【作者】 张芬；

【导师】 丁诗华； 郑曙明；

【作者基本信息】 西南大学 ， 水产养殖， 2008， 硕士

【摘要】 神经肽Y(Neuropeptide Tyrosing,NPY)是一种由36个氨基酸组成的多肽,广泛分布于中枢神经系统和外周神经系统中,尤其在端脑、视前区、视顶盖以及下丘脑含量丰富。在下丘脑内又以弓状核、室旁核、室周核、视交叉上核、视交叉后区等分布密集。NPY具有调节摄食、心血管功能、生殖、应激、免疫应答、激素调节、肥胖、性逆转等的生理效应,另外NPY对哺乳动物和鱼类生理节律和性行为也具有调节作用。本研究以草鱼为试验对象,经PCR条件的优化,运用了RACE技术克隆草鱼神经肽Y(NPY)基因cDNA的全长序列,并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。同时,本研究还检测了饥饿不同时间段(24h、48h、72h、73h、75h)以及在饥饿72h重新投饵后第1h、3h的草鱼脑组织中NPY表达水平的变化,揭示了NPY对摄食的调节作用,为鱼类营养学研究提供科学依据。1.在对多种鲤科鱼类的NPY基因进行了多重比对后设计特异性引物,通过RACE技术以草鱼脑组织的mRNA为模板扩增获得草鱼NPY cDNA的全序列信息。从NFY cDNA全序列扩增结果得到草鱼NPY全长cDNA为765bp,包括一个5′非编码区序列长度为51个核苷酸的(5′UTR),起始密码子(ATG),编码96个氨基酸的开放阅读框(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(3UTR),3′非编码区序列包括423个核苷酸。经同源性比较分析,可以确定试验结果为NPY cDAN序列。2.草鱼NPY的cDNA编码的氨基酸多肽链全长96个氨基酸,其中有28个信号肽氨基酸,分子量为10987,等电点pI为5.94。在氨基酸组成上,亮氨酸(Leu)是含量最高的氨基酸(11.36%);其次是丙氨酸(Ala),占9.38%;再次是谷氨酸(Glu)和苏氨酸(Thr),均占7.29%;含量最低的氨基酸为缬氨酸(Val),约1.04%。从整体上来看,碱性氨基酸(赖氨酸Lys、精氨酸Arg)和酸性氨基酸(天冬氨酸Asp、谷氨酸Glu)分别占9.38%和12.5%。草鱼NPY的二级结构由α-螺旋、β-折叠、转角和随机卷曲四种形式组成,α-螺旋所占比例最高,β-折叠的比例最低。3.同源性分析显示,鲤科鱼类的NPY编码区氨基酸序列具有较高的同源性,其中草鱼NPY与鲤鱼NPY均含有96个氨基酸,且其氨基酸水平上的同源性达100%。它与中华倒刺鲃、斑马鱼、金鱼NPY氨基酸序列同源性分别为97.9%、93.8%、95.8%。此外,草鱼NPY蛋白与其他物种同源性较鲤科鱼类低,如与斑点叉尾鮰、舌齿鲈、虹鳟、石纹电鳐、斜带石斑鱼等的NPY氨基酸序列的同源性分别为68.4%、68.4%、66.7%、61.5%、65.3%。与七鳃鳗NPY氨基酸序列的同源性最低,只有41.1%。这些结果说明鲤科鱼类NPY在进化过程中保持高度保守性。4.本试验设计了7个试验组,分别为24h、48h、72h、73h、75h饥饿组,以及73h、75h重新投饵组(73R、75R),用于检测饥饿对草鱼脑组织NPY表达的影响。结果显示随着饥饿时间增加草鱼脑组织中NPY表达含量升高,饥饿24h、48h差异不显著(P＞0.05);饥饿72h时脑组织中NPY表达含量显著升高(P＜0.05),饥饿73h、75h与72h差异不显著。在饥饿72h对试验动物进行重新投饵,结果表明在投饵后1h、3h(73R、75R)脑组织NPY表达含量急剧下降,与饥饿73h、75h比较差异显著(P＜0.05)。结果表明NPY是一种调节草鱼每天摄食量和食欲的脑部生理学传感器。更多还原

【Abstract】 Neropeptide Tyrosing(NPY)is a polypeptide containing 36 amino acid residues,is abundantly and widely distributed in both the central nervous system(CNS)and the peripheral nervous system (PNS),especially in telencephalon,preoptic area,optic tectal slices and hypothalamus.In hypothalamus,the NPY is concentrated in arcuate nucleus(ARC),paraventricular nucleus(PEVN), periventricular nucleus,suprachiasmatic nucleus(SCN),and optic chiasma postzone and so on.NPY is implicated in various physiological effects such as food intake,reproduction,cardiovascular function,reproduction,stress,immunologic response,immunologic response,fatness,sex reversal and so on.Moreover,NPY is involved in the regulation of circadian rhythms and sexual behavior in mammals as well as in fish.In this study,a full length cDNA of grass carp NPY was cloned by RACE methods and analyzed by bioinformatics.At the same time,its potential amino acid sequence was deduced and the corresponding protein structure was predicted and analysed.Meanwhile,the change of expression from brain tissue of grass Carp in the different stage time(24,48,72,73,75 h)of starvation and in the 1h and 3h after refeed revealed the NPY’s effect in regulation of food intake,and the results provided the scientific evidence for fish nutriology.1.Based on multiple alignment of a number of Cypr(?)n(?)dae NPY genes,the(?)ull-length cDNA of grass carp was amplificated by the RACE technique using the total RNA isolatedo from grass carp brain as template.The length of the cDNA is 765bp,which includes a 5’-untranslated region(5’UTR, 51bp),the start codon(ATG),an open reading frame of 291 bp coding for a protein of 96 amino acids,the stop codon(TGA),3’-untranslated region(3’ UTR,423bp)containing a polyadenylation signal(AATAAA)and the poly A tail.Comparsion of the sequence homology between NPY cDNAs from various animals indicated that the cDNA fragment obtained in the study encodes a NPY protein.2.The grass carp NPY pro-polypeptide of 96 amino acid residues includes 28 signal peptide amino acids.Its molecular weight(Mw)is 10987 and its isoelectnic point(pI)is.5.94.Analysis of amino acid composition showed that the highest content of amino acids is leucine(11.36%),the second highest content is alanine(9.38%),the third highest contents are glycine and threonine (7.29%),and the lowest content is valine(1.04%).The proportions of acidic amino acids and basic amino acids are 12.5%and 9.38%,respectively.The protein contains four main secondary structures:α-helix;β-sheet,turn and randona coil.Alpha helix is the dominant secondary structure andβ-sheet is the lowest.3.Analysis of phylogenetic tree showed that Cyprinidae fishes NPY proteins are highly homologous.The NPY proteins of grass carp and Cyprinus carpio have 96 amino acids and exhibit homology of 100%.Similarly,amino acid sequence of grass carp NPY protein also has high homology compared with other Cyprinidae fishes such as spinibarbus sinensis,goldfish and zebrafish(97.9%,93.8%,95.8%amino acid homology,respectively).Furthermore,it has relatively low homology with other species such as channel catfish,european seabass,rainbow trout,marbled electric ray and orange-spotted grouper(68.4%,68.4%,66.7%,61.5%and 65.3%amino acid homology,respectively),but there is lowest homology when compared with lampetra fluviatilis (only 41.1%).All of results suggested that Cyprinidae fishes NPYs are highly conservated in the evolutional process.4.After different periods(24h,48h,72h,73h and 75h)of feed deprivation and refeed at 73h or 75h after feed deprivation(73R,75R),the levels of NPY mRNA in the brain tissue were determined. The result showed that the expression levels of the NPY were increasing follow the time extension of feed deprivation(FD).The expression levels of NPY were highest in 72h FD fish(0h-72h),and continued for 1-3 h after 72h FD;whereas,refeeding reversed the increase of the NPY mRNA levels only in 1-3 h after refeeding(73R,75R),exhibiting a significant difference(P＜0.05)when compared with the levels of 73h and 75h FD.Taken together,these results further supported that NPY is a physiological transducer of brain involved in the regulation of daily appetite and feeding in grass carp.更多还原