Mutant Phenotype Analysis And Interacting Proteins Identification Of The Heart Development Candidate Gene Fbx15 In Zebrafish

Fbxl5 (F-box and leucine-rich repeat protein 5) gene is Located on chromosome No.23 of zebrafish and has 4186bp, which contains 11 exons and 10 introns。 All genes on the genome is up to 77.7kb, encoding a 693 amino acid long protein,which contains an F-box domain and four LRR domain. F-box protein is a kind of protein that can recognize special substrate in the ubiquitin-proteasome pathway.Ubiquitin-proteasome pathway (UPP)play a role in a variety of heart development process. Based on previous work,This research paper further study the role of Fbxl5 gene in the ubiquitin-proteasome pathway-mediated in embryonic heart development of the zebrafish.Fbxl5 gene which our laboratory cloned is a heart development of candidate genes, in order to further study the function of the Fbxl5 gene, we injected morpholino into zebrafish embryos in the laboratory. The study found that interference in zebrafish embryos by morpholino, the heart of the zebrafish embryo as a mild deformity, heart development cyclization is not completely linear; This shows that zebrafish1 s Fbxl5 gene can affect the heart development of zebrafish.This research paper focuses on the Fbxl5 gene lead to the possible mechanism of zebrafish heart mutation phenotype. In our laboratory, through the yeast two-hybrid Foxp4 genes may be direct interaction with Fbx15 gene lead to mutation phenotype occurred. Firstly, through the CO-IP experiment results prove Fbxl5 gene may be interact with Foxp4 gene. Ubiquitin-proteasome pathway (UPP)play a role in a variety of protein degradation process. Customarily, the F-box domain and Skpl connect to the SCF complex get together. When the protein is degraded via the ubiquitin-proteasome pathway, ubiquitin can be transfered to the covalent substrate by ubiquitin-conjugating enzyme (E2). Then ubiquitination substrate is degraded by the 26 S proteasome, and the SCF complex can promote the substrate interactubiquitin conjugating enzyme (E2) better, I think FbxlS as an F-box protein family, also take part in the ubiquitin-proteasome pathway, then, we prove Fbxl5 gene interact with Skpl gene. Fbxl5 interfere some protein through the ubiquitin-proteasome pathway, these proteins can mediate the migration of zebrafish heart development center tube, making the zebrafish heart showing a biventricular phenotype. In addition, our laboratory’s luciferase reporter system analysis experiments, in cells enhance Fbxl5 expression makes Nkx2.5 enhancer activity; at the protein level, as the expression of Fbxl5 enhanced expression of Nkx2.5 also increased, so we think Fbxl5 is able to participate in the regulation of Nkx2.5 expression.on the basis of preliminary work in the laboratory, this research paper initially revealed possible molecular mechanism of Fbxl5 gene through Foxp4 gene, ubiquitin-proteasome pathway and Mkx2.5,the key target gene of Cardiac development to regulation of heart development of zebrafish. Laid the foundation of Fbxl5 gene regulation chamber development lead to the occurrence of zebrafish cardiac phenotype for further study.