本文選題：克隆 + RACE��； 參考：《桂林醫(yī)學(xué)院》2011年碩士論文

【摘要】：第一部分人親磷酸酯酶A1基因的克隆及序列分析 目的克隆人親磷酸酯酶A1(phosphatidic acid-preferring phospholipase A1,PA-PLA1)基因,獲mRNA全長序列,并通過分析所編碼氨基酸的序列特征為從分子水平上研究PA-PLA1生理功能提供理論依據(jù)。方法從人的肝臟組織中提取RNA,逆轉(zhuǎn)錄為cDNA。PCR擴(kuò)增該基因片段,cDNA末端快速擴(kuò)增技術(shù)擴(kuò)增該基因末端,克隆測序后獲得PA-PLA1基因mRNA全長序列。Vector NTI 8.0拼接序列并進(jìn)行序列比對,DNASTAR構(gòu)建系統(tǒng)樹。登錄NCBI用人類基因組數(shù)據(jù)庫做BLAST分析,ORF finder預(yù)測PA-PLA1的編碼框。結(jié)果PA-PLA1基因mRNA全長為2923bp,編碼區(qū)長2238bp,編碼一個(gè)由745個(gè)氨基酸殘基組成的蛋白多肽,分子量為83141道爾頓,等電點(diǎn)為5.64。全序列與人脂肪酶家族序列差異較大,與KIAA0725蛋白及P125蛋白具一定同源性。氨基酸序列中具有與酶活性相關(guān)的保守序列Ser408-His-Ser410-Leu-Gly412, Glu448-Ala475區(qū)可形成卷曲螺旋區(qū)域,且N末端可形成具DDHD結(jié)構(gòu)域。結(jié)論獲得人PA-PLA1基因mRNA全長序列。序列分析研究顯示人PA-PLA1的功能可能與細(xì)胞內(nèi)信號傳導(dǎo)有關(guān)。 第二部分PA-PLA1基因表達(dá)與胰島素分泌關(guān)系的初步研究 目的研究PA-PLA1基因在小鼠胰島β細(xì)胞株MIN6中的表達(dá)與胰島素分泌的關(guān)系。方法以不同濃度及不同時(shí)間分別對MIN6細(xì)胞行葡萄糖刺激胰島素分泌實(shí)驗(yàn),放免法測定細(xì)胞上清液中胰島素分泌量,實(shí)時(shí)熒光定量PCR檢測相應(yīng)條件下PA-PLA1 mRNA的表達(dá)水平,線性相關(guān)分析PA-PLA1 mRNA表達(dá)水平與胰島素分泌的相關(guān)性。結(jié)果在一定葡萄糖濃度范圍內(nèi),PA-PLA1 mRNA水平隨著葡萄糖濃度升高和刺激時(shí)間的延長而升高,并與細(xì)胞胰島素分泌量呈現(xiàn)明顯的正相關(guān),不同葡萄糖濃度和不同刺激時(shí)間的PA-PLA1 mRNA水平與胰島素水平的相關(guān)系數(shù)r分別為0.8839(P0.01)和0.981 (P0.01)。結(jié)論小鼠胰島β細(xì)胞株MIN6中PA-PLA1基因的表達(dá)與胰島素分泌有關(guān)并呈正相關(guān)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.
【學(xué)位授予單位】：桂林醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346

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本文編號：1949173