Cloning And Sequence Analysis Of CDNA Encoding Gonadotropin-releasing Hormone From Rice Field Eel (Monopterus Albus Zuiew)

In vertebrates,gonadotropin-releasing hormone(GnRH) is an critical signal molecule in the hypothalamus-pituitary-gonadal axis,which plays important roles in the development and maintenance of reproductive functions.It is well-known a decapeptide to modulate the synthesis and release of the gonadotropins from the pituitary and regulate the sexual determination and differentiation. Rice field eel {Monopterus albus) is a prevalent breed in China and is used intensively for study on sexual determination and sexual reversal in the aquaculture due to its special sexual reversal.Although the mechanism of the sexual reversal of M.albus is still not clear,some researches indicate that its GnRH plays a key role in sexual reversal of M.albus.Few reports touched upon the molecular mechanism of GnRH in the study of reproductive development and sexual reversal in M.albus.Study on M.albus GnRH gene can help to understand the molecular mechanism of sexual reversal and the harmonious functionary mechanism of hypothalamus-pituitary-gonadal axis in M.albus, furthermore,this work can lay the base for producing an effective reproductive technology of M.albus by genetic engineering.In the research the cDNA encoding chicken Gonadotropin-releasing hormone-II (cGnRH-II) from M.albus was cloned and sequenced,at the same time , its potential amino acid sequence was deduced and the corresponding protein structure was predicted and analysed, primary results are as follows: 1 .Full-length cDNAs sequence of cGnRH- II from M.albusBased on multiple alignment of a number of Perciformes cGnRH-II genes,two special 3' cDNA ends primers were designed for amplification of the 3'cDNA ends of cGnRH-II genes and the sequence was acquired from the cDNA templet of the brain of M.albus.Two special 5'cDNA ends primers were designed according to 3'cDNA ends sequence, so the 5' cDNA ends sequence was acquired from the cDNA templet of the brain of M.albus.Then two special primers were designed for amplification of the full-length cDNAs sequences according to 5' and 3'cDNA ends of M.albuscGnRH-II gene.The full-length cDNA of M.albus cGnRH-II is 617bp with 168bp 5'untranslated region and 200bp 3' untranslated region.The cDNA included an open reading frame of 249bp encoding the 83 amino acid residues of cGnRH- II precursor. 2.Deduced amino acid sequence analysis of M.albus cGnRH- II geneFrom the subcloning and sequence of 3'RACE and 5'RACE products,the complete sequence of the cDNA encoding M.albus cGnRH-II peptide was identified and determined to be 617bp in length.The cDNA comprises a long 5'untranslated region,an open reading frame(83 amino acid residues),a stop codon(TGA),and a 3'untranslated region,including a polyadenylation signal and a poly(A) tail.The open reading frame is 249bp in length encoding the 83 amino acid residues,including an signal peptide composed of 21 amino acids, cGnRH-II composed of 10 amino acids and a GnRH-associated peptide composed of 49 amino acids,which was connected to GnRH by a Gly-Lys-Arg sequence.So far the cGnRH- II precursor had the same architecture as that of other vertebrates reported. 3.Homology analysis of M.albus cGnRH-II geneThe cDNA sequence, including non-coding regions,acquired from M.albus demonstrated high similarity with those from Perciformes, comparatively, amino acid similarity is higher than nucleotide similarity. Similarity of amino acid sequences between the M.albus and other Perciformes such as Dicentrarchus labrax, Morone saxatilis, Oreochromis niloticus, Sparus aurata remain fairly high(86%,85%,88%,86%,respectively).Cornparison of the amino acid sequence of M.albus cGnRH- II precursor from cDNA to that of other teleost fishes such as Anguilla japonica, Oryzias latipes, rainbow trout, Danio rerio and Rutilus rutilus,shows 68%,77%,75%,70%,71% amino acid similarity,respectively.Similarities are low to 53%,63%,61% with Eublepharis macularius, Rana catesbeiana, Homo sapiens.The amino acid sequence of signal peptide,cGnRH- II in combining with the Gly-Lys-Arg sequence is highly conserved during evolution when compared with the corresponding regions of other cGnRH- II precursors of some vertebrates. However, the associated peptide region of cGnRH- II are markedly divergent.Consequently,the encoded characteristic of cGnRH-II cDNA,the cGnRH-II decapeptide and the processing site(Gly-Lys-Arg) is fully conserved during the long period of evolution of vertebrates, but the signal peptide and the GnRH-associated peptide of each of cGnRH- II in different vertebrates show a differential degree of structural conservation.In conclusion,amino acid sequence of cGnRH-II precursor is fairly conserved in all vertebrates.4.Deduced amino acid sequence analysis and protein structure prediction of M.albus cGnRH-II geneThe predicted M.albus cGnRH- II protein is 83 aa long and has a Mw of 9.375kDa and a p1 of...