| Siberian sturgeon (Acipenser baerii) is one of the oldest fish on earth and aneconomic fish also, so it has high research value. In recent years, some researchershave focused on embryonic development of Siberian sturgeon and its lateral linesystem.CXCR7is a receptor of SDF-1, and it is expressed in different tissues of Homosapiens, Xenopus laevis, Danio rerio, but not in flies (Drosophila melanogaster). Inzebrafish, it has recently been shown that CXCR7is required for posterior lateral lineprimordium migration, and expressed in trailing cell of primordium andproneuromasts L2and L3in a32hpf embryo, as well as in the trail ofinterneuromastic cells. But there is no research on CXCR7in Siberian sturgeon lateralline system and embryonic development periods.The full-length cDNAs of CXCR7a and CXCR7b were isolated from Siberiansturgeon using RT-PCR,3′and5′RACE. The size of CXCR7a cDNA was2325bp,which included a1086bp open reading frame (ORF) encoding a putative protein of362amino acids, a207bp5′untranslated region (5′UTR) and a1032bp3′untranslatedregion (3′UTR) with a poly(A) signal sequence. The size of siberian sturgeonCXCR7b cDNA was2423bp, which included a1110bp open reading frame (ORF)encoding a putative protein of370amino acids, a209bp5′untranslated region(5′UTR) and a1104bp3′untranslated region (3′UTR) with a poly(A) signal sequence.The deduced amino acid sequence of CXCR7a shared82.6%,81.4%,80.9%and81.0%similarity with CXCR7of Homo sapiens, Mus musculus, Xenopus laevis andDanio rerio, respectively. The deduced amino acid sequence of CXCR7b shared82.9%,82.3%,80.8%and82.5%similarity with CXCR7of Homo sapiens, Musmusculus, Xenopus laevis and Danio rerio, respectively. And the deduced amino acid sequence of CXCR7a shared96.5%similarity with CXCR7b. The phylogenetic treesof CXCR7a and CXCR7b among Siberian sturgeon and other vertebrate species wereconstructed by neighbour-joining method and maximum parsimony respectively,which showed Siberian sturgeon CXCR7clustered with Danio rerio CXCR7. Also, itpredicted that CXCR7a and CXCR7b are cell surface receptors with seven transme-mbrane domains structurally homologous to chemokine G-protein coupled receptors(GPCRs) using the TMHMM Server version2.0. Three-dimensional structures ofCXCR7s of Acipenser baerii, Homo sapiens and Danio rerio were built using RasMol,which showed that they have similar structure. Furthermore, it was confirmed thatCXCR7a and CXCR7b were from different gene copies through the analysis ofgenomic DNA.Real-time PCR is highly sensitive, relatively easy to perform assay for quantifyingmRNA abundance. In this study, the transcription levels of5commonly usedreference genes(β-actin, GAPDH, SDHA, EF-1α,18S rRNA) were determined atdifferent stages (cleavage period, blastula stage, wide neural plate stage, optic vesiclestage, tail bud separation stage, otic placode formation, muscular effect stage,heart-beat period, pre-hatching stage, hatching stage,1day post-hatch) in order toselect the most stable gene, and according to geNorm, SDHA was the most stablereference gene.In order to explore the expression of CXCR7a and CXCR7b in various stages ofembryonic development stages and1day larval after hatching, SDHA was used toassess the expression of CXCR7a and CXCR7b mRNA across various stages fromcleavage period to1day post-hatch, and the lowest and highest expression stages ofCXCR7a was cleavage period, optic vesicle stage respectively, the lowest and highestexpression stages of CXCR7b was blastula stage, midgastrula stage respectively.Whole mount in situ hybridization(WISH) was used to explore the distribution ofCXCR7mRNA in the tail touches the head and1day post-hatch, showed that CXCR7is expressed in jaw, eyes and truck to tail region, which showed that the distribution ofCXCR7mRNA varies with different developmental stages. All these results providenew basic information to further study the role of CXCR7in embryonic developmentof Siberian sturgeon and lateral line system. |
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