【作者】 周杰；

【导师】 俞菊华；

【作者基本信息】 南京农业大学 ， 水产养殖， 2016， 硕士

【摘要】 脂肪是生物体重要的储能物质,其分解产物脂肪酸又是生物体重要的结构物质和活性物质。脂蛋白脂肪酶能够分解脂肪,在生物体脂肪代谢及其相关脂类调控中发挥着重要的作用。在鱼类中存在着两种脂蛋白脂肪酶(LPL1、LPL2)。目前对于鲤鱼LPL结构及功能方面的研究还没有报道,所以本文选取鲤鱼为研究对象,阐明了鲤鱼LPL基因的表达及其功能情况。利用RT-PCR的方法分离了鲤鱼LPL1和LPL2编码区cDNA全长序列,克隆结果表明,鲤鱼LPL1和LPL2开放阅读框(ORF)分别为1524 bp和1503 bp,分别编码507个和500个氨基酸。同源分析结果表明,LPL1和LPL2氨基酸序列相似性为45.51%。氨基酸功能位点分析表明,LPL1、LPL2的N-糖基化位点分别为85N、401N和400N,肝素结合域分别为321R-323N和319R-321N,催化活性位点分别为174S、198D、283H和172S、196D、281H,二聚体形成的保守疏水残基位点分别为218A、230G、237G和216A、228G、235G。系统进化分析表明,鲤鱼LPL1、LPL2与不同纲动物的LPL1、LPL2之间的遗传距离与分类地位基本一致,进化树很好的显示了鱼纲不同科、目的系统发育水平。利用实时荧光定量PCR检测了 iPL1、LPL2在鲤鱼不同组织中的表达情况,结果显示,LPL1和LPL2在不同组织中均有表达,在肝脏中表达量最高,其次为心脏、脂肪、肌肉、脑、中肾,在前肠中表达量最低,在所有组织中,LPL1的表达量始终高于LPL2的表达量。利用酶联免疫法和BCA全蛋白测定法测定了鲤鱼心脏、脂肪、肌肉、肝脏等组织中的脂蛋白脂肪酶含量,结果发现,这与LPL在mRNA水平上的表达量一致。LPL1和LPL2原核表达蛋白成功地在大肠杆菌中获得且使用Ni柱纯化。使用对硝基苯酚法(pNP)测定了 LPL1和LPL2酶活,结果表明,LPL1和LPL2酶活的最适温度均为35℃,最适pH均为8.0,在最适温度和pH条件下,LPL1和LPL2的酶活分别为22.69U/g、17.4U/g。很显然,两者序列差异导致了蛋白酶活不同。更多还原

【Abstract】 Fat is an important energy storage substance in the body,and its decomposition product fatty acid is an important structural material and active substance.Lipoprotein lipase can break down fat,and it plays an important role in the regulation of lipid metabolism and lipid metabolism.There are two kinds of lipoprotein lipase(LPL1,LPLZ)in fish.At present,the research on the structure and function of comnon carp LPL has not been reported,so this paper selected carp as the research object,the expression and function of LPL gene in common carp were clarified.Using the method of RT－PCR to separate the LPL1 and LPL2 encoding cDNA full length sequence,The cloning results showed that the open reading frame of LPL1 and LPL2 were 1524bp and 1503bp,encoding 507 and 500 amino acids respectively,and the amino acid similarity was 45.51%.The analysis of functional sites of amino acids about LPL1 and LPL2 showed that the N-glycosylation sites were 85N,404N and 400N,the heparin binding domain were respectively 321R-323N and 319R-321N,the catalytic active sites respectively were 174S,198D,283H and 172S,196D,281H,and the dimer fomation conserved hydrophobic residues sites were 218A,230G,237G and 216A,228G,235G.Phylogenetic analysis showed that the genetic distances between LPL1 and LPL2 of common carp and the different classes of LPL2 and LPL1 are basically consistent with the classification status.The phylogenetic tree showed a good level of fish in different families and objective system.The expression of LPL2 and LPL1 in different tissues of common carp was detected by real-time fluorescence quantitative PCR The results showed that LPL1 and LPL2 were expressed in different tissues,and the expression of LPL1 and LPL2 in the liver was the highest,followed by heart,fat,muscle,brain,kidney,and in the foregut was the lowest.In all tissues the expression of LPL1 was higher than that of LPL2.The content of LPL in heart,fat,muscle,liver and other tissues of common carp was determined by the method of enzyme-linked immunosorbent assay and BCA total protein.It vas found that the results was consistent with the mRNA expression levels of LPL.The recombinant mature peptides of LPL1 and LPL2 were successfully expressed and purified in E.coli,The results of the analysis of the method of p-nitrophenol showed that the optimum temperature for LPL1 and LPL2 enzyme activity was 35℃,and the optimum pH was 8.Under the optimum temperature of 35℃and pH8 conditions,the enzyme activity of LPL1 and LPL2 were 22.69U/g and 17.4U/g respectively.It was clear that the difference between the two sequences resulted in different protease activities.更多还原