【作者】 王艳红；

【导师】 陈宏；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2015， 博士

【摘要】 动物骨骼肌形成和发育机制一直是研究的热点,有关骨骼肌生长和发育的基因调控已有很多报道,且主要集中在检测单个基因的表达或者单个通路的功能。然而,骨骼肌生长发育的表观遗传学调控或者转录后调控报道相对较少。近年来,mi RNA越来越成为研究热点,mi RNA在转录后水平调控基因表达,并且在各种生物过程中发挥着重要作用。尽管肌肉mi RNAs表达谱在多种动物上已有报道,山羊肌肉组织mi RNA表达谱以及mi RNA功能报道还比较少并且不全面。徐淮山羊具有性早熟、产仔多、板皮质地优良、耐粗饲等特点,是黄淮地区的主要肉羊品种。为了揭示mi RNAs对不同发育阶段徐淮山羊骨骼肌发育的调控作用,本研究利用高通量测序技术首先揭示了山羊不同发育期肌肉mi RNA转录组和m RNA转录组,整合这两个转录组结果,得到mi RNA调控肌肉发育的网络,并从中初步筛选出部分肌肉发育相关mi RNA。进一步研究了这些mi RNA的遗传变异与山羊生产性状的关联性,并结合它们表达的组织特异性,进一步确定mi R-424-5p为主要候选mi RNA。并结合生物信息学和细胞生物学方法筛查了mi R-424-5p的靶基因,初步揭示其作用机制。主要获得了以下研究结果:1、徐淮山羊背最长肌小RNA测序及mi RNA的鉴定本研究构建了徐淮山羊90天胎羊和6月龄青年羊肌肉组织小RNA文库并利用新一代高通量测序技术进行了测序。对文库进行生物信息学分析,发现两文库中小RNA在组成、种类和长度分布上有显著差异。通过与mi Rbase数据库比对,共获得464个已知mi RNA,可以归为270个mi RNA家族,大部分的mi RNA家族在物种之间都是保守的。对已知mi RNA进行碱基偏好性分析发现首位碱基和第9位碱基U出现的频率最高。通过比较两个发育阶段的mi RNA表达谱,检测到336个差异表达的mi RNA,其中317个mi RNA表达量在青年山羊组织中显著下调,这说明肌肉组织中的mi RNA表达量处在动态变化中。同时利用mi RNA所特有的发夹结构预测出83个新mi RNA,其中有40个差异表达,与胎羊肌肉组织文库相比,青年羊肌肉组织文库中有33个新mi RNA表达量下调。2、徐淮山羊背最长肌转录组的测定利用高通量测序技术测定了徐淮山羊90天胎羊和六月龄青年羊肌肉组织m RNA转录组。通过分析,在胎羊文库和青年羊文库中分别检测到15,960和14,981个基因。比较两个文库中基因表达量的差异,发现6432个基因在两文库中显著差异表达。对差异表达的基因进行GO分析,发现差异基因显著富集到分子功能(34个)、细胞组成(27个)和生物进程(55个)中。同时,差异基因共比对上256个KEGG条目,其中41个差异显著。大量的差异基因富集到胚胎期肌生成、细胞增殖和分化相关通路(如WNT),且参与这些通路的大部分基因都在青年羊肌肉组织中下调。在两个组织中共检测到743个新转录本,其中37.8%具有编码能力。同时,本研究中共检测到4种可变剪接,即外显子跳读,内含子保留,可变的5’端,可变的3’端。3、mi RNA转录组谱和m RNA转录组谱整合分析整合两个发育阶段肌肉组织mi RNA转录组和m RNA转录组分析结果,将差异mi RNA靶基因预测结果同m RNA转录组中与对应mi RNA表达呈负相关的差异基因进行比对取其交集,得到差异靶基因。对差异靶基因进行KEGG分析,筛选出10个差异最显著的通路,再从这些通路中筛选出12个mi RNA,即mi R-668-3p、mi R-323a-3p、mi R-665-3p、mi R-541-3p、mi R-411c-5p、mi R-129-3p、let-7e、mi R-424-5p、mi R-432、mi R-495-3p、mi R-767、mi R-29a,这些mi RNA参与多条通路,且位于节点处。进一步分析发现,mi R-424-5p在这10个通路中都存在,并且处于网络的多个节点上,因此在肌肉发育过程中可能发挥重要作用,作为下一步研究的主要候选mi RNA。4、肌肉发育相关mi RNA组织差异表达谱测定从前述调控网络中筛选出的12个肌肉发育相关mi RNA中选出10个,另外再筛选10个预测在肌肉中表达量最高的新mi RNA进行组织(背最长肌、心、肝、脾、肺、肾、肠)表达谱研究。结果表明,mi R-129-3p、mi R-424-5p、mi R-432和mi R-665-3p在肌肉中相对表达量较高,其中mi R-424-5p和mi R-432在两个发育阶段肌肉组织中表达量显著高于其他组织,表现出很高的组织特异性。检测的10个新mi RNA中,Novelmi R-47在肌肉组织中特异性表达。5、肌肉发育关键mi RNA前体基因遗传变异分析对于筛选出的mi RNA进一步进行了遗传变异分析。我们共检测了20个mi RNA在三个山羊品种(徐淮山羊、海门山羊及波尔山羊)共计270个个体中的遗传变异。结果发现,检测的mi RNA前体基因中,只有5个发现了变异位点,其余14个均未发现变异位点。在5个多态基因座中,mi R-424-5p、mi R-411c-5p、Novelmi R-47和mi R-541-3p的多态位点均位于其前体序列之外。将5个基因多态性与生长性状关联分析发现,mi RNA-424-5p多态性与三个品种山羊体高和胸围显著相关(P<0.05),提示它在山羊发育过程中有重要作用。6、mi RNA-424-5p靶基因的验证综合第四章和第五章的结果,结合现有肌肉发育调控的研究进展,在预测的mi R-424-5p靶基因中,选定AKT3、SMAD3和CHEK1三个肌肉发育关键基因作为mi R-424-5p的候选靶基因,利用双荧光素酶报告基因法对其进行验证。构建了山羊mi R-424-5p过表达载体,并构建了分别含有AKT3、SMAD3和CHEK1 3’UTR及其对应mi R-424-5p靶点缺失突变体的萤火虫荧光素酶报告基因载体。检测结果显示,mi R-424-5p不与SMAD3和CHEK1的3’UTR结合,不影响报告基因荧光素酶的活性(P>0.05),它们不是mi R-424-5p的靶基因;mi R-424-5p显著抑制含AKT3 3’UTR的报告基因荧光素酶的活性(P<0.01),AKT3可确定为mi R-424-5p的靶基因。7、mi RNA-424-5p对肌肉生长发育调控研究本研究将mi R-424-5p过表达载体(pc DNA3.1-pri-mi R-424-5p)转染C2C12细胞后分别用生长培养基(GM)或分化培养基(DM)培养,然后对C2C12细胞中的mi RNA-424-5p以及肌肉细胞增殖(Ki67)和分化标志基因(MHC、Mef2c、Myo D和Myf5)的表达情况进行定量检测。结果表明mi RNA-424-5p在C2C12细胞中成功过表达且差异极显著(P<0.01)。与对照组相比,转染了pc DNA3.1-pri-mi R-424-5p质粒的C2C12细胞中Mef2c和Myo D表达量上升且差异极显著(P<0.01),Myf5表达量也显著上升(P<0.05),说明mi RNA-424-5p对C2C12细胞分化有促进作用。更多还原

【Abstract】 Goat is an important agricultural animal for meat production. Knowledge about the molecular developmental mechanisms of skeletal muscle formation is of vital interest. However, over the past few years, studies on skeletal muscle growth and development of goat were limited, and mainly examined the expression and function(s) of single genes or single developmental pathways. Functional studies have demonstrated that micro RNAs(mi RNAs) regulate gene expression at the post-transcriptional level and play an important role in various biological processes. Although studies on mi RNAs expression profiles have been performed in various animals, relatively limited information about goat muscle mi RNAs has been reported. To investigate the mi RNAs involved in regulating different periods of skeletal muscle development, global identification of the known and novel mi RNAs involved in goat skeletal muscle development is essential. In addition, the m RNA profiles were analyzed in this study. Based on these, we briefly analyzed the regulation network of mi RNA-m RNA, and the regulatory mechanism of some muscle-related mi RNA, which will significantly enhance our understanding the effect of mi RNAs on muscle development for goat.The main results are as follow: 1.Identification and profiling of micro RNAs from developing caprine skeletal muscleTwo small RNA libraries from fetal goat and six month-old goat muscle tissue were constructed using Solexa high-throughput technology. 464 known mi RNAs and 83 novel mi RNA candidates were identified. Characterization of the frequency of the position-specific nucleotide composition along the length of mi RNA revealed that nucleotide positions 1 and 9 are significantly enriched for U relative to other bases. Family analysis showed that most of the identified mi RNA families have been shown to be conserved in a variety of species. Furthermore, by comparing the mi RNA profile, 336 differentially expressed mi RNAs were identified. Most of the differentially expressed mi RNAs were higher expressing in fetal goat muscle tissue, suggest that the skeletal muscle mi RNAs composition is dynamically altered during the different development stages. By comparing the novel mi RNA profile, 40 differentially expressed novel mi RNAs were identified, among which 33 were down regulated in juvenile goat muscle tissue. 2.Global transcriptional profiling of longissimus thoracis muscle tissue in fetal and juvenile domestic goat using RNA sequencingWe used comparative transcriptional profiling based on RNA sequencing of longissimus thoracis muscle tissue obtained from fetal and six month-old domestic goats to identify genes that are differentially expressed. Gene annotation revealed that 15,960 and 14,981 genes were expressed in the fetal and six month-old goat libraries, respectively. We detected 6,432 differentially expressed genes, and when considering GO-terms found 34, 27 and 55 terms to be significantly enriched in molecular function, cellular component, and biological process, respectively. Pathway analysis revealed that differentially expressed genes were enriched in 256 KEGG terms, among which 41 were significantly enriched. Further more, large numbers of differentially expressed genes enriched in embryonic myogenesis or cell proliferation and differentiation-related pathways(such as WNT), and most of the genes involved in these pathways were down-regulated in the six month-old goats library. The identified novel transcript units, including both non-coding and coding RNAs, as well as alternative splicing events increase the level of complexity of regulation mechanisms during muscle tissue formation and differentiation. 3.Integration of mi RNA profiling and m RNA profiling analyses reveals network of mi RNA regulationAfter small RNA sequencing and RNA-Seq(Transcriptome) analysis of the paired samples, the integrated analysis of differentially-expressed mi RNAs and their predicted target genes were performed. The result showed that most individual mi RNAs had multiple gene targets and each target was regulated by multiple mi RNAs. According to mi RNA-m RNA-KEGG annotation, 10 pathways with the smallest P-value were screened. It was very interesting that among the 7 networks, mi R-424-5p were very active in that it regulates largest numbers of DE-targets and at the center of the networks. 4.Analysis of expression profiles of muscle-related mi RNAThrough integrated analysis the mi RNA profile and m RNA profile, we obtained the regulatory network for differentially expressed mi RNA and differentially expressed genes. We screened out some muscle-related mi RNAs which in the node of the net, analysed their expression profiles, including some novel mi RNA. Result showed that mi R-129-3p, mi R-424-5p, mi R-432 and mi R-665-3p were higher expressed in muscle tissue compared to other tissues or organs, of which mi R-424-5p and mi R-432 were the highest expressing in muscle tissue on both the two development stages. We detected that Novelmi R-47 is muscle tissue specificity expressed. 5.Genetic variations analysis of muscle-related mi RNAsGenetic variations of 20 above-mentioned muscle-related mi RNAs were detected by PCR-SSCP and DNA sequencing techniques in 270 individuals of three populations(Chinese Haimen goat, Chinese Xuhuai white goat and Boer goat). Result showed that 5 of the 20 detected mi RNAs have SNPs. Of the 5 polymorphic loci in this study, the polymorphic loci of mi R-411c-5p, mi R-424-5p, Novelmi R-47 and mi R-541-3p were located in the precursor sequence, Only mi RNA-767 polymorphic site is located in the pre-mi RNAs. Polymorphic information content distribution was the same in the three species, only different in different locus. Besides mi RNA-411c-3p, the other four sites were moderately polymorphic. 6.Verification of target genes for the mi R-424-5pWe construct the overexpression vector of mi R-424-5p(pc DNA3.1-pri-mi R-424-5p). Then we predict the target genes of the mi R-424-5p using bioinformatics software, namely AKT3, SMAD3, CHEK1, and construct the expression vector of the 3’UTR for the three target genes: wild type(p GL3-control-AKT3/SMAD3/CHEK1) and mutant type(p GL3-control-mut-AKT3/SMAD3/CHEK1). Moreover, the three predicted target genes of mi R-424-5p were verified using dual fluorescence report assay experiment. The results indicated that AKT3 gene is a target of mi R-424-5p. 7.The regulating effect of mi RNA-424-5p to muscle cellIn this study, C2C12 cells were cultured in growth medium(GM) or differential medium(DM) after transfected with pc DNA3.1-pri-mi R-424-5p. Then expression of marker genes of cell proliferation(Ki67) and muscle differentiation(MHC, Mef2 c, Myo D and Myf5) was analyzed by q RCR. The results showed that Myo D,Myf5 and Mef2 c were up-regulated and the difference was significant or extremely significant(P<0.05 or P<0.01). That means mi R-424-5p plays a positive role in the differentiation of C2C12 cells.更多还原