摘要
胰岛素样生长因子I(Insulin-like growth factor-I,IGF-I)是20世纪50年代发现的一类多肽生长因子,它由70个氨基酸组成,它的分子量约为7500道尔顿。IGF-I主要存在于血液中,绝大部分由肝脏合成,胰腺、胃、小肠、大肠、肾、鳃、生殖腺、脑、心脏和眼等组织中也有IGF-I的表达,这些肝外组织分泌的IGF-I主要以旁分泌或自分泌的形式发挥作用。它不仅具有调节细胞代谢,促进细胞生长、分裂和抑制细胞死亡等多种生理功能,它还是生长激素功能的主要介导因子,能促进蛋白质的合成,抑制蛋白质的降解,调节河海洄游性鱼类的渗透压,在鱼类的生长和生殖生理中具有重要意义。
岩原鲤(Procypris rabaudi(T.chang))是长江上游名贵土著经济底栖鱼类,目前主要分布于宜昌以上的四川及重庆境内长江水系的干支流中,具有巨大的养殖开发前景。长期以来,针对岩原鲤的研究都集中在形态、分类等生物性特征上,很少有从分子水平对岩原鲤的研究。本实验从分子水平研究岩原鲤,既可以填补岩原鲤分子水平研究的欠缺,又可以为保护该鱼类种质资源、保护三峡库区的生物多样性做出贡献,另外还可以发掘重庆地区名优水产养殖品种。
本研究运用RACE技术克隆了岩原鲤IGF-I基因,对其核苷酸序列及推导的氨基酸序列以及预测的蛋白质高级结构进行了分析,并对其分泌以及激素对其影响进行了研究和分析,主要研究结果如下:
1.从岩原鲤肝胰脏中提取总RNA。以总RNA为模板,利用AMV反转录酶和试剂盒自带的oligo dT-3 sites adaptor primer为引物,合成第一链cDNA。在接下来的PCR中,以第一链cDNA为模板,人工合成的寡聚核苷酸为引物,用Taq DNA聚合酶进行扩增反应,从而得到3'端序列。用5'端磷酸化标记的基因特异性反转录引物反转录mRNA得到第一条cDNA链。然后用RNase H将RNA和杂合链中的RNA链裂解。接着在T4 RNA连接酶催化下,使单链cDNA环化成首尾连接物,用基因特异引物进行PCR扩增5'端。以两者结果拼接出岩原鲤IGF-I的全长序列。实验中得到的岩原鲤IGF-I拼接全长cDNA为612bp,包括一个长度为8个核苷酸的5'非编码序列(5'untranslated region,5'UTR),起始密码子(ATG),编码区486bp,编码161个氨基酸的开放阅读框(Open Reading Frame,ORF)、终止密码子(TGA)和一个包括加尾信号(AATAAA)、poly(A)信号的3'非编码区序列(3'-untranslatedregion),3'非编码区序列包括118个核苷酸。经同源性比较,可以确定实验结果为IGF-IcDNA序列
2.岩原鲤IGF-I的cDNA编码区核苷酸长度为486bp,编码161个氨基酸,其蛋白质肽链包含一个氨基酸序列为MSSGHFFQGHWCDVFKCTMRCLSCTHTLSLVLCVLALTPATLEA的信号肽(46个氨基酸)、切割位点在Ala46和Gly47之间。岩原鲤IGF-I编码区蛋白的分子量MW=17.88kDa,等电点(pI)=8.97,电荷(pH=7.0)=11.5。其二级结构主要有4种形式组成,即α-螺旋、随机卷曲、β-转角和延伸链,随机卷曲比例最高,占整个图形的59.63%,其次依序为α-螺旋、延伸链以及β-转角,这三者所占比例差距极大,分别为25.47%、11.8%和3.11%。
3.系统进化分析显示,鲤科鱼类的IGF-I蛋白很相似,如鲤鱼与岩原鲤这两种鲤科鱼类的IGF-I蛋白仅有一个氨基酸不同,其他全部一样,同源性高达99.38%。用岩原鲤IGF-I蛋白与其他鱼类比对,如鲮鱼、黑鲷、斑马鱼等,其同源性均在95%以上,说明鲤科鱼类IGF-I在进化过程中保持着高度保守性。
4.通过注射17β-雌二醇,对肝胰脏组织IGF-I表达进行的分析显示,在注射17β-雌二醇的剂量较低时,17β-雌二醇的注射量会显著影响IGF-I的表达,岩原鲤IGF-I的表达随注射量增大而增大,当过量注射时,岩原鲤IGF-I的表达随注射量增大而回落,直至与空白组表达无显著差异。
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.
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