transformed into E.coli cells. The recombinant peptide was induced by IPTG and purified by affinity chromatography on a Ni_NTA resin column. A purified recombinant protein was obtained and had a anticipated molecular weight 12.8KDa. so it may lay the foundation for the biological mechanism of p8.