| The growth of teleosts is mainly regulated by the growth axis of growth hormone(GH)/insulin-like growth factor(IGF).Growth hormone receptors(GHR),IGF and insulin-like growth factor receptor(IGFR)are important factors of the growth axis.In order to explore the roles of GHR,IGF-2 and IGF-IR genes in the growth and development of the Odontobutis potamophila.In this study,firstly,the O.potamophila was chosen as research object,and the rapid amplification of cDNA ends(RACE)approach was used to clone the full-length cDNA of growth hormone receptor(GHR),insulin-like growth factor 2(IGF-2),as well-as IGF-IR.Secondly,qRT-PCR was used to quantify the tissue-specific expression and different developmental stages from embryo to larva expression patterns of GHR,IGF-2 and IGF-IR genes.Meanwhile,qRT-PCR was further used to compare the mRNA expression of GHR,IGF-2 and IGF-IR genes in three tissues and five developmental stages between males and females of O.potamophila.At last,we identified the SNP locus of growth related genes of O.potamophila,and analyzed the correlation between the SNPs sites and the nine growth traits in the O.potamophila.The main results are shown as follows:1.The full-lengths of GHR,IGF-2 and IGF-IR cDNA in O.potamophila were cloned using homologous alignment and rapid amplification of cDNA ends(RACE).Sequence analysis revealed that the full lengths of GHR,IGF-2 and IGF-IR cDNA were 2179 bp,1586 bp and 4889 bp,respectively.And the putative lengths of open reading frame(ORF)were predicted as 1830 bp,642 bp and 1650 bp,encoding 609,213 and 549 amino acids,respectively.The lengths of the 5'UTR were 251 bp,48 bp and 2590 bp respectively,while the lengths of 3'UTR were 638 bp,896 bp and 649 bp,respectively.The molecular weights were predicted as 98.59 KDa,24.45 KDa,61.24 KDa;the isoelectric points were 4.80,10.00 and 5.50.respectively.The structure of GHR gene contained a signal peptide with 21 amino acids,an extracellular region with 227 amino acids,a single transmembrane region with 22 amino acids and an intracellular region with 339 amino acids.The intracellular region included conservative cysteine residues and FGDFS motifs,while the intracellular domain has two conservative boxes.The IGF-2 coding region contains a signal peptide of 47 amino acids,5 conservative structural domains named A,B,C,D,E.Besides,6 cysteine residues are involved in the formation of three pairs of disulfide bonds.The IGF-1R coding region contains a predicted transmembrane domain(TMD)of 22 aa,a TyrKc domain of 267 amino acids and two low complex domains of 40 amino acids.The homology comparison revealed that consistencies of GHR,IGF-2,IGF-1R genes in O.potamophila and other species were 28.51?70.53%,35.96?88.84%and 26.17?89.00%,with close relatives were the Boleophthalmus pectinirostris,Siniperca chuatsi and hates calcarifer.Phylogenic tree analysis indicated that all fish species were classified in one branch,while both-of reptiles and mammals formed an independent one.2.qRT-PCR analysis indicated that GHR,IGF-2 and IGF-1R genes were widely expressed in all selected tissues,which are liver,muscle,spleen,heart,intestine,gonad,gill,brain and kidney.The highest expression of GHR and IGF-2 was detected in the live,followed by muscle,gonad and brain,while IGF-1R had the highest expression in brain,then followed by gonad,heart and muscle.Moreover,GHR,IGF-2 and IGF-1R genes were ubiquitously expressed at all developmental stages from embryo to larva.The highest expression of GHR,IGF-2 was in gastrula,and the highest expression of IGF-1R was in morula.In addition,the qRT-PCR analysis showed that mRNA expressions of GHR and IGF-2 in males were significantly higher than those of females in 90 d,150 d,210 d,270 d,330 d in liver tissue,and the body length and body weight of males O.potamophila were higher than females,and the growth speed of males were also faster than that of females.There was no significant difference in the mRNA expression of GHR in muscle and brain between males and females,while the expression level of IGF-2 significantly differed between male and female in muscle of 150 d,210 d and brain of 210 d.Compared with GHR and IGF-2,the IGF-1R expression of O.potamophila had significant sexual differences in liver and brain,while there was no significant difference in muscle.Among the total growth stages,GHR,IGF-2 and IGF-1R of O.potamophila in 210 d had the highest expression in brain compared with other tissues.Growth rate analysis of O.potamophila also indicated that both male and female reached the fastest growth rate at 210?270 d.3.The direct sequencing method was used to identifiy and analyze the SNPs of growth-related gene in O.potamophila.The results showed that three SNPs loci(SNPs 1777T>C,1704G>A,1208G>A)were found in IGF-1R gene.The correlation analysis between SNPs within IGF-1R gene and nine growth traits of O.potamophila showed that the SNP 1208G>A was significantly associated with specially important traits including OL,BL,TW,CPL,BWT and BWH.The OL,CPL and BWT of AG genotype fish were extremely and significantly higher than those of the GG genotype fish(P<0.01),and the BL,TW and BWH of the AG genotype fish were also significantly higher than those of the GG genotype fish(P<0.05).As for the other two SNPs(1777T>C,1704G>A),there was no significant association between them and the nine growth traits. |
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