Cloning, Expression Analysis And SNP Locus Development Of HTR4, TGF-β1 And CIRP Genes Of Takifugu Obscurus

Takifugu fasciatus is a north temperate zone anadromous economic fish whose market supply and demand contradiction are mainly alleviated by artificially bred populations.However,due to long period of domestication,inbreeding has inevitably occured in the captive breeding population.The weakness of cold resistance also brings a low survival rate during winter.From the view of molecular assisted breeding,three genes related to growth and cold resistance of T.fasciatus,namely 5-hydroxyserotonin receptor 4,transforming growth facto β1 and cold-induced binding protein,were cloned.Their structural functions,expression characteristics and related SNPs were seriously studied,which has laid a theoretical foundation for molecular assisted breeding of T.fasciatus.The results are as follows:1.Cloning of HTR4,TGF-β1 and CIRP in T.fasciatusRACE technique was used to clone 5-hydroxyserotonin receptor 4(HTR4),transforming growth factor β1(TGF-β1)and cold inducible RNA-binding protein(CIRP)in T.fasciatus.The c DNA sequence of Tf HTR4 was 2,302 bp,including 662 bp at the 5 ’end,470 bp at the 3’ end,1,170 bp at the open reading frame(ORF).The theoretical molecular weight of Tf HTR4 was 43,392.36,theoretical isoelectric point was 9.31,and theoretical molecular formula was C1957H3087N527O517S35.The c DNA sequence of Tf TGF-β1 was 2,217 bp,including 51 bp at the 5 ’end,1,014 bp at the 3’ end and 1,152 bp at the ORF.The theoretical molecular weight of Tf TGF-β1 was 44,233.70,theoretical isoelectric point was 8.34,and theoretical molecular formula was C1957H3089N531O588S24.The c DNA sequence of Tf CIRP was 775 bp,including 106 bp at the 5 ’end,144 bp at the 3’ end,and 525 bp at the ORF.The theoretical molecular weight of Tf CIRP was 18,926.32,theoretical isoelectric point was 9.00,and theoretical molecular formula was C801H1226N254O274S4.Tf HTR4 has a 288 AA(amino acid)rhodopsin receptor protein domain at the middle of ORF and a low complexity region(LCR)from 371 AA to 385 AA.Tf TGF-β1 has a 237 AA TGF-β1 prepeptide domain at the front of ORF,and a 97 AA TGF-β1 subdomain at the end of the sequence.Tf CIRP has a 288 AA ancient conserved RNA recognition motif of about 74 AA at the front of ORF,and two LCRs between 93 ～ 122 AA and 126 ～ 152 AA.The full-length sequence of all three c DNA sequences have the highest similarity with that of Takifugu rubripes.2.Detecting the expression pattern of HTR4,TGF-β1 and CIRPThe developmental process of T.fasciatus can be divided into five stages(stage I,II,III,IV and V)according to the gonadal developmental status.q RT-PCR was used to detect the relative expression of HTR4,TGF-β1 and CIRP in eight different tissues(heart,liver,brain,spleen,kidney,muscle,midgut,gill)from five developmental stages.HTR4 gene is expressed in all eight examined tissues,and was bound to be predominantly in brain and midgut.The temporal distribution showed a model in all tissues,that is ‘relatively high in stage I,slightly decreased in stage II,stopped in stage III,recovered in stage IV,and lowest in stage V’.TGF-β1 is dectected in all eight tissus and highest expressed in kidney.It had the temporal distribution characteristics of ‘high in stage I and V,low in stage III,and depended on the tissues for stage II and IV’.Among the tissues of each period,the relative expression level of CIRP in the brain was significantly higher than all other tissues.No significant difference was dectected among the tissues in the other four stages.3.Excavating of SNPs for T.fasciatus HTR4,TGF-β1 and CIRPEffective SNP locus was found in each of the three genes through PCR amplification and multiple aligment.The SNP c.1202 C>G locus on HTR4 was significantly correlated with the condition factor of T.fasciatus,implying individuals with CC genotype are significantly fatter than others.The SNP g.3908 C>A site on TGF-β1 is related to several growth traits of T.fasciatus.The CC type individuals are shorter in head and body,but wider,fatter and flatter than individuals with AA type.Individuals with AA genotype at the locus of g.567 in CIRP had a 6.67% improvement in cold-resistance compared with TT individuals.