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利用cDNA末端快速扩增技术(Rapid amplification of cDNA ends,RACE)克隆鉴定出斜带石斑鱼(Epinephelus coioides)卷轴受体3(Frizzled 3,gFZD3)基因的cDNA序列全长,使用ClustalX(1.81)和MEGA4.0软件分别进行了序列同源比对和系统进化树分析;利用RT-PCR检测gFZD3在成鱼不同组织中的表达和gFZD3在胚胎发育及卵巢发育过程中的表达模式。研究显示:gFZD3cDNA序列全长为2 278bp,其中开放读码框为2 079bp,编码692个氨基酸;序列同源性分析表明,gFZD3的氨基酸序列具有FZD家族保守的功能结构域;脊椎动物的FZD3系统进化树分析显示,斜带石斑鱼与大黄鱼(Larimichthys crocea)的亲缘关系最近;在成鱼的组织分布中,只在脑区和性腺中检测到gFZD3表达信号,其中在卵巢中表达量最高;在胚胎发育过程中,在未受精卵和受精卵中gFZD3的表达量最高,随着胚胎的发育呈逐渐下降趋势;在卵巢发育过程中,gFZD3的表达在早期卵巢中相对较低,在随后的III和IV期卵巢中较高。研究结果表明,FZD3序列在脊椎动物中具有较高的保守性;斜带石斑鱼gFZD3的表达具有明显的组织特异性,并在胚胎和卵巢发育中,可能主要作用于胚胎发育的早期和卵巢发育的后期。本研究为进一步研究Wnt信号通路在斜带石斑鱼生殖调控中的作用提供了参考。
Abstract：In this study,the cDNA of orange-spotted grouper(Epinephelus coioides)frizzled 3(FZD3)gene was isolated with its expression analyzed.The full length cDNA sequence of grouper FZD3(gFZD3)gene was isolated by using rapid-amplification of cDNA ends(RACE)method.The amino acid alignment and phylogenetic tree construction were performed by using ClustalX(1.81)and MEGA4.0,respectively.Tissue distribution of gFZD3 transcript was examined thorough RT-PCR.The abundance of gFZD3 transcript during embryonic and ovarian developments was detected by qRT-PCR.The isolated gFZD3 cDNA was 2 255 bp in length and contained a 2 079 bp ORF encoding a deduced protein of 692 amino acids.Amino acid sequence analysis revealed that gFZD3 presented all typical structural characteristics of FZD family.Phylogenetic analysis showed that gFZD3 clustered with the evident subclade of FZD3,and showed a close relationship to that of Larimichthys crocea.In orange-spotted grouper,gFZD3 was found to express only in brain and two gonads,ovary and testis,with the highest abundance of its transcript detected in ovary.During embryonic development,FZD3 is highly expressed in unfertilized and fertilized eggs,but such expression gradually decreased at subsequent embryonic development stages.Furthermore,the abundance of its transcriptwas low at early ovarian stage,and high at subsequent III(cortical-alveolus)and IV(vitellogenic)ovarian stages.In conclusion,gFZD3 was isolated from orange-spotted grouper and FZD3 was highly conservative among different species in structure.The specific expression of gFDZ3 in brain and gonads suggested an evident tissues-specific distribution pattern of gFZD3.The expression of gFZD3 during the embryonic and ovarian developments indicated that gFDZ3 may play important roles at early stages of embryonic development and late stages of ovarian developments of orange-spotted grouper.The present study supplied the preliminary information for the further evaluation of Wnt pathway in fish reproduction.
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基本信息:
DOI：10.16441/j.cnki.hdxb.20150384
中图分类号:S917.4
引用信息:
[1]陈华谱,李臻,邓思平,等.斜带石斑鱼卷轴受体3基因的分离及表达分析研究[J],2017,47(01):68-75.DOI:10.16441/j.cnki.hdxb.20150384.
基金信息:
广东省海洋渔业科技推广专项(A201408A06);; 海南省科技合作专项(KJHZ2015-08)资助~~