Cloning And Gene Expression Of Insulin-like Growth Factor-I CDNA From Procypris Rabaudi (T.chang)

Insulin-like growth factor-Ⅰ(IGF-Ⅰ)is one kind of polypeptide growth factor discovered in 1950s.It makes up of a 70-amino acid peptide.Its molecular weight abaout 7500 Dalton..IGF-Ⅰmainly exists in blood and most of them are synthesized in the liver.IGF-Ⅰis also produced in a wide variety of tissues,such as pancreas,stomach,small intestines,large intestines,kidney,gill,gonad, brain,heart and eyes and acts locally in paracrine and autocrine manners IGF-Ⅰmay regulate the cell metabolism,stimulate cell growth,promote cell differentiation and inhibit apoptosis.It also is the main growth hormone mediated factor,witch can promote protein synthesis,inhibit protein degradation,mediate the osmotic pressure of river-sea migration fish.It is very important to growth and propagation of fish..Procypris rabaudi(T.chang)is a famous and precious economical demersal fish in the upper Yangtze River.Now it mainly distributes in main and branch of upper Yangtze River..These former researches on Procypris rabaudi are only about morphology,classification and rarely about molecule level.This study about Procypris rahaudi is on molecule level,may contribute to protect the wild resource of the species and species diversity in The Three Gorges and to find out the famous high quality aquaculture species.This research uses RACE technology cloning IGF-Ⅰgene from Proeypris rabaudi,and analyses the nucleotide sequences and deduces high structure of the protein based on nucleotide sequences,we also research the effect of 17β-estradiol on IGF-Ⅰ,the main research reasons are as follows:1.Total RNA was extracted from Procypris rabaudi.'s hepatopancreas.Using total RNA as template synthesis the first chain cDNA.The cNDA was used to perform PCR with a special reverse transcription primer and one of special primer.So the sequence of the 3'cDNA ends was acquired from the cDNA templet of the hepatopancreas of Procypris rabaudi.The phosphorylation. of 5'end primer was used to synthesis the frist chain eDNA that was used to amplified the 5'.end sequences of rock carp IGF-Ⅰ.We get the full-length cDNA of IGF-Ⅰby splicing the two part of sequences(3'end and 5'end sequence).The full-length cDNA of IGF-Ⅰof Procypris rabaudi is 612 bp,including 5'untranslated region(5'UTR)8bp,,encoding 161 amino acids residues of the open reading frames(Open Reading Frame,ORF)486 bp,a start codon(ATG),a stop codon(TGA)and 3'-untranslated region 118bp(3'UTR)that including a polyadenylation signal(AATAAA)and a poly (A)tail.2.Procypris rabaudi.IGF-ⅠcDNA open reading frame is 486 bp,encoding 161 amino acids residues,the deduced amino acids residues including a signal peptide composed of 46 amino acids residues(MSSGHFFQGHWCDVFKCTMRCLSCTHTLSLVLCVLALT PATLEA),cutting sites is between Ala46 And Gly47.the molecular weight of Rock carp IGF-Ⅰprotein is 17.88kDa,its isoelectric point(pI)is 8.97,its Charge(pH=7.0)is 11.5.IGF-Ⅰprotein contains four main secondary structures:α-helix,random coil,β-turn and extended strand,The dominant secondary structure is random coil,and its proportions in IGF-Ⅰis 59.63%.,The folow isα-helix,extended strand andβ-turn,the proportion of them in IGF-Ⅰis 25.47%,11.8%and 3.11% respectively.3.Phylogenetic analysis showed that the IGF-Ⅰprotein of Cyprinid fishes is very similar.There is only one amino acid residues is different beteeen common carp and Rock Carp.The similarity is 99.38%.Comparision of the amino acid sequence of rock carp IGF-Ⅰto that of other fshes such as mud carp,black sea bream,zebrafish,shows more than 95%of amino acid similarity.The results show that there is a highlyconserved of fishes IGF-Ⅰin the in the process of evolution.4.Through injecting rock carp with17β-estradiol,analysising the hepatopancreas organizations IGF-Ⅰexpression shows that the IGF-Ⅰexpression was affected obviously at a low dosage 17β-estradiol. The more 17β-estradiol be used,the more obviously effects would be appeared.But when performing excessive dosage,the rock carp IGF-Ⅰexpression would be turn to lower,until the IGF-Ⅰexpression is no significant difference with blank control.