Spatial/temporal Expression Pattern And Primary Function Study Of VEGF And VEGFR In Amphioxus(Branchiostoma Japonicum)

Vascular endothelial growth factor (VEGF) and its receptor VEGFR are theimportant molecules to promote angiogenesis. Both of them can stimulate theproliferation and migration of vascular endothelial cells, promote the formation ofblood vessels, and change vascular permeability. Only single copy of BjVEGF andBjVEGFR respectively, has been found in amphioxus by global genome analysis. Inthis thesis, we will emphasized on sequence cloning and analysis, inferring theirphylogenetic scenarios, and exploring their possible functions.BjVEGF has an open reading frame (ORF) of1047bp in length, coding a proteinwith348amino acids (39kDa), which contains a PDGF domain by protein structureanalysis. BjVEGF has a sequence identity of30%-34%with vertebrate VEGFs,whilethe identity in the PDGF domain is higher (46%-48%).It has higher sequence identitywith VEGF-C and VEGF-D than with VEGF-A and VEGF-B. The intron phaseanalysis also shows that BjVEGF is similar to VEGF-C and VEGF-D.The fifth extronof BjVEGF and the fifth and sixthis extrons of vertebrate is homologous.Besidethis,BjVEGF and VEGFs have same intron phase. The synteny analysis shows thatBjVEGF has similar foci with that of human VEGFB，VEGFC and VEGFD, exceptfor VEGFA. Moreover, BjVEGF was clustered at the root of VEGF-C and VEGF-Dbranch, suggest that BjVEGF may be functional as VEGFC and VEGFD invertebrates.There is a single copy of BjVEGFR in genome, most part of the gene is cloned,covering nearly all of coding sequence. The partial ORF, which is3873bp in length,can code a protein with of1290amino acids(143kDa). Protein structure analysisshows that BjVEGFR contains seven immunoglobulin like domains(Ig) out of cellmembrane, a transmembrane area and a tyrosine kinase domain in cell membrane.Sequence analysis shows that BjVEGFR has33%-35%sequence identity withVEGFR in vertebrate, while the Ig domains of BjVEGFR shows lower identity of27%-29%with the corresponding region of VEGFR1and tyrosine kinase domain ofBjVEGFR shows higher identity of59%-61%compared to that of VEGFR1, suggestextracellular region of BjVEGFR（Ig domain region）is under strong selection. Theintron phase analysis shows that BjVEGFR has similar intron phase with vertebrateVEGFRs,though there are some extrons combind or divide. The synteny analysisshows that BjVEGFR has similar foci with the four kind of vertebrate VEGFRs. TheVEGFR phylogenetic tree reconstructed with whole sequences shows that theBjVEGFR was located at the root of vertebrate VEGFRs’ branches, suggest it is the ancestor of vertebrate VEGFR.The spatiotemporal patterns of BjVEGF and BjVEGFR are determined by thereal-time quantitative fluorescence PCR and in situ hybridization. Real-timequantitative PCR shows that VEGF and VEGFR have expessed at2-8cells stageduring the embryo development. The expression level rises through development, themax value was detected at blastula stage, then gradually reduced after neutrula stage.However, both of their expression level is very low in adult, but still concentrated inthe gills, hepatic cecum and digestive tract. Whole mount in situ hybridization showsthat BjVEGF and BjVEGFR have strong signal cover the embryo before the gastrulastage, but they become lower level after neutrula stage, mainly located in theendoderm and mesoderm of amphioxus embryo. A track of weak signal in digestiveand gills was detected at laval stage. Slice in situ hybridization shows that the signalof BjVEGF and BjVEGFR concentrated in the gills, hepatic cecum, ovarian anddigestive tract. Combined with real-time quantitative PCR and in situ hybridizationresults, we can find that BjVEGF and BjVEGFR have the similar expression patternsin both of embryo and adult, respectively. More importantly, the signal areco-localized, which suggests that BjVEGF and BjVEGFR have the potentialinteraction ability, but this point still needs to be determined by proteins interactionassay and co-localization assay of BjVEGF and BjVEGFR proteins.When infected with bacteria of Aeromonas hydrophilia and Staphyloccocusaureus Rosenbach, respectively. VEGFR expression level rises, while VEGFexpression level is unchanged through real-time quantitative fluorescence PCRanalysis, suggesting VEGFR may be related to immune function, which should still beconfirmed by bacteria binding or inhibition assay in vitro.