【作者】 王静；

【导师】 李宏基； 杨国宇；

【作者基本信息】 河南农业大学 ， 基础兽医学， 2009， 硕士

【摘要】 脂肪诱导转录物(FIT)和Lipin都是与脂肪代谢有关的基因,FIT是对脂肪组装成脂滴起关键作用的基因,是内质网膜蛋白,参与脂类代谢的氧化作用。Lipin是一种关键的脂肪调节酶,作为蛋白质在哺乳动物细胞中调节脂肪代谢。为了研究Lipin和FIT蛋白的生物学功能,本试验以EST片段为基础,利用同源序列克隆原理设计引物,对猪各种组织提取的总RNA进行RT-PCR,通过RT-PCR技术克隆得到FIT(FIT1和FIT2)和Lipin(Lipin1,2和3)的cDNA序列,利用生物信息学工具预测其相关生物学性质,并对其组织进行分布初探,试验分两个系列。一、猪Lipin蛋白家族的克隆和组织分布Lipin1基因包含一个完整的开放阅读框架(open reading frame, ORF)为2-2686bp,全长为2692bp,由894个氨基酸残基组成,其中酸性氨基酸为189个,碱性氨基酸为122个,分子量为98.91kDa,等电点为6.40。结构域预测含有LNS2结构域,位置在678-834aa,核定位NLS预测含有“KKRRKRRRK”结构,含有HAD-样结构域“DIDGT”催化模序,Lipin1基因与小鼠、人和大鼠的cDNA序列同源性分别为82.8%、85.0%、82.8%,预测的氨基酸序列与小鼠、人和大鼠的同源性分别为88.8%、89.9%和88.7%。Lipin2基因全长为2693bp,ORF为11-2686bp,由891个氨基酸残基组成,其中酸性氨基酸为190个,碱性氨基酸为126个,分子量为98.89kDa,等电点为5.32。LNS2结构域位置在680-836aa,核定位NLS预测含有“KKKKRRRKK”结构,含有HAD-样结构域“DIDGT”催化模序,Lipin2基因与小鼠、人和大鼠的cDNA序列同源性分别为83.0%、86.7%、82.9%,预测的氨基酸序列的同源性分别为87.4%、90.3%和87.4%。Lipin3基因变体1全长为2872bp,ORF为149-2728bp,由859个氨基酸残基组成,其中酸性氨基酸为165个,碱性氨基酸为105个,分子量为93.59kDa,等电点为5.42。LNS2结构域位置在648-804aa,核定位NLS预测含有“KKKRRRRKPRRKE”结构,含有HAD-样结构域“DIDGT”催化模序,Lipin3基因变体1与小鼠、人和大鼠的cDNA序列同源性分别为75.6%、83.4%、78.6%,预测的氨基酸序列的同源性分别为75.9%、81.6%和77.4%。变体2全长为2848bp,ORF为149-2704bp,由851个氨基酸残基组成,其中酸性氨基酸为163个,碱性氨基酸为104个,分子量为92.74kDa,等电点为5.42。LNS2结构域位置在640-796aa,核定位NLS预测含有“KKKRRRRKPRRKE”结构,含有HAD-样结构域“DIDGT”催化模序,变体2与小鼠、人和大鼠的cDNA序列同源性分别为75.6%、83.2%、78.5%,预测的氨基酸序列的同源性分别为76%、80.9%和77.4%。组织分布结果显示:Lipin1在脾、肺、肾、膀胱、大脑、小脑、胃、空肠、肌肉和脂肪等多种组织中都有分布,而Lipin2和Lipin3仅在脾、肺、回肠和肌肉中有分布。二、猪FIT蛋白家族的克隆和组织分布利用同源序列克隆原理设计克隆引物,对猪各种组织提取的总RNA进行RT-PCR,将PCR产物与pMD19-T载体连接后转化Ecol.JM109感受态细胞,筛选阳性克隆、测序,并进行序列分析,克隆的猪FIT1基因包含完整的开放阅读框架,长为1310bp,ORF为400-1272bp,编码290个氨基酸,其中酸性氨基酸为22个,碱性氨基酸为39个,分子量为32.17kDa,等电点为10.47,信号肽为1~35aa,含有6个跨膜结构域。猪FIT1的核酸序列与人、牛、大鼠和小鼠的同源性分别为92.1%、93.2%、87.4%和88.1%,氨基酸序列的同源性分别为97.2%、96.6%、94.8%和95.5% ,为进一步研究FIT1的生物学功能奠定了基础。克隆的猪FIT2基因包含完整的开放阅读框架,长为1776bp,ORF为82-870bp,编码262个氨基酸,其中酸性氨基酸为28个,碱性氨基酸为31个,分子量为29.64kDa,等电点为8.91,信号肽为1~30aa,含有4个跨膜结构域。FIT2的核酸序列与小鼠、大鼠和人的同源性分别为85.0%、85.7%和88.1%,氨基酸序列的同源性分别为87.4%、87.4%和92.0%,为进一步研究FIT2的生物学功能奠定了基础。组织分布结果显示:FIT1主要在肌肉和脂肪组织有分布,其它组织分布量太低,而FIT2在心、肝、脾、肾、大脑、小脑、空肠、回肠、甲状腺、肌肉和脂肪等组织都有分布。更多还原

【Abstract】 Fat-induced transcript (FIT) and Lipin are related to fat metabolism gene.FIT is assembled into lipid droplets of fat play a key role of the gene, is a endoplasmic reticulum protein involved in oxidation of lipid metabolism. Lipin is a key regulatory enzymes of fat,it has been identified as a protein that regulates fat metabolism in mammalian cells.In order to study the biological function of Lipin and FIT proteins, EST fragment of this test is based on the principle of the use of cloned sequences primers were designed for a variety of organizations pig of total RNA extracted for RT-PCR, through RT-PCR technology has been cloned FIT (FIT1 and FIT2) and Lipin (lipin1, 2 and 3) cDNA sequences, using bioinformatics tools are available to predict the nature of their biology and distribution of their organizations.The study was divided into two series.Ⅰ.Cloning and tissues distribution of pig Lipin proteins.The cloned sequences containing the open reading frame of Lipin1 gene consist of 2692bp, and ORF is 2-2686bp and encodes 894 amino acids, of which 189 acidic amino acid, basic amino acid for 122, molecular weight 98.91kDa, isoelectric point of 6.40. Forecast containing LNS2 domain, the location of the 678-834, NLS nuclear localization prediction with "KKRRKRRRK" structure, with HAD-like domain "DIDGT" catalytic motif.Identity analysis showed that the lipin1 nucleotide sequences shared 82.8%, 85.0%, 82.8% homology with that of mouse, human and rat, the deduced amino acid sequences shared 88.8%,89.9% and 88.7% homology with that of mouse, human and rat.The cloned sequences containing the open reading frame of Lipin2 gene consist of 2693bp, and ORF is 11-2686bp and encodes 891 amino acids, of which 190 acidic amino acid, basic amino acid for 126, molecular weight 98.89kDa, isoelectric point of 5.32. Forecast containing LNS2 domain, the location of the 680-836, NLS nuclear localization prediction with " KKKKRRRKK " structure, with HAD-like domain "DIDGT" catalytic motif.Identity analysis showed that the Lipin2 nucleotide sequences shared 83.0%、86.7%、82.9%, homology with that of mouse, human and rat, the deduced amino acid sequences shared 87.4%、90.3%和87.4%homology with that of mouse, human and rat. The cloned sequences containing the open reading frame of Lipin3 transcript variant1 gene consist of 2872bp, and ORF is 149-2728bp and encodes 859 amino acids, of which 165 acidic amino acid, basic amino acid for 105, molecular weight 93.59kDa, isoelectric point of 5.42. Forecast containing LNS2 domain, the location of the 648-804, NLS nuclear localization prediction with " KKKRRRRKPRRKE " structure, with HAD-like domain "DIDGT" catalytic motif.Identity analysis showed that the Lipin3 transcript variant1 nucleotide sequences shared 75.6%、83.4%、78.6%, homology with that of mouse, human and rat, the deduced amino acid sequences shared 75.9%、81.6%和77.4% homology with that of mouse, human and rat. The cloned sequences containing the open reading frame of Lipin3 transcript variant2 gene consist of 2848bp, and ORF is 149-2704bp and encodes 851 amino acids, of which 163 acidic amino acid, basic amino acid for 104, molecular weight 92.74kDa, isoelectric point of 5.42. Forecast containing LNS2 domain, the location of the 640-796, NLS nuclear localization prediction with "KKKRRRRKPRRKE " structure, with HAD-like domain "DIDGT" catalytic motif.Identity analysis showed that the Lipin3 transcript variant1 nucleotide sequences shared75.6%、83.2%、78.5%, homology with that of mouse, human and rat, the deduced amino acid sequences shared 76%、80.9%和77.4% homology with that of mouse, human and rat.Tissue distribution results showed that: Lipin1 in a variety of tissues are distributed, but only in Lipin2 and Lipin3 spleen, lung, ileum, and muscle are distributed.Ⅱ.Cloning and tissues distribution of pig FIT proteins.Cloning primers were designed according to the cloning principle of homologous sequence. Total RNA extracted from all kinds of tissue of pig and was amplified by RT-PCR, the PCR products were ligated into the pMD19-T vector, and then transformed into Ecol. JM109 competent cells. The positive clone was identified and the sequence was sequenced and analyzed. The cloned sequences containing the open reading frame of pig FIT1 consist of 1310bp, and ORF is 400-1472bp and encodes 290 amino acids,of which 22 acidic amino acid, basic amino acid for 39, molecular weight 32.17kDa, isoelectric point of 10.47,signal peptide for 1 ~ 35aa, containing six transmembrane domain. Identity analysis showed that the pig FIT1 nucleotide sequences shared 92.1%、93.2%、87.4%和88.1% homology with that of human, bovine, rat and mouse, the deduced amino acid sequences shared 97.2%、96.6%、94.8%和95.5% homology with that of human,bovine, rat and mouse. It provides the experiment basis for further researching its biological function.The cloned sequences containing the open reading frame of pig FIT2 consist of 1776bp, and ORF is 82-870bp and encodes 262 amino acids,of which 28 acidic amino acid, basic amino acid for 31, molecular weight 29.64kDa, isoelectric point of 8.91,signal peptide for 1 ~ 30aa, containing four transmembrane domain. Identity analysis showed that the pig FIT2 nucleotide sequences shared 85.0%、85.7%和88.1% homology with that of mouse, human and rat, the deduced amino acid sequences shared87.4%、87.4%和92.0% homology with that of mouse, human and rat. It provides the experiment basis for further researching its biological function.Tissue distribution results showed that: FIT1 pig is highly expressed in the cerebellum, in the lung, stomach, ileum and a small amount of expression in the brain, FIT2 mainly in the ileum and is highly expressed in the brain.更多还原