暗纹东方鲀TGF-β1基因的克隆、表达特征及生长性状相关SNP位点筛选

Molecular cloning,expression analysis and growth traitsralated SNP loci screening of TGFβ1 gene in Takifugu fasciatus

为探究暗纹东方鲀(Takifugu fasciatus)TGF-β1基因与其生长性状的关联及作用,采用RACE技术克隆了暗纹东方鲀TGF-β1基因,分析了其基因结构与表达特征,并通过直接PCR扩增法对TGF-β1基因进行多片段比对,在此基础上筛选出与生长性状相关的SNP位点。研究发现TGF-β1基因cDNA全长共2217 bp,包含5′UTR 51 bp,3′UTR 1014 bp,ORF 1152 bp,编码383个氨基酸。氨基酸序列比对分析表明,暗纹东方鲀TGF-β1基因与红鳍东方鲀(Takifugu rubripes)同源性最高(92.82%)。进化树分析表明,暗纹东方鲀TGF-β1基因编码氨基酸序列与红鳍东方鲀聚为一支,与鲈形目鱼类亲缘关系最近(68.46%~73.33%)。qRT-PCR检测显示,TGF-β1基因在暗纹东方鲀8种不同组织(心脏、肝脏、脑、脾脏、肾脏、肌肉、中肠和鳃)都有表达,其中肾脏表达量最高,心脏和脾脏表达量次之。通过SNP位点不同基因型与个体生长性状的相关性分析,发现TGF-β1上的SNP g.3908 C>A位点与暗纹东方鲀多项生长性状有关,其中CC型是优势基因型,且CC型个体体宽、肥满度较高。研究结果证实TGF-β1基因与暗纹东方鲀生长性状有一定的相关性,可为暗纹东方鲀分子标记辅助育种提供基础资料。

Takifugu fasciatus is an important farmed fish in China with high nutritional and economic value.The natural germplasm resources of T.fasciatus are getting depleted with the deterioration of living environment.Artificial breeding has become a necessary measure to alleviate the contradiction between supply and demand of T.fasciatus in the market and to slow down the decrease of wild resources of T.fasciatus in natural environment.In the absence of rejuvenation of wild resources and updating of genetic information of T.fasciatus,inbreeding occurred in the population.The degradation of germplasm is manifested in many aspects such as uneven growth,gradual decline of individual resistance and slow growth rate.TGFβ1,a multieffect cytokine of the transforming growth factorβfamily,is related to cell proliferation and differentiation,cell proliferation inhibition,tissue formation,apoptosis regulation and immune regulation,and also plays an important role in the growth and development of fish.In order to investigate the association and role of TGFβ1 gene with growth traits in T.fasciatus,healthy T.fasciatus were selected to clone the fulllength cDNA sequence of TGFβ1 gene by RACE technology.Further,to characterize the expression characteristics of TGFβ1 gene in 8 different tissues(heart,liver,brain,spleen,kidney,muscle,midgut and gill)by qRTPCR.Finally,we selected 300 T.fasciatus at the same period.And then we measured their 10 quantitative characters and calculated one proportional character(condition factor).We also developed and analyzed the SNP loci on TGFβ1 of T.fasciatus by PCR direct sequencing and correlated the growth traitsrelated SNP loci screening of TGFβ1 gene in T.fasciatus by sequence multiplexed analysis.The results showed:1)the cDNA sequence of TGFβ1 was 2217 bp,composing of 51 bp at the 5′end,1014 bp at the 3′end and 1152 bp at the ORF,encoding 383 amino acids.Amino acids sequence alignment analysis showed that TGFβ1 of T.fasciatus had high homology of 92.82%with T.rubripes.The phylogenetic tree analysis showed that T.fasciatus TGFβ1 clustered with T.rubripes.The fulllength cDNA sequence of TGFβ1 gene of T.fasciatus was similar to that of most pelagic fish,and had the closest relationship with Perciformes and the farthest relationship with Gekko japonicus.2)TGFβ1 gene expression data showed that TGFβ1 gene was expressed in different levels in 8 tissues of T.fasciatus.The expression level of TGFβ1 was the highest in kidney,followed by heart and spleen,while the expression was lower in the other 5 tissues with no significant difference from each other(P<0.05).3)TGFβ1 gene SNP loci of T.fasciatus were developed,and a mutation in the nucleotide sequence at position 3908 in TGFβ1 was found,named SNP g.3908 C>A.After comparison,it was found that SNP g.3908 C>A was located on the intron of TGFβ1 in T.fasciatus,and did not participate in the transcription and translation of TGFβ1.Correlation analysis between different genotypes of SNP loci and growth traits showed that SNP g.3908 C>A on TGFβ1 was related to many growth traits of T.fasciatus,among which the CC type was the dominant genotype,and CC type individuals had higher body width and plumpness.The results confirm that TGFβ1 gene has a correlation with the growth traits of T.fasciatus,which is expected to provide reference materials for molecular markerassisted breeding of T.fasciatus.