人亲磷酸酯酶A1基因的克隆及其功能的初步研究

发布时间：2018-05-29 02:37

本文选题：克隆 + RACE　；参考：《桂林医学院》2011年硕士论文

【摘要】：第一部分人亲磷酸酯酶A1基因的克隆及序列分析目的克隆人亲磷酸酯酶A1(phosphatidic acid-preferring phospholipase A1,PA-PLA1)基因,获mRNA全长序列,并通过分析所编码氨基酸的序列特征为从分子水平上研究PA-PLA1生理功能提供理论依据。方法从人的肝脏组织中提取RNA,逆转录为cDNA。PCR扩增该基因片段,cDNA末端快速扩增技术扩增该基因末端,克隆测序后获得PA-PLA1基因mRNA全长序列。Vector NTI 8.0拼接序列并进行序列比对,DNASTAR构建系统树。登录NCBI用人类基因组数据库做BLAST分析,ORF finder预测PA-PLA1的编码框。结果PA-PLA1基因mRNA全长为2923bp,编码区长2238bp,编码一个由745个氨基酸残基组成的蛋白多肽,分子量为83141道尔顿,等电点为5.64。全序列与人脂肪酶家族序列差异较大,与KIAA0725蛋白及P125蛋白具一定同源性。氨基酸序列中具有与酶活性相关的保守序列Ser408-His-Ser410-Leu-Gly412, Glu448-Ala475区可形成卷曲螺旋区域,且N末端可形成具DDHD结构域。结论获得人PA-PLA1基因mRNA全长序列。序列分析研究显示人PA-PLA1的功能可能与细胞内信号传导有关。第二部分PA-PLA1基因表达与胰岛素分泌关系的初步研究目的研究PA-PLA1基因在小鼠胰岛β细胞株MIN6中的表达与胰岛素分泌的关系。方法以不同浓度及不同时间分别对MIN6细胞行葡萄糖刺激胰岛素分泌实验,放免法测定细胞上清液中胰岛素分泌量,实时荧光定量PCR检测相应条件下PA-PLA1 mRNA的表达水平,线性相关分析PA-PLA1 mRNA表达水平与胰岛素分泌的相关性。结果在一定葡萄糖浓度范围内,PA-PLA1 mRNA水平随着葡萄糖浓度升高和刺激时间的延长而升高,并与细胞胰岛素分泌量呈现明显的正相关,不同葡萄糖浓度和不同刺激时间的PA-PLA1 mRNA水平与胰岛素水平的相关系数r分别为0.8839(P0.01)和0.981 (P0.01)。结论小鼠胰岛β细胞株MIN6中PA-PLA1基因的表达与胰岛素分泌有关并呈正相关性。
[Abstract]:Part I cloning and sequence Analysis of Human Phosphatase A1 Gene Objective to clone the human phosphatophilic enzyme A1(phosphatidic acid-preferring phospholipase A1-PA-PLA1) gene and obtain the full-length mRNA sequence, and to provide theoretical basis for studying the physiological function of PA-PLA1 at molecular level by analyzing the sequence characteristics of the encoded amino acids. Methods RNAs were extracted from human liver tissue and amplified by cDNA.PCR. After cloning and sequencing, the full-length mRNA sequence of PA-PLA1 gene. Vector NTI 8.0 splicing sequence was obtained, and the sequence alignment was carried out to construct the system tree. Log on to NCBI and use human genome database to do BLAST analysis for ORF finder prediction PA-PLA1 coding box. Results the total length of PA-PLA1 gene mRNA was 2923bpand the coding region was 2238bp. it encodes a polypeptide composed of 745amino acid residues with a molecular weight of 83141 Dalton and a isoelectric point of 5.64. The whole sequence was different from that of human lipase family and had some homology with KIAA0725 protein and P125 protein. A conserved sequence Ser408-His-Ser410-Leu-Gly412 was found in the amino acid sequence. The Glu448-Ala475 region could form a crimp helix region and the N-terminal region could form a DDHD domain. Conclusion the full-length mRNA sequence of human PA-PLA1 gene was obtained. Sequence analysis showed that the function of human PA-PLA1 may be related to intracellular signal transduction. A preliminary study on the relationship between PA-PLA1 Gene expression and Insulin secretion Objective to study the relationship between the expression of PA-PLA1 gene and insulin secretion in mouse islet 尾 cell line MIN6. Methods MIN6 cells were treated with glucose to stimulate insulin secretion at different concentrations and at different times. Insulin secretion in supernatant was measured by radioimmunoassay and the expression of PA-PLA1 mRNA was detected by real-time fluorescence quantitative PCR. Linear correlation analysis showed the correlation between PA-PLA1 mRNA expression and insulin secretion. Results the level of PA-PLA1 mRNA increased with the increase of glucose concentration and the prolongation of stimulation time, and was positively correlated with the amount of insulin secretion. The correlation coefficient between PA-PLA1 mRNA level and insulin level at different glucose concentration and different stimulation time was 0.8839 (P0.01) and 0.981 (P0.01), respectively. Conclusion the expression of PA-PLA1 gene in mouse islet 尾 cell line MIN6 is positively correlated with insulin secretion.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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