长江刀鲚mor-4k13基因的分离鉴定及表达分析

分布于长江的刀鲚(Coilia nasus)具有洄游和定居两个生态型,生殖洄游是区别两者的主要表征。为了探索嗅觉受体(OR)基因是否参与了刀鲚的生殖洄游过程,本文采用RACE技术从洄游型刀鲚中获得了mor-4k13基因,该基因全长1 098 bp,编码区长963 bp,单外显子结构,可编码320个氨基酸。预测表明,mor-4k13基因编码的蛋白质,为7个疏水性的α-螺旋跨膜结构,属G-蛋白偶联受体,有胆固醇和油酸两个配体。MOR-4K13蛋白与已报道的其他鱼类OR蛋白的同源性在40%~68%之间,其中,与近缘种大西洋鲱(Clupea harengus)嗅觉受体蛋白同源性高达68%。采用Real-time PCR方法对10个组织或器官所作的荧光定量分析显示,mor-4k13基因在定居型刀鲚嗅囊和性腺中高表达,在肌肉、眼球、胃壁、肝和鳃中低表达,心肌中几乎不表达。mor-4k13基因在洄游型刀鲚嗅囊中的表达量总体高于定居型,且洄游型雄性刀鲚嗅囊中此基因的表达量约是其雌性嗅囊中的3倍。这表明mor-4k13基因不仅与嗅觉功能和性腺发育相关,也可能与生殖洄游习性相关,不同性别的个体间也存在着嗅觉能力的差异。

Isolation, Identification and Expression of mor-4k13 Gene in Japanese Grenadier Anchovy (Coilia nasus) from the Yangtze River

Coilia nasus have two migratory and sedentary ecotype in the Yangtze River, and spawning migration is a major difference between the two characterization. In order to explore olfactory receptors gene is involved in the process of spawning migration of C. nasus, InStheSexperiment, the MOR-4K13 gene that coded olfactory receptor was cloned in olfactory rosette RNA of migratory C. nasus using RACE. The MOR-4K13 gene was 1098bp in length, the open reading frame which encoded 320 amino acid residues was 963bp in length, and the gene was single exon. The protein had seven hydrophobic and alpha helix transmembrane structure, belonged to the G-protein-coupled receptor superfamily, and had two ligands: cholesterol and oleic acid. The homology in MOR-4K13 protein of C. nasus and OR protein of other fishes which had been reported was 40% to 68%, which the homology of closely related species Clupea harengus was 68%. Based on qRT-PCR among 10 tissues and organs of the settlement population, the result revealed that the MOR-4K13 gene was expressed strongly in female olfactory rosette and ovary than others. The expression quantity in female olfactory rosette and gonad was high expression quantity and weakly in muscle ,eye, stomach, liver and gills, but almost not in the heart. In addition, the MOR-4K13 gene overall expression quantity in migratory C. nasus olfactory rosette above the sedentary, but the expression quantity in male olfactory rosette was 3 times higher than that in female olfactory rosette in the migratory population, and the expression quantity in female olfactory rosette in the settlement population was 3 times higher than that in male olfactory rosette in the migratory population. In summary, the MOR-4K13 gene not only was related to olfactory function, but also participated in gonadal development or associated with reproductive migration, existed in gender gap in the olfactory ability.