Molecular Cloning,Expression And Sequence Analysis Of GnRH In American Shad Alosa Sapidissima

Americanshad,Alosa sapidissimabelonged to Clupeomorpha,Clupeiformes,Clupeidae and Alosa.As the same Alosinae species of theAlosa sapidissima and Tenualosa reevesii,they are similar inmorphological characteristics and life habit.Both of them are anadromousspecies with very high nutritional value and economic value.So theAmerican shad can be cultured,popularized and as a substitute forTeaualosareevesiwhich is in an endangered situation.Gonadotropin releasing hormone(GnRH)is a highly conserved ten peptide hormone as a key hormone in vertebrate neural endocrine system,and it plays an important role in regulating axis.In this study,based on the homologous cloning and RACE techniques,the full-lengthcDNA of three types of GnRH in American shad(Alosa sapidissima)wereobtained.We use bioinformatic softwares and quantitative real-time PCR to study eachrespectively:(1)The full-lengthof Alosa sapidissima GnRH1 cDNA was 462 bp,composed of 150 bp by of5'UTR,258 bp by of ORF,and 61 bp by of 3'UTR,and it could encode 86 amino acids as residues with the signal peptide is 22 amino acidswhich estimated molecular mass of 9.52 kDa,and the isoelectric point is 6.80.(2)The full-lengthof Alosa sapidissima GnRH1 cDNA was 809 bp,composed of 224 bp by of5'UTR,255 bp by of ORF,and 327 bp by of 3'UTR,and it could encode 85 amino acids as residues with the signal peptide is 24 amino acids which estimated molecular mass of 9.82 kDa,and the isoelectric point is 8.66.(3)The full-lengthof Alosa sapidissima GnRH1 cDNA was 887 bp,composed of 111 bp by of5'UTR,315 bp by of ORF,and 458 bp by of 3'UTR,and it could encode 105 amino acids as residues with the signal peptide is 21 amino acids which estimated molecular mass of 12.29 kDa,and the isoelectric point is 8.18.Each GnRH cDNA encodeda signal peptide(SP),GnRH and a GnRH-associated peptide(GAP),which wasconnected to GnRH by a Gly-Lys-Arg sequence.Phylogenetic analysis classifymultiple molecular forms of GnRHs into three branches: cGnRH-II branch,sGnRH branch and the fish specific GnRH branch.The tissue and sex specific expression ofthese three genes were determined using real time PCR.They all showed the highestexpression levels in brain for both sexes.All these three GnRH genes exhibited sex-specific expressionpattern with females expressed higher mRNA levels than that of males.In the study of periodic expression,the expression of GnRH1 mRNA increased from November to the following year in March,reached the maximum value in the year of April,and then declined rapidly and remained at a low level;The expression of GnRH2 mRNA in different months,also in March and in the mRNA expression in a high level,after the rapid decline,and maintained at a low level of GnRH2.However,the expression level of GnRH3 mRNA did not show a significant change in gonad development.