双锯鱼(Amphiprion)胚胎不同发育阶段和成鱼不同组织中内参基因的筛选及应用

SCREENING AND APPLICATION OF REFERENCE GENE IN ANEMONE FISH(AMPHIPRION)EMBRYOS IN DIFFERENT DEVELOPMENT STAGES AND ADULT TISSUES

双锯鱼(Amphiprion)亦称为海葵鱼,又称为小丑鱼,是一类经济价值较高的海水观赏鱼。目前国内在双锯鱼的胚胎发育、人工饲养以及形态学观察等方面已经取得了有效成果,但在分子生物学水平上对其基因表达的研究较少。为了筛选出适用于双锯鱼胚胎不同发育阶段以及成鱼组织的内参基因,分析酪氨酸酶(TYR)基因的表达情况,以眼斑双锯鱼(A.ocellaris)和白条双锯鱼(A.frenatus)为材料,利用实时定量聚合酶链式反应(qPCR)对18SrRNA、GAPDH(甘油醛-3-磷酸脱氢酶基因)、Ef-1α(转录延伸因子基因)和β-actin(β-肌动蛋白基因)这4个候选内参基因的表达水平进行检测,同时通过geNorm和Norm Finder软件对其稳定性进行评估,最后以合适的内参基因作为参考,研究TYR mRNA的表达水平。结果表明,在双锯鱼胚胎的不同发育阶段,18S和β-actin的表达量相对于其他基因较为稳定;在双锯鱼的不同组织中,稳定性依次为18S>β-actin>Ef-1α>GAPDH。以18S作为内参基因时分析发现TYR基因的表达在双锯鱼胚胎发育过程中呈先上升后下降的趋势,在体节期表达量最高;在双锯鱼各组织中均有表达,且在眼、尾和红皮中表达量最高。

Anemone fish(Amphiprion),or known as clownfish,is a class of economically valuable seawater ornamental fish.Outstanding results have been achieved in the embryo development,artificial breeding,and morphological observation of anemone fish,but few studies are made on its gene expression at molecular biological level.To screen out the reference genes that are suitable for different development stages of anemone fish embryos and the adult fish tissues and to analyze the expression of tyrosinase(TYR)genes,we conducted this experiment using Fales-Clown anemone fish(Amphiprion ocellaris)and tomato clownfish(Amphiprion frenatus)as the materials,for which quantitative real-time polymerase chain reaction(qPCR)was applied to determine the expression levels of four candidate reference genes:18S rRNA gene,GAPDH,Ef-1αandβ-actin.Meanwhile,the stability of these four candidate reference genes is evaluated with software geNorm and Norm Finder.Finally,an appropriate reference gene was used to study the expression level of TYR mRNA.Results show that the expression of 18S andβ-actin was relatively stable in anemone fish embryos at different development stages.In different tissues of anemone fish,18S was the most stable.When 18S was used as the reference gene,the expression of TYR gene was first increased and then decreased during the development of anemone fish embryos and the highest expression level was found at the body segmentation stage.The TYR gene was expressed in all tissues and the expression level peaked in eye,tail,and red skin.A reference gene suitable for the anemone fish was selected and applied to analyze the expression of TYR gene in different stages of embryo and adult tissues.