Functional Study Of Zebrafish Blood Developmental Candidate Gene Loc 558117

Blood development is a complex process that is regulated by a series of transcription factors. Although some hematopoiesis transcription regulatory factors have been identified, the new genes and the molecular mechanism remain to be identified. The regulatory networks of hematopoiesis system between zebrafish and mammals are highly conserved. Because of its distinctive advantage, zebrafish has become one of the most important models in developmental biology of vertebrates. A human blood cell developmental gene candidate TRIM45 was cloned by our lab, and here the functional of TRIM45 is studied by using the zebrafish as an animal model.The zebrafish loc558117 gene is the homolog of TRIM45, which contains a ring finger, two B-boxes, and aα-helical Coiled-Coil. RT-PCR analysis suggested that the mRNA levels of Hematopoiesis markers TRIM33、SMAD5、GATA2、BMP2B are significantly down-regulated in the loc558117 knock-down fish, suggesting that loc558117 may be involved in zebrafish blood cell development.Embryos at various stages were examined by whole-mount in situ hybridization with digoxin labeled antisense RNA probe of loc558117. The results revealed that loc558117 was expressed at each stage of zebrafish development. In order to study its protein expression,491 bp of the Loc558117 gene was amplified by PCR and cloned into pET-28a vector and then transferred into the recombinant expression plasmid. Its fusion protein was induced by IPTG. After purifying by HIS-IDA sephrose, the fusion protein was used to immune the New Zealand white rabbits and prepared an anti-loc558117 polyclonal antibody with a good titer and specification checked by Western blot.The further results showed that in the adult zebrafish, the loc558117 protein was expressed in blood, heart, eyes, eggs and brain, with a higher expression in the blood.Knocking down its expression in zebrafish embryos with loc558117-MO caused severe malformation such as the dim of the blood-color, the slower formation of melanin and the failure of the cardiac looping. Furthermore, the expression of the hematopoiesis marker genes TRIM33, SMAD5 and GATA2 examined by whole-mount in situ hybridization was also decreased in the mutants.These results showed that the loc558117 gene is involved in early hematopoiesis and plays an important role during the development of the blood cells, which lay a foundation for further studies on the role of the gene in the hematopoiesis.