黄颡鱼GnRHR基因的克隆和表达及CRISPR/Cas9构建GnRHR基因敲除突变体

Molecular cloning,and expression of GnRHR gene and establishment of GnRHR mutant by CRISPR/Cas9 in yellow catfish Pelteobagrus fulvidraco

为研究促性腺激素释放激素受体(gonadotropin releasing hormone receptor,GnRHR)对黄颡鱼Pelteobagrus fulvidraco性别分化和性腺发育的调控作用,采用逆转录PCR、氨基酸序列多重比对、系统进化树分析及实时定量PCR方法,对黄颡鱼GnRHR基因的序列、表达和进化进行了研究。结果表明:扩增到的黄颡鱼GnRHR cDNA序列长2894 bp,包含239 bp的5′非翻译区,1155 bp的开放阅读框和1500 bp的3′非翻译区,预测编码384个氨基酸、7次跨膜结构域蛋白;氨基酸序列多重比对显示,黄颡鱼GnRHR与硬骨鱼类GnRHR的同源性较高,其与斑点叉尾鮰Ictalurus punctatus、斑马鱼Danio rerio、鲤Cyprinus carpio、银鲫Carassius auratus GnRHR氨基酸序列的一致性分别为96%、87%、83%和82%,与哺乳动物的一致性较低,为42%~44%;系统进化分析显示,黄颡鱼GnRHR与鲤、虹鳟等硬骨鱼类的GnRHR聚为GnRHRⅡB分支;实时定量PCR显示,黄颡鱼GnRHR mRNA主要在端脑、中脑、下丘脑、垂体和精巢中高表达,但在卵巢和其他组织中几乎不表达;通过CRISPR/Cas9基因编辑技术构建和筛选出了黄颡鱼GnRHR突变体,成功获得了各种不同的黄颡鱼GnRHR F0代突变体。研究表明,GnRHR可能参与调控黄颡鱼雄性发育,黄颡鱼GnRHR F0代突变体的获取为探索GnRHR的功能和机制提供了基础资料。

To elucidate the regulation mechanism of gondadotropin releasing hormone receptor(GnRHR)in sexual differentiation and gonad development,the sequence,phylogenetic pattern and expression of GnRHR gene in yellow catfish Pelteobagrus fulvidraco were analyzed by reverse transcription PCR,multiple alignment of amino acid sequence,phylogenetic tree and real-time quantitative PCR analysis.It was found that the yellow catfish GnRHR cDNA was cloned to be 2894 bp in length,containing a 1155 bp open reading frame(ORF)encoding a putative protein of 384 amino acids,a 239 bp 5′untranslated region(UTR)and a 1500 bp 3′UTR.Protein conserved domain prediction showed that the yellow catfish GnRHR protein contained seven transmembrane domains(TMs).Multiple sequence alignment analysis of amino acid sequence of yellow catfish GnRHR homologues revealed that GnRHR shared high similarities with GnRHRs of other fishes,including channel catfish Ictalurus punctatus(96%),zebrafish Danio rerio(87%),common carp Cyprinus carpio(83%)and crucian carp Carassius auratus(82%).However,yellow catfish GnRHR had low similarities with mammalian GnRHRs ranging from 42%to 44%.Phylogenetic analysis indicated that yellow catfish GnRHR were clustered with typeⅡB GnRHRs of other teleost.Real time quantitative PCR analysis demonstrated that yellow catfish GnRHR transcript was mainly expressed in telencephalon,mesencephalon,hypothalamus,pituitary and testis,without expression in ovary and other tissues.Furthermore,yellow catfish GnRHR mutants were generated by the CRISPR/Cas9 system and several different yellow catfish GnRHR F0 mutants were obtained.The findings indicated that yellow catfish GnRHR played potential role in male development of yellow catfish,and that provided important information for further research of the function of yellow catfish GnRHR and the molecular mechanisms underlying yellow catfish reproduction.