本文關(guān)鍵詞： 豬 PRMT1 基因克隆 表達(dá)分析　出處：《信陽師范學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：蛋白精氨酸甲基轉(zhuǎn)移酶1(protein arginine methyltransferases 1,PRMT1)屬于I類PRMTs,其催化活性在蛋白甲基轉(zhuǎn)移酶家族中是最高的,在哺乳動物的胚胎和成年各種器官廣泛表達(dá),負(fù)責(zé)精氨酸的不對稱二甲基化。PRMT1具有重要的生物學(xué)功能,參與細(xì)胞信號轉(zhuǎn)錄傳導(dǎo)、RNA剪接、DNA修復(fù)、轉(zhuǎn)錄調(diào)控等生物學(xué)過程。本研究從豬PRMT1基因的克隆及表達(dá)著手,利用小鼠C2C12成肌細(xì)胞模型,結(jié)合RACE、qRT-PCR、免疫印跡、細(xì)胞轉(zhuǎn)染、免疫熒光、過表達(dá)等生物學(xué)手段,克隆豬PRMT1基因的cDNA全長及啟動子序列,并分析其時(shí)空表達(dá)及對C2C12成肌細(xì)胞增殖分化中的影響。主要結(jié)果如下:1.豬PRMT1基因的cDNA全長及啟動子序列克隆克隆得到豬PRMT1基因的cDNA全長序列1381 bp,包含1116 bp的CDS區(qū),編碼372個氨基酸,起始密碼子位于25-27 bp處,終止密碼子位于1138-1140 bp處,5'端非翻譯區(qū)共24 bp,3'端非翻譯區(qū)共241 bp。豬PRMT1為酸性蛋白,蛋白不穩(wěn)定且是親水性蛋白。獲得包含豬PRMT1基因上游5'側(cè)翼區(qū)和外顯子1和部分內(nèi)含子1807 bp啟動子序列,該序列上具有Myod1、Myog和MEF2C為骨骼肌中特異表達(dá)的轉(zhuǎn)錄因子結(jié)合元件,包含一個GC含量為65.2%長度為469 bp CpG島。2.豬PRMT1基因的時(shí)空表達(dá)分析研究梅山豬胚胎65 d及出生后3 d、4 m的大腦、脾臟、心臟、肺、肝臟、背最長肌不同組織中的表達(dá)情況。分析結(jié)果表明:PRMT1基因在豬的不同組織中廣泛表達(dá),胚胎65 d大腦中表達(dá)量最高肝臟中最低;出生后3 d肝臟中表達(dá)最高脾臟中最低;出生后4 m大腦中表達(dá)最高背最長肌中最低。用qRT-PCR分析豬PRMT1基因在大白豬和梅山豬背最長肌中表達(dá),結(jié)果表明:PRMT1基因在大白豬和梅山豬中的表達(dá)模式相似,均在胚胎65 d齡高表達(dá),出生后3 d表達(dá)量顯著減少(P0.05)。兩品種相互對比可知:在出生后3 d兩品種間背最長肌中長白豬PRMT1的表達(dá)極顯著高于梅山豬(P0.01),在胚胎期、出生后1 m中的長白豬表達(dá)量顯著高于梅山豬(P0.05);但在出生后4 m中的長白豬表達(dá)量顯著低于梅山豬(P0.05)。3.豬PRMT1對C2C12細(xì)胞增殖分化的影響增殖和分化的C2C12細(xì)胞免疫熒光表明PRMT1主要在細(xì)胞質(zhì)中表達(dá),且在分化的C2C12細(xì)胞中表達(dá)升高;在C2C12細(xì)胞中超表達(dá)豬PRMT1基因后,C2C12細(xì)胞增殖受到抑制,成肌轉(zhuǎn)錄因子MyoG、Mef2a、MyoD及細(xì)胞周期調(diào)控因子的CDK抑制蛋白Cdkn1a的表達(dá)量顯著上調(diào)(P0.05),成熟骨骼肌相關(guān)分子標(biāo)記物MLC2、MCK的表達(dá)量上調(diào)但差異不顯著(P0.05),細(xì)胞周期調(diào)控因子Ccnd1與對照相比卻顯著下調(diào)(P0.05)。說明過表達(dá)PRMT1后C2C12細(xì)胞的增殖受到抑制,并啟動了分化,PRMT1對C2C12細(xì)胞增殖分化發(fā)揮作用可能是通過直接或間接調(diào)節(jié)成肌轉(zhuǎn)錄因子MyoD等表達(dá)來實(shí)現(xiàn)的。
[Abstract]:Protein arginine methyltransferase 1 protein arginine methyltransferases 1 (PRMT1) belongs to class I PRMTs. Its catalytic activity is the highest in the proteomethyltransferase family and is widely expressed in mammalian embryonic and adult organs. PRMT1, which is responsible for asymmetric dimethylation of arginine, plays an important biological role in RNA splicing and DNA repair. This study was based on the cloning and expression of porcine PRMT1 gene, using mouse C2C12 myoblast model, combined with RACE-qRT-PCR, Western blot. The full-length and promoter sequences of porcine PRMT1 gene were cloned by cell transfection, immunofluorescence, overexpression and other biological methods. The temporal and spatial expression of C2C12 and its effect on the proliferation and differentiation of C2C12 myoblast were analyzed. The main results were as follows:. 1. The full-length cDNA and promoter sequence of porcine PRMT1 gene were cloned and cloned. The full-length cDNA sequence of porcine PRMT1 gene was 1381bp. The CDS region contains 1116 BP and encodes 372 amino acids. The initial codon is located at 25-27 BP and the termination codon is located at 1138-1140 BP. The 5'-terminal untranslated region was 241bp.Porcine PRMT1 was acidic protein. The protein was unstable and hydrophilic. The 5'flanking region and exon 1 and partial intron 1807 BP promoter sequence of porcine PRMT1 gene were obtained, which contained Myod1. Myog and MEF2C are transcription factor binding elements specifically expressed in skeletal muscle. Analysis of Spatio-temporal expression of porcine PRMT1 Gene in Meishan Pig embryo at 65 days and 3 days after birth. Four meters of brain, spleen, heart, lung, liver and longissimus dorsi muscle were expressed in different tissues. The highest expression level was found in the brain of the embryo at day 65, and the lowest in the liver. The highest expression of spleen was the lowest in the liver 3 days after birth. The expression of PRMT1 gene in the longissimus dorsi muscle was detected by qRT-PCR analysis in the longissimus dorsi muscle of white pigs and Meishan pigs. The results showed that the expression pattern of the 1% PRMT1 gene was similar in white pigs and Meishan pigs, both of which were highly expressed at the age of 65 days. The expression of PRMT1 in longissimus dorsi was significantly higher in longissimus dorsi than in Meishan pig 3 days after birth. P0.01). At embryonic stage, the expression level of Landrace in 1 m postnatal was significantly higher than that in Meishan P0.05; But the expression level of Landrace pigs at 4 m after birth was significantly lower than that in Meishan pigs (P0.05). Effect of porcine PRMT1 on proliferation and differentiation of C2C12 cells. Immunofluorescence of C2C12 cells showed that PRMT1 was mainly expressed in the cytoplasm of C2C12 cells. And the expression was increased in differentiated C2C12 cells. After overexpression of porcine PRMT1 gene in C2C12 cells, the proliferation of C2C12 cells was inhibited and myogenic transcription factor MyoGnMef2a was observed. The expression of CDK inhibitory protein Cdkn1a in MyoD and cell cycle regulator was significantly up-regulated by P0.05, and the mature skeletal muscle related molecular marker MLC2. The expression of MCK was up-regulated, but the difference was not significant (P0.05). Compared with the control group, the cell cycle regulator Ccnd1 significantly down-regulated P0.05, which indicated that the proliferation of C2C12 cells was inhibited after overexpression of PRMT1, and the differentiation of C2C12 cells was initiated. The effect of PRMT1 on the proliferation and differentiation of C2C12 cells may be mediated by direct or indirect regulation of the expression of muscle transcription factor (MyoD).
【學(xué)位授予單位】：信陽師范學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S828;Q78

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本文編號：1486335