暗纹东方鲀leptin基因的克隆、SNP筛选及其与生长性状的关联分析

为明确暗纹东方鲀Takifugu obscurus瘦素(Leptin)基因的基本性质,并探究leptin基因的多态性及其与暗纹东方鲀生长的相关性,随机选择6尾健康的3月龄暗纹东方鲀幼鱼作为基因克隆的试验样本,采用RT-PCR方法克隆leptin基因的cDNA序列,随机选择同期繁殖、同塘养殖的178尾8月龄鱼,采用直接测序法筛选leptin基因中与生长相关的SNP位点。结果表明:克隆得到的leptin cDNA序列长为661 bp,包括459 bp的开放阅读框,共编码152个氨基酸,Leptin氨基酸序列含19个氨基酸的信号肽序列,推测该蛋白为亲水的稳定蛋白;暗纹东方鲀Leptin氨基酸序列与红鳍东方鲀Leptin氨基酸序列一致性最高,为96.7%,与不同种属的其他鱼类相似性较低,与草鱼氨基酸序列的一致性仅为25.25%,但不同物种的Leptin存在较高的三级结构相似性;在暗纹东方鲀leptin基因上筛选得到2个分型稳定的SNP位点(T24G、T193A),其中,T24G位点不同基因型个体间的体质量存在显著性差异(P<0.05),GG基因型个体体质量显著小于TG和TT基因型(P<0.05);将2个SNP位点的不同基因型进行组合,经关联分析获得优势双倍型D5,D5型个体的3个生长性状值最高,体质量显著高于D2、D4型个体(P<0.05),极显著高于D6型个体(P<0.01),体长和体全长显著高于D6型个体(P<0.05)。研究表明,T24G位点的TG和TT基因型及双倍型D5是暗纹东方鲀优势生长的基因型,可作为暗纹东方鲀辅助育种的候选分子标记。

Cloning,SNP screening and association with growth traits of leptin gene in river puffer Takifugu obscurus

In order to clarify the basic properties of leptin gene in river puffer Takifugu obscurus,and to explore the polymorphism of the leptin gene and its correlation with the growth of the puffer,the cDNA sequence of the leptin gene was cloned in 6 samples of 3 month old puffer by RT-PCR.Then the SNP loci related to growth in the leptin gene were screened in 178 samples of 8 month old river puffer by direct sequencing method.The results showed that the cloned leptin cDNA sequence was 661 bp in length,including an open reading frame of 459 bp,encoding a total of 152 amino acids(hydrophilic stable protein),containing a signal peptide sequence of 19 amino acids.There was high tertiary structural similarity(96.7%)between river puffer T.obscurus and other fish species,with the maximal similarity of Leptin amino acid sequence between river puffer T.obscurus and tiger puffer Takifugu rubripes and weak similarity of Leptin amino acid sequence between puffer T.obscurus and other species(including grass carp Ctenopharyngodon idella 25.25%).Two SNP loci with stable typing(T24G,T193A)were screened on the leptin gene of river puffer T.obscurus,in which there was significantly different body weight in the individuals with different genotypes at the T24G locus(P<0.05),significantly smaller in GG genotype than that in TG and TT genotype.The association analysis of combination of the different genotypes of the two SNP loci revealed that the dominant double-type D5 individuals had the maximal average value of the three growth traits,and significantly higher body mass than that in D2 and D4 individuals(P<0.05),very significantly higher than that in D6 individuals(P<0.01).There was significantly higher body length and total length in D5 individuals than that in D6 individuals(P<0.05).The findings indicate that TG and TT genotype in T24G locus and double-type D5 can be used as candidate molecular markers for assisted breeding of river puffer T.obscurus.