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...

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.