【作者】 孙伟；

【导师】 雷初朝；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2013， 硕士

【摘要】 microRNA(miRNA)是一类进化保守的大小在18-25个核苷酸的内源性非编码小分子RNA,广泛存在动植物体内,对机体的生长发育、衰老和死亡等生物过程起着重要的作用。研究表明microRNA在骨骼肌的生长发育过程中发挥着重要调控作用。本文以在骨骼肌中特异性表达的microRNA-1和microRNA-206为主要研究对象,筛选其靶标基因,并研究它们在猪骨骼肌发育中的作用。本研究的主要结果如下：1.利用TargetScan和PicTar等生物信息学软件预测miRNA-1和miRNA-206的靶标基因,结果发现有258个候选靶标基因。利用DAVID数据库对258个候选靶标基因进行GO和KEGG pathway分析,筛选出与肌肉发育相关的分泌型卷曲相关蛋白1(SFRP1)和分泌型卷曲相关蛋白2(SFRP2)基因为miRNA-1和miRNA-206的共有的候选靶标基因作为本论文研究重点。2.拼接猪SFRP1的mRNA序列,扩增SFRP1和SFRP2基因mRNA的3’UTR序列。以Psi-Check2作为骨架载体,构建Psi-Check2-SFRP1和Psi-Check2-SFRP2载体,与体外合成的miRNA-1mimics和miRNA-206mimics共转染PIEC细胞36h,并进行双萤光素酶检测。结果发现,SFRP1和SFRP2基因实验组的萤光素酶表达量比对照组均显著下调(P<0.01),表明miRNA-1和miRNA-206可能通过SFRP1和SFRP2基因3’UTR的结合位点相互作用,抑制SFRP1和SFRP2基因的表达。3.利用桥联PCR技术对猪SFRP1基因的结合位点进行突变,并以Psi-Check2作为骨架载体,构建SFRPl基因突变载体,转染PIEC细胞36h,并进行双萤光素酶检测。结果发现,与对照组相比,突变载体的双萤光素酶的活性没有显著变化(P>0.05),表明SFRPl基因的这个突变位点就是miRNA-1和miRNA-206的靶标结合位点。4.由于SFRP1在PIEC细胞中表达量较低,体外扩增SFRP1的CDS区域,构建Psi-Check2-SFRP1CDS-SFRP13’UTR载体,与体外合成的miRNA-1mimics和miRNA-206mimics共转染PIEC细胞36h,收集细胞,检测SFRP1基因在RNA和蛋白质水平的表达。实时荧光定量PCR(real-time PCR)结果表明,实验组miRNA-1和miRNA-206的表达量与对照组没有显著差异(P>0.05)；Western blot结果表明,实验组(转染SFRP1过表达载体和miRNA-1和miRNA-206minics)比对照组的SFRP1蛋白表达水平显著降低,表明SFRP1是miRNA-1和miRNA-206的共同靶标基因,且miRNA-1和miRNA-206主要在翻译水平下调SFRP1的表达。5.利用real-time PCR方法,检测SFRP1和SFRP2基因在通城成年母猪组织(9个组织),以及通城猪(27个时间点)和长白猪(23个时间点)骨骼肌生长发育过程中的表达量。结果显示：SFRP1在肺、肾、小肠和胃中高度表达,在大肠、肝和脾中度表达,在心脏、背肌和腿肌中低度表达；SFRP2在胃、小肠、肺和大肠中表达较高,在背肌和腿肌中中度表达,在心和脾中则是低度表达。SFRP1和SFRP2基因在通城猪和长白猪胚胎期的骨骼肌的表达量均显著高于出生后的表达量,表明SFRP1和SFRP2基因可能主要参与骨骼肌初级和次级纤维的形成。SFRP1和SFRP2基因在通城猪和长白猪两个品种间肌肉发育过程中的表达模式并不完全一致,推测SFRP1和SFRP2可能与不同猪品种间的肌肉表型差异有关。更多还原

【Abstract】 MicroRNAs (miRNAs), widely existed in plants and animals, were a class of evolutionarily conserved endogenous non-coding small RNA molecules, about18-25nucleotides in length. They played an important role in organism growth and development, aging and death process. Previous studies had shown that microRNAs had an important regulatory function in the skeletal muscle growth and development process. In this study, we focused on microRNA-1and microRNA-206which were specific expressed in skeletal muscle as the main object to identify their target genes and explore the function of these two miRNAs in skeletal muscle development. The results of our study are following:1. The target genes of miRNA-1and miRNA-206were predicted by Two bioinformatics software, TargetScan and PicTar. Results showed that there were258target genes could be predicted by both prediction programs. GO and KEGG pathway analysis of the258predicted target genes were performed using the DAVID database. Two targeted genes, Secreted Frizzled-Related Protein1(SFRP1) and Secreted Frizzled-Related Protein2(SFRP2) genes which were related to skeletal muscle development, were selected as the candidate target genes for further research.2. The mRNA sequence of porcine SFRP1gene was assembled and the mRNA3’UTR sequences of SFRP1and SFRP2were amplified. With Psi-Check2as the skeleton vector to construct the Psi-Check2-SFRP1and Psi-Check2-SFRP2vectors, PIEC cells were co-transfected with these two vectors and miRNA-1or miRNA-206mimics, respectively. After transfection for36h, the activity of Dual-Luciferase were detected. The results showed that compared with the negative control group, the Dual-Luciferase activity of the transfected miRNA-1mimics or miRNA-206mimics group were significantly decreased (P<0.01), and indicated that the seed sequence of miRNA-1and miRNA-206might be interaction with the3’UTR binding sites of the SFRP1and of SFRP2genes, and could inhibit the expression of these two genes.3. The bridging PCR method was used to knockout the miRNA-1and miRNA-206binding sites of SFRP13’UTR which was predicted by bioinformatics methods. With Psi-Check2as the skeleton vector to construct the Psi-Check2-SFRP1mutation vector, after had transfected PIEC cells for36h, the activity of Dual-Luciferase were detected. The results showed that compared with the NC control group, the Dual-Luciferase activity of mutation vector had no significantly changed, and indicated that this site might be the target binding site of miRNA-1and miRNA-206.4. Because the expression level of SFRP1gene in PIEC cells was very low, the CDS of porcine SFRP1gene was amplified, and the Psi-Check2-SFRP1CDS-SFRP13’UTR vector was constructed. PIEC cells were co-transfected with this vector and miRNA-1or miRNA-206mimics. After had transfected for36h, the mRNA and protein were extracted to detect the mRNA and protein level of SFRP1. The real-time quantitative PCR (RT-PCR) results showed that compared with the NC group, the SFRP1mRNA expression levels (co-transfected the SFRP1over-expression vector and miR-1/206mimics) had no significant difference (P>0.05). The Western-blot analysis showed that compared with the NC group, SFRP1protein expression levels at the experimental group were significantly decreased. It was indicated that SFRP1was the common target of miRNA-1and miRNA-206and the expression of SFRP1were mainly regulated by these two mircoRNAs at the translate level.5. The spatial-temporal expression profiles of SFRP1and SFRP2genes were carried out using real-time PCR. The spatial expression levels of9different tissues in adult Tongcheng pigs and the temporal expression levels in developmental skeletal muscle at Tongcheng (27time points) and Landrace pigs (23time points) were detected. The results showed that the SFRP1gene were highly expressed in the small intestine, stomach, kidney and lung, moderately expressed in liver, the large intestine, spleen and weakly expressed in the Longissimus muscles and leg muscles. And the SFRP2gene were abundantly expressed in the stomach, lung, colon and small intestine, moderately expressed in the Longissimus muscles and leg muscles and weakly expressed in the heart and spleen. The expression levels of SFRP1and SFRP2genes in the embryonic skeletal muscle development stages were significantly higher than that in the postnatal stages birth in both Tongcheng pigs and Landrace pigs. These results indicated that SFRP1and SFRP2genes may involved in the formation of primary and secondary fibers of the skeletal muscle. Between Tongcheng and Landrace pigs, the expression pattern of SFRP1and SFRP2showed significantly differences. It suggested that SFRP1and SFRP2might be related to the muscle phenotypic differences between these two pig breeds.更多还原