Characterization Of Growth-related Genes From Trachinotus Ovatus And Their Expression Responses To The Feed Types

Trachinotus ovatus,golden pompano now has been one of the most important marine aquaculture products in coastal region of south china due to well taste and fast growth.‘Hypothalamus-pituitary-liver axis’ mainly regulates expression of a handful of growthrelated hormone genes to promote proliferation of whole body cell in order to realize organism growth,which is the main regulation axis during vertebrate growth.Growth hormone(GH),growth hormone receptor(GHR)and insulin-like growth factor(IGF)are really crucial hormones in this axis,they are involved in the regulation of wide range of cellular functions,such as growth,increase food conversion rate,promote synthesis metabolism,regulate osmotic pressure and reproductive behavior regulation.Besides,kisspeptin also plays a critical regulation role in body growth,energy metabolism and reproduction.Thus,this expriment screens and clones four growth related genes,GH,GHRs,IGFs and Kiss,from Trachinotus ovatus feed by different feed types to study those genes expression reflection on various feed types.This article screens GH、GHRs、IGFs and Kiss gene sequence from transcriptome,and clone their c DNAs by RACE.Then use bioinformatics software to analyze their genomics informations.Further resaerch on those gene tissue distribution by q RT-PCR.Based on those growth related genes sequence from Trachinotus ovatus genome sequencing,analysis and predictions on gene structure,promoter and microsatellite loci are carried out.In addition,use fluorescent probe technique to research on microsatellite loci polymorphism in 30 Trachinotus ovatus individuals.Considering that nutrition affect gene expression,Trachinotus ovatus are feed by three different foodtypes,Loligo chinensis 、 Trachurus iaponicus and commercial pelleted feed(including 36% crude protein)in order to those gene expression reflection on various foodtypes.The followings are specific research results.(1)The full-length c DNA of To GH(Gen Bank accession No.MF795138)is 850 bp and encodes 201 amino acid residues.To GH amino acid sequence has a signal peptide and two typical GH/SL/PRL family conserved domains.The 5’-flanking region contains consensus sequences for one pit-1a,three TATA boxes,three GRE,two TRE,two HNF-3,and two ERE.To GH have 12 microsatellite loci sites that did not show SSR polymorphism.The genomic structure of To GH(Gen Bank accession No.MF795139)was determined to span approximately 4.0 kb and be composed of six exons and five introns.The ORFs of To GHR1(Gen Bank accession No.MF677853)and To GHR2(Gen Bank accession No.MF677854)spans 1869 bp and 1758 bp,which code 622 aa and 585 aa,respectively.The result of a multiple-sequence alignment analysis suggested that To GHRs were highly homologous with GHRs of other vertebrate and shared a highly conserved FGEFS motif and two conserved boxes.To GHR1 has 12 SSR sites and 3 SSR sites has SSR polymorphism.To GHR2 has 1 SSR site that does’ t have SSR polymorphism.The 5′-flanking regions of both To GHRs contained transcription factor binding sites such as SP1,C/EBPs,and GATA-1 and so on.The full length c DNAs of To IGF-1(Gen Bank accession No.MH300904)、To IGF-2(Gen Bank accession No.MH300905)and To IGF-3(Gen Bank accession No.MH300906)are respectively 1718bp、1658bp and 2272 bp which code 185 aa,215aa and 194 aa,respectively.The construction of amino acid sequences of To IGFs are same as other species,which is consisted of 5 parts,signal peptide,B domain,C domain,A domain,D domain and E domain.To IGF1 dose’t have SSR polymorphism;To IGF2 and To IGF3 respectively have 3 and 1 SSR polymorphism sites.The full length c DNA of To Kiss1(Gen Bank accession No.MG843840)is 505 bp which code 104 aa.The amino acid sequence of To Kiss1 has a signal peptide and one kisspeptin-10 domain.To Kiss1 does’ t have SSR polymorphism sites.(2)The results of tissue expression position show that To GH was highly expressed in the intestine,brain and muscle tissues compared to other tissues evaluated.In liver,muscle,and heart,To GHR1 showed high expression.To GHR2 expression level was highest in liver and muscle.To IGF1 expression level was highest in liver,and was middle in spleen,muscle and spermary,and was low in ovary,blood,stomach,fin,gill,intestine and brain.To IGF2 expression level was highest in gill,and was middle in intestine,stomach,brain,spermary,ovary and liver,and was low in muscle,spleen,fin,gill and blood.To IGF3 expression level was highest in gill,ovary and spermary,and was middle in brain and fin,and was low in muscle,intestine,kidney,stomach,blood.To IGF3 didn’t express in liver and spleen.To Kiss m RNA expression level was highest in brain,and was middle in intestine,stomach,spleen,muscle and heart.The results of gene tissue distribution show that To GH,To GHRs,To IGFs and To Kiss distribute not only in growth related tissue,such as the brain,muscle and liver,but also in reproductive tissues,osmotic pressure regulating related tissue and ingestion related tissue.This is similar to other bony fish,it reflects that the To GH,To GHRs,To IGFs and To Kiss has extensive biological function,need further mining and utilization.(3)By feed three different feed types,compared to the pelleted feed and the iced squid groups,the iced fish group had higher relative levels of hepatic To GH expression(P < 0.05).The relative levels of intestinal To GH expression were significantly different between the iced squid and pelleted feed groups(P < 0.05),but there were no significant differences between iced fish group with the other groups.To IGFs show different expression levels in different dietary groups.Liver To IGF1 m RNA expression level between the iced squid group and the iced fish group didn’t have significant difference,but liver To IGF1 m RNA expression level in the pelleted group is higher than it in the iced squid group and the iced fish group.To IGF-2 m RNA expression level was highest in the pelleted group,and was middle in the iced fish group,and was low in the iced squid group.Intestinal To IGFs m RNA have highest expression level in the pelleted group.Besides,To IGF1 expression level in the iced fish group was obvious higher than To IGF1 expression level in the iced squid group.To IGF2 expression level in the iced squid group was obvious higher than To IGF2 expression level in the iced fish group.There was no significant difference between the iced fish group and the iced squid group in To IGF3 m RNA expression level.Intestinal To Kiss-1 m RNA expression level was highest in the pelleted group,and was middle in the iced fish group,and was low in the iced squid group.There was no significant difference among these groups in liver.