七彩神仙鱼催乳素基因的克隆、定位及表达分析

Cloning,Localization and Expression of Prolactin Gene in Discus Fish(Symphysodon aequifasciatus)

【目的】七彩神仙鱼(Symphysodon aequifasciatus)具有特殊的亲代抚育行为,克隆并定位七彩神仙鱼催乳素基因,分析其在亲代抚育中的表达模式,为理解催乳素在七彩神仙鱼亲代抚育中的作用提供依据。【方法】通过BLASTP对七彩神仙鱼全基因组进行分析,鉴定出1个催乳素基因,命名为dfprl。基于dfprl基因的CDS区,设计克隆和PCR引物,通过RACE技术克隆获得dfprl基因全长,并利用生物信息学对dfprl基因进行结构分析,对其氨基酸序列进行理化性质和进化分析。对不同抚育阶段七彩神仙鱼的脑、性腺和皮肤进行转录组分析,探究dfprl基因在亲代抚育中的表达特征,并利用原位杂交技术对dfprl基因在七彩神仙鱼皮肤中的表达进行定位。【结果】dfprl全长为1282 bp,cDNA序列开放阅读框长度为639 bp,共编码212个氨基酸,5'-UTR为309 bp,3'-UTR为334 bp。dfprl蛋白存在PRL家族的典型结构域Hormone_1,dfprl蛋白与慈鲷科其他鱼类PRL蛋白相似性较高,与尼加拉瓜湖始丽鱼(Archocentrus centrarchus)对应的氨基酸序列同源性最高、为96.70%。亲鱼进入抚育阶段后,dfprl在性腺和皮肤中的表达水平逐渐上升,抚育结束时表达水平下降。原位杂交结果显示,dfprl在皮肤粘液细胞中表达,且与七彩神仙鱼催乳素受体(dfprlr)表达位点重叠,在抚育早期阶段高表达。【结论】七彩神仙鱼dfprl基因高度保守,存在稳定的Hormone_1结构域。七彩神仙鱼dfprl基因在亲代抚育阶段的皮肤中高表达,且在亲代抚育阶段的皮肤粘液细胞中与dfprlr表达位点重合,表明dfprl可能通过作用于dfprlr进而促进七彩神仙鱼独特抚育行为的发生。

【Objective】Discus fish(Symphysodon aequifasciatus)has special parental care behavior.In the study,prolactin(PRL)gene from discus fish was cloned and localized and its expression pattern during parental care was analyzed,with an aim to provide a basis for understanding the function of PRL in parental care of discus fish.【Method】A prolactin gene named dfprl was identified by analyzing the whole genome data of the discus fish through BLASTP.The CDS region of dfprl genes were used to design the primer of gene cloing and PCR.The full length of dfprl gene was cloned by RACE technology,and the structure of dfprl gene was analyzed by bioinformatics and its amino acid sequence was analyzed by physicochemical properties and phyletic evolution.Transcriptome sequencing was performed on brain,gonad and skin of discus fish at different rearing stages to explore the expression pattern of dfprl gene during parental care.The dfprl gene in the skin of discus fish was located by paraffin-fluorescence probe-FISH.【Result】The full length of dfprl was 1282 bp,with 639 bp cDNA sequence encoding 212 amino acids,309 bp 5'-UTR and 334 bp 3'-UTR.The dfprl protein has the typical domain of PRL family,the Hormone_1 domain.dfprl protein is highly similar to PRL protein of other Cichlidae,and the amino acid sequence homology of dfprl with Archocentrus centrarchus prolactin(AcPRL)was the highest(96.70%).The expression level of dfprl in gonad and skin increased gradually after the parent fish entered the rearing stage,and decreased at the end of the rearing stage.The paraffin-fluorescence probe-FISH results showed that dfprl was expressed in skin mucous cells,and it was coincident with the expression site of prolactin receptor(dfprlr)and was highly expressed in the early stage of parental care.【Conclusion】The dfprl gene is highly conserved and has stable Hormone_1 domain.dfprl gene was highly expressed in the skin during the parental care stage,.and the expression sites of dfprl and dfprlr overlapped in skin mucous cells during the parental care stage of discus fish,suggesting that dfprl might be involved in the occurrence of unique parental care behavior of discus fish.