不同体色锦鲤体色观察、基因表达以及过表达pnp4a对体色发育的影响

Observation of Body Colour and Gene Expression in Koi of Different Body Colours,and the Effects of Overexpression of pnp4a on Body Colour Development

锦鲤色彩鲜艳,游姿飘逸,观赏价值高,被称作“会游泳的艺术品”。鱼类体色主要是由皮肤或鳞片上的色素细胞的种类和分布决定的。本文对黑色、白色、黄色和红色4种不同体色锦鲤的鳞片和皮肤的色素细胞组成、分布及形态结构等进行了比较观察,结果显示,鳞片和皮肤的色素细胞组成一致,4种锦鲤均存在着丰富的虹彩细胞,在黑色锦鲤中还观察到了黑色素细胞,黄色锦鲤和红色锦鲤还分布着黄/红色素细胞,在白色锦鲤中只观察到虹彩细胞。利用透射显微镜观察了4种体色锦鲤的虹彩细胞的超微结构,虹彩细胞主要有3种不同形态:梭形、短棒状和空泡状;采用实时荧光定量PCR检测了8个虹彩细胞发育相关基因在4种不同体色锦鲤皮肤中的表达情况,结果显示,8个虹彩细胞发育相关基因在不同体色锦鲤皮肤中均有表达,但表达存在差异,其中alk、pka、sox10、pax3、foxd3和pnp4a基因在红色和黄色锦鲤中相对表达量较高,ltk和alx4a基因在白色锦鲤中相对表达量最高。为进一步证实pnp4a基因在虹彩细胞中的作用,构建了pnp4a过表达重组质粒,并将其显微注射至AB斑马鱼受精卵中,过表达斑马鱼中虹彩细胞明显多于对照组,进一步证实了pnp4a在鱼类体色形成尤其是在虹彩细胞发生方面发挥着重要作用。

Koi is a brightly coloured,elegant fish with high ornamental value,and is known as a“swimming work of art”.Fish body colour is mainly determined by the type and distribution of pigment cells on the skin or scales.In this paper,the composition,distribution and morphology of pigment cells in the scales and skin of four different body colours,namely,black,white,yellow and red koi,were compared.The composition of pigment cells in the scales and skin was consistent,and rich iridescent cells were present in all four koi,with melanophores in black koi,and xanthophores and erythrophores in yellow and red koi.Only iridophores were observed in white koi.The ultrastructure of iridophores in all four koi colours was observed by transmission microscopy,and the iridophores mainly had three different morphologies:pike-shaped,short rod-shaped and vacuolated.Real-time fluorescence quantitative PCR was used to detect the expression of eight iridocyte development-related genes in the skin of four different body colors of koi carp,the results showed that eight genes related to iridophores development were expressed in the skin of different body colours,whereas there were differences in expression,among which the relative expression of alk,pka,sox10,pax3,foxd3 and pnp4a genes was higher in red and yellow koi,while the relative expression of ltk and alx4a genes was the highest in white koi.In order to further confirm the role of the pnp4a genes in iridophores development,the recombinant plasmid of overexpression of pnp4a gene was constructed,and was microinjected into the fertilized eggs of AB zebrafish.In order to further confirm the role of pnp4a gene in fish body colour formation,especially in iridophores,we constructed a pnp4a overexpression recombinant plasmid and microinjected it into the fertilized eggs of zebrafish(AB).