CONSTRUCTION OF BRAIN cDNA LIBRARIES AND MOLECULAR CLONING AND EXPRESSION ANALYSIS OF <I>GNRH</I> GENE IN MARBLED EEL(<I>ANGUILLA MARMORATA</I>)

Abstract

Marbled eel(Anguilla marmorata) is the secondary protected 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 constructed by CloneminerTM kit from brain of marbled eel.The titer of the amplified cDNA library was 4.3×106 cfu/mL,the total of recombinants was 5.16×107 cfu,and the percentage of recombinant efficiency was about 99.6%,the exogenous inserts of the recombinants was 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 and cGnRH-II) cDNA sequences were isolated from cDNA library by PCR.Sequence analysis showed that mGnRH cDNA contained an open reading frame(ORF) of 276 bp and encoded 91 amino acid residues,which consisting of a 22-amino acid signal peptide precursor(1—22 amino acid residues),mGnRH decapeptide(23—32 amino acid residues),3-amino acid signal processing site(33—35 amino acid residues),and a 56-amino acid GnRH-associated peptide(36—91 amino acid residues).cGnRH-II cDNA open reading frame(ORF) contained 264 bases encoded 87 amino acid residues,which consisting of a 24-amino acid signal peptide precursor(1—24 amino acid residues),cGnRH-II decapeptide(25—34 amino acid residues),3-amino acid signal processing site(34—36 amino acid residues),and a 50-amino acid GnRH-associated peptide(37—87 amino acid residues).The homology analysis showed that the percentage of mGnRH and cGnRH-II precursor sequence identity with 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 RT-PCR showed that cGnRH-II and mGnRH gene expression had no obvious differences between female and male marbled eel individuals,but mGnRH gene could be expressed in more tissues than cGnRH-II.mGnRH were detected in forebrain,midbrain,hindbrain,pituitary,hypothalamus,liver,heart,spleen,kidney,intestine,gill,ovary and testis,while expression of cGnRH-II was mainly limited to forebrain,midbrain,hindbrain,ovary and testis.The present work provided evidence of two GnRH in Marbled eel reproductive system and suggested an important role of mGnRH in reproduction.