褐牙鲆变态期间骨骼发育及其相关基因Sox9,Bmp4和Bmp2的表达分析

褐牙鲆作为一种重要的经济鱼种,有一个特殊的变态发育时期,其间骨骼形态和位置都发生很大改变。本研究利用三重染色技术,对褐牙鲆变态发育时期不同阶段的仔鱼整体骨骼进行染色,并对各阶段仔鱼及部分成鱼组织,采用特异性引物对骨骼发育的相关基因Bmp2,Bmp4和Sox9基因片段进行了克隆和半定量分析。研究表明,随着仔鱼变态发育,部分软骨逐渐转化为硬骨,头部筛骨呈不对称分化,额骨和顶骨位置发生改变,口形态改变,鳃丝软骨成骨,脊椎逐渐发育成熟,随之侧线位置发生改变。Sox9以及Bmp2、Bmp4基因在这时期中存在着差异表达,对骨骼发育有一定的调控作用。成鱼中,Bmp2和Sox9只在鳃、肠道和肝脏中有表达,且鳃中表达量较高。Sox9出膜后表达量突然增加,头部表达量较高,变态发育中开始递减,在变态高峰期明显减少;而Bmp4表达量则是出膜后明显减少,随后在变态高峰期表达量有增加,总体趋势表达量减少。

Skeleton development of Japanese flounder(Paralichthys olivaceus) and expression analysis of related genes (Sox9, Bmp4 and Bmp2) during metamorphosis

As an important economic species, Japanese flounder ( Paralichthys olivaceus ) had a special period of abnormal growth, during which the bones witnessed great morphogenetic changes. The triple-staining technique was used to examine the bone development of Japanese flounder at different stages of metamorphosis. For larval fish and some adult fish tissues, specific primers were used to do cloning and semi-quantitative analysis on bones related to gene fragments Bmp2 , Bmp4 and Sox9 . Research showed that during the abnormal development of larvae, some cartilages gradually transformed into hard bones; ethmoid showed asymmetric division; the location of frontal and parietal bones changed; morphological changes happened to the mouth; gill cartilages changed into bones, and spinal cord gradually matured, then the location of lateral line changed. Sox9 , Bmp2 and Bmp4 of this period showed differential expressions, which had a regulation effect on the skeleton development. For adult fish, Bmp2 and Sox9 only expressed in gill, intestine and liver, with the highest expression in gill. Sox 9 expression increased heavily after the fish coming out of the membrane, with a relatively high expression in the head, and decreased during metamorphosis, with an obvious reduction during the peak metamorphosis; while Bmp4 expression decreased heavily after the fish coming out of the membrane, and increased during the peak metamorphosis, but with the whole trend of reduced expression.