GTP环化水解酶1(GTP cyclohydrolase1,Gch1)作为蝶啶代谢通路中的限速酶,在动物体色形成过程中发挥重要作用.为探究Gch1基因在虹鳟(Oncorhynchus mykiss)体色形成中的作用,利用RACE技术成功克隆出虹鳟Gch1基因的全长cDNA,并对其进行了生物信息学分析,同时采用qRT-PCR技术检测了该基因在虹鳟、金鳟及其杂交F1代体色发生不同时期和不同组织中的相对表达量.结果显示,虹鳟Gch1基因cDNA全长1 688 bp,包含开放阅读框714 bp,共编码237个氨基酸.同源性比对及系统进化分析显示,虹鳟与大磷大马哈鱼(Oncorhynchus tshawytscha)同源性最高,亲缘关系最近,且GTP-cyclohydrol结构域在脊椎动物中高度保守.虹鳟与金鳟同一时期的表达分析发现,Gch1基因在16细胞期、神经期、体节期、7 dph(days post hatch,日龄)、2 mph(month post hatch,月龄)和3 mph背部皮肤中的表达差异显著.在各组织中,Gch1基因在虹鳟与金鳟的背部皮肤和眼以及金鳟的心脏中高表达,且在金鳟背部皮肤中的表达量显著高于虹鳟背部皮肤.在杂交F1代中,Gch1直到原肠期才表达,在大部分时期中的表达与双亲存在显著差异且高于虹鳟.上述结果表明,Gch1基因可能在金鳟黄色体色形成过程中发挥重要作用.研究结果可为进一步研究虹鳟体色形成机制提供基础资料.
Cloning,sequence analysis and expression of Gch1 gene in Oncorhynchus mykiss
Guanosine triphosphate cyclohydrolase 1(Gch1)is a key rate-limiting enzyme in the pteridine metabolic pathway and plays an important role in the formation of animal body color.To explore the role of Gch1 gene in the body color formation of Oncorhynchus mykiss,the full-length cDNA sequence of Gch1 gene of O.mykiss was obtained by rapid amplification of cDNA ends(RACE)technique,and its bioinformatics analysis was carried out.Furthermore,the relative expression of Gch1 gene in 17 different periods and 10 different tissues of wild-type rainbow trout(WT),yellow mutant rainbow trout(YM),and hybrid F1 generation was detected by quantitative real-time PCR(qRT-PCR).The results showed that the full-length cDNA of Gch1 gene was 1 688 bp,including a 714 bp open reading frame,encoding 237 amino acids.Amino acid sequence analysis showed that the molecular weight of Gch1 protein was 26.74 kD,the theoretical isoelectric point was 6.03,the instability index was 40.96,the hydrophilic coefficient was-0.200,which indicated that Gch1 protein was an unstable hydrophilic protein.Homology analysis showed that O.mykiss had the highest homology with Oncorhynchus tshawytscha,and the GTP cyclohydro 1 domain was highly conserved in vertebrates.Phylogenetic analysis showed that O.mykiss had the closest genetic relationship with D.tshawytscha and the farthest genetic relationship with poultry and mammals.qRT-PCR results showed that Gch1 gene was expressed in WT and YM at different levels from embryo to post hatch stages,and it was higher than that in the embryonic stage at all post hatch stages,but this gene was not expressed until the gastrula stage in the hybrid F1 generation.Comparative analysis of WT and YM at the same stages showed that the expression of Gch1 gene was significantly different in 16-cell stage,neural,somites,7 dph(days post hatch),2 mph(month post hatch),and 3 mph dorsal skin(P＜0.05).In various tissues,Gch1 gene was highly expressed in the dorsal skin and eyes of WT and YM,and in the heart of YM,and the expression in the dorsal skin of YM was significantly higher than that in WT(P＜0.05).In the hybrid F1 generation,the expression of Gch1 gene was higher in the post-emergence period than that in the embryonic stage.Gch1 gene was barely expressed in the fertilization,4-cell,16-cell,mulberry,and blastocyst stages,and was significantly different from both parents and higher than that in O.mykiss in most of the periods.The results indicate that Gch1 gene may play an important role in the formation of the yellow body color of YM.The results of this study provide a reference for further study on the mechanism of body color formation in O.mykiss.
万方数据
维普
