Molecular cloning,expression analysis and growth traits-ralated SNP loci screening of TGF-β1 gene in Takifugu fasciatus
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.TGF-β1,a multi-effect cytokine of the transforming growth factor β family,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 TGF-β1 gene with growth traits in T.fasciatus,healthy T.fasciatus were selected to clone the full-length cDNA sequence of TGF-β1 gene by RACE technology.Further,to characterize the expression characteristics of TGF-β1 gene in 8 different tissues(heart,liver,brain,spleen,kidney,muscle,midgut and gill)by qRT-PCR.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 TGF-β1 of T.fasciatus by PCR direct sequencing and correlated the growth traits-related SNP loci screening of TGF-β1 gene in T.fasciatus by sequence multiplexed analysis.The results showed:1)the cDNA sequence of TGF-β1 was 2 217 bp,composing of 51 bp at the 5'end,1 014 bp at the 3'end and 1 152 bp at the ORF,encoding 383 amino acids.Amino acids sequence alignment analysis showed that TGF-β1 of T.fasciatus had high homology of 92.82%with T.rubripes.The phylogenetic tree analysis showed that T.fasciatus TGF-β1 clustered with T.rubripes.The full-length cDNA sequence of TGF-β1 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)TGF-β1 gene expression data showed that TGF-β1 gene was expressed in different levels in 8 tissues of T.fasciatus.The expression level of TGF-β1 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)TGF-β1 gene SNP loci of T.fasciatus were developed,and a mutation in the nucleotide sequence at position 3908 in TGF-β1 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 TGF-β1 in T.fasciatus,and did not participate in the transcription and translation of TGF-β1.Correlation analysis between different genotypes of SNP loci and growth traits showed that SNP g.3908 C>A on TGF-β1 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 TGF-β1 gene has a correlation with the growth traits of T.fasciatus,which is expected to provide reference materials for molecular marker-assisted breeding of T.fasciatus.