Comparative Analysis Of Urotensin Ⅱα In Danio Rerio And Pristella Maxillaris

Urotensin Ⅱ(UⅡ)is a 11-amino acid cyclic neuropeptide synthesized in Dahlgren cells,stored in and secreted from the urophysal of fish caudal neurosecretory system(CNSS).UⅡ is able to regulate cardiovascular contraction and is an effective vasoconstrictor.In addition,UⅡ has influence on cardiovascular,kidney,diabetes and it plays a biological and pharmacological role in the urogenital system,gastrointestinal tract,pancreas,liver and adrenal gland.However,most studies focused on in vitro function of UⅡ,the in vivo function of it is unclear.Pristella maxillaries is native to South America such as Brazil and Peru.It is now widely raised as a aquatic ornamental fish because of transparence and easy reproducing.Pristella maxillaries has the advantage of small individual,massive spawning and short reproductive cycle.It can be clearly observed muscle,bones and spinal cord because of its adult transparent.It has a great advantage to use this species as a new model animal for function research,but the molecular biology research of Pristella maxillaries is unknown.The 769 bp sequence of Pristella maxillaries UⅡα cDNA was cloned by RACE based on the partial UⅡα transcript obtained from transcriptome analysis.The cDNA contained a79 bp 5’-untranslated region(5’-UTR),a 369 bp coding region,and a 321 bp 3’-untranslated region(3’-UTR),respectively.The open reading frame(ORF)encoded a 122 amino acid prepro UⅡα.6-conserved amino acid cyclopeptide has been found in amino acid alignment of different species.The phylogenetic tree showed that UⅡα gene of Pristella maxillaries was shared high sequence identity with Danio rerio.RT-qPCR(Real-time Quantitative polymerase chain reaction)revealed expression of UⅡα mRNA was predominantly expressed in CNSS while there was only trace expression in skin,ovary,testis,heart,liver,brain,kidney,muscle,intestines,eye and gill,which occurred both in Pristella maxillaries and Danio rerio.The results of in situ hybridization showed that UⅡα mRNA was mainly expressed around Dahlgren cells in adult fish CNSS.The whole-mount in situ hybridization showed that the expression of UⅡα mRNA was2019 5undetected at the tail of embryo until 3dpf in Pristella maxillaries and Danio rerio.The expression of UⅡα mRNA was obvious in 5dpf and it was similar to that in adult Pristella maxillaries.UⅡα mRNA in Danio rerio was higher than that in Pristella maxillaries at the same period.The UⅡα promoter sequence of Danio rerio was cloned,and the UⅡα coding region of Pristella maxillaries was cloned.The two fragments were ligated into the plasmid by the recombinant technique of TOL2.The plasmid is 3062 bp,which is induced by zebrafish UⅡα promoter and encodes a total of 399 amino acids,including UⅡα of Danio rerio and mCherry fluorescent fusion protein.The fusion protein overexpression plasmid of UⅡα-mCherry vector induced by Danio rerio UⅡα promoter was constructed in order to construct a UⅡα mutant for further research of invivo UⅡα.It is the first time of cloning UⅡα from Pristella maxillaris and comparing the distribution of UⅡα in different tissues of Pristella maxillaries and Danio rerio.The UⅡαmRNA was first labeled in adult Pristella maxillaris CNSS,and Danio rerio,Pristella maxillaries embryos.It showed that Pristella maxillaris was superior to Danio rerio in the research of UⅡα mRNA distribution because of its transparence,which may contribute to the functional analysis of endogenous UⅡα in Pristella maxillaris.