CONSTRUCTION OF BRAIN cDNA LIBRARIES AND MOLECULAR CLONING AND EXPRESSION ANALYSIS OF GNRH GENE INMARBLED EEL(ANGU ILLA MARMORATA)

Marbled eel(Anguilla marmorata) is the secondary rotected animal in China. In order to save the genetic information of this rarity and clone the function genes on growth, development and reproduction, a cDNA library was con2 structed by CloneminerTM kit from brain ofmarbled eel.The titer of the amp lified cDNA library was 4.3?06 cfu/mL, the total of recombinantswas 5.16?07 cfu, and the percentage of recombinant efficiencywas about 99.6%, the exogenous inserts of the recombinantswas from 0.43kb to 3.2kb and the average size was 1532 bp.These results showed that cDNA library had excellent quality.Two types of GnRH(mGnRH andcGnRH-II) cDNA sequenceswere isolated from cDNA library by PCR.Sequence analysis showed thatmGnRH cDNA contained an open reading frame(ORF) of 276 bp and encoded 91 amino acid residues,which consisting of a 222amino acid signal pep tide precursor(1-22 amino acid residues),mGnRH decapep tide(23-32 amino acid residues), 32amino acid signal processing site(33-35 amino acid residues), and a 562 amino acid GnRH-associated pep tide(36-91 amino acid residues).cGnRH-II cDNA open reading frame(ORF) contained 264 bases encoded 87 amino acid residues, which consisting of a 242amino acid signal pep tide precursor(1-24 amino acid residues),cGnRH-II decapep tide(25-34 amino acid residues), 32amino acid signal processing site(34-36 amino acid residues), and a 502amino acid GnRH2associated pep tide(37-87 amino acid residues).The homology analysis showed that the percentage ofmGnRH andcGnRH-II precursor sequence identitywith Japanese eel Anguilla japonica is 98%,with fishes of Salmoniformes, Perciformes and Pleuronectiformes is 73%-78%.However, it was relative low with fishes of Cypriniformes(63%-67%).Phylogenetic tree analysis ranked the fish GnRH as three distinct groups,mGnRH and sbGnRH group,cGnRH-II group and sGnRH group, respectively.Expression analysis by RT2PCR showed thatcGnRH-II andmGnRH gene expression had no obvious differences between female and male marbled eel individuals, butmGnRH gene could be expressed in more tissues thancGnRH-II.mGnRH were detected in forebrain,midbrain, hindbrain, pituitary, hypothalamus, liver, heart, sp leen, kidney, intestine, gill, ovary and testis,while expression ofcGnRH-IIwas mainly limited to forebrain,midbrain, hindbrain, ovary and testis.The present work provided evidence of two GnRH inMarbled eel reproductive system and suggested an important role ofmGnRH in reproduction.