猪PRMT1基因的克

发布时间：2018-02-03 03:50

本文关键词： 猪 PRMT1 基因克隆表达分析　出处：《信阳师范学院》2017年硕士论文　论文类型：学位论文

【摘要】：蛋白精氨酸甲基转移酶1(protein arginine methyltransferases 1,PRMT1)属于I类PRMTs,其催化活性在蛋白甲基转移酶家族中是最高的,在哺乳动物的胚胎和成年各种器官广泛表达,负责精氨酸的不对称二甲基化。PRMT1具有重要的生物学功能,参与细胞信号转录传导、RNA剪接、DNA修复、转录调控等生物学过程。本研究从猪PRMT1基因的克隆及表达着手,利用小鼠C2C12成肌细胞模型,结合RACE、qRT-PCR、免疫印迹、细胞转染、免疫荧光、过表达等生物学手段,克隆猪PRMT1基因的cDNA全长及启动子序列,并分析其时空表达及对C2C12成肌细胞增殖分化中的影响。主要结果如下:1.猪PRMT1基因的cDNA全长及启动子序列克隆克隆得到猪PRMT1基因的cDNA全长序列1381 bp,包含1116 bp的CDS区,编码372个氨基酸,起始密码子位于25-27 bp处,终止密码子位于1138-1140 bp处,5'端非翻译区共24 bp,3'端非翻译区共241 bp。猪PRMT1为酸性蛋白,蛋白不稳定且是亲水性蛋白。获得包含猪PRMT1基因上游5'侧翼区和外显子1和部分内含子1807 bp启动子序列,该序列上具有Myod1、Myog和MEF2C为骨骼肌中特异表达的转录因子结合元件,包含一个GC含量为65.2%长度为469 bp CpG岛。2.猪PRMT1基因的时空表达分析研究梅山猪胚胎65 d及出生后3 d、4 m的大脑、脾脏、心脏、肺、肝脏、背最长肌不同组织中的表达情况。分析结果表明:PRMT1基因在猪的不同组织中广泛表达,胚胎65 d大脑中表达量最高肝脏中最低;出生后3 d肝脏中表达最高脾脏中最低;出生后4 m大脑中表达最高背最长肌中最低。用qRT-PCR分析猪PRMT1基因在大白猪和梅山猪背最长肌中表达,结果表明:PRMT1基因在大白猪和梅山猪中的表达模式相似,均在胚胎65 d龄高表达,出生后3 d表达量显著减少(P0.05)。两品种相互对比可知:在出生后3 d两品种间背最长肌中长白猪PRMT1的表达极显著高于梅山猪(P0.01),在胚胎期、出生后1 m中的长白猪表达量显著高于梅山猪(P0.05);但在出生后4 m中的长白猪表达量显著低于梅山猪(P0.05)。3.猪PRMT1对C2C12细胞增殖分化的影响增殖和分化的C2C12细胞免疫荧光表明PRMT1主要在细胞质中表达,且在分化的C2C12细胞中表达升高;在C2C12细胞中超表达猪PRMT1基因后,C2C12细胞增殖受到抑制,成肌转录因子MyoG、Mef2a、MyoD及细胞周期调控因子的CDK抑制蛋白Cdkn1a的表达量显著上调(P0.05),成熟骨骼肌相关分子标记物MLC2、MCK的表达量上调但差异不显著(P0.05),细胞周期调控因子Ccnd1与对照相比却显著下调(P0.05)。说明过表达PRMT1后C2C12细胞的增殖受到抑制,并启动了分化,PRMT1对C2C12细胞增殖分化发挥作用可能是通过直接或间接调节成肌转录因子MyoD等表达来实现的。
[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).
【学位授予单位】：信阳师范学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828;Q78

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