【作者】 孙桂荣；

【导师】 康相涛；

【作者基本信息】 河南农业大学 ， 动物营养与饲料科学， 2011， 博士

【摘要】 miRNA是近几年发现的对基因表达转录后调控的小分子RNA。目前已经鉴定鸡的miRNA大部分来源于胚胎、体节和干细胞。而对于鸡后期发育相关的miRNA鉴定和表达研究较少。每个miRNA的靶基因的个数很多,同时多个miRNA调控一个基因的表达,他们交叉在一起形成了复杂的网络调控。因此研究miRNA对基因表达调控的影响,分析其与靶基因的关系,必须在机体的整体水平进行研究。本研究对鸡下丘脑不同发育阶段(1日龄和36周龄)固始公鸡进行miRNA和mRNA高通量测序,筛选出差异表达的miRNA和mRNA,并对其表达情况和潜在功能进行分析和预测。同时采用系统生物学的方法将mRNA和miRNA表达谱进行综合分析,以期阐明miRNA对动物采食、生长发育中的调控机制。为采食和生长发育的的研究提供新的思路和有益的线索。本研究获得以下主要结果:实验一、Solexa技术构建鸡下丘脑miRNA表达谱采用solexa技术对1日龄和36周龄鸡下丘脑miRNA进行高通量测序测序,并进行生物信息分析。结果表明1日龄和36周龄组成的小RNA的片段长度主要分布在18~30个核苷酸(nt)的范围内,符合miRNA的长度特点。但是1日龄和36周龄下丘脑组织文库中miRNA长度分布存在差异,在1日龄中长度主要分布为22nt的小RNA占44.35%,其次是23nt的小RNA占26.37%,两者占总序列的70.72%,但是在36周龄中表达量最高的是23nt,占23.06%,其次是22nt的小RNA占26.37%,两者占总序列的49.43%。其他小RNA分布在36周龄和1日龄鸡中差异较大。以1日龄下丘脑组织为对照,在36周龄下丘脑中筛选到179个差异表达miRNA,其差异倍数大多集中在1-2倍。其中22个miRNA上调表达,157个miRNA下调表达。在两个文库中筛选到363个潜在新的候选miRNA,其中85个在两个文库中都有,112和166个分别在1日龄和36周龄中特异表达。179个差异显著的miRNA预测到4362个靶基因,KEGG和GO分析过程中基因大部分参与细胞代谢,和调控细胞代谢有关。实验二、下丘脑发育相关的miRNA的验证及组织时空特异性表达采用定制茎环反转录引物,利用qPCR对miR-21、miR-9、miR-92、miR-181a在下丘脑中的表达进行了定量检测,结果表明qPCR检测的这几个基因的变化倍数与高通量测序检测结果不完全相同,但变化方向一致。而miR-9、miR-181a和miR-92在鸡的同一时期不同组织部位的表达有组织特异性,不同的miRNA的在不同组织中的表达规律不一致。利用TargetScan 5.1与PicTar两种计算方法对miR-9、miR-181a和miR-92进行靶基因预测,交集靶基因有160,137和157个。miR-9靶基因在KEGG通路分析中,显著的通路有细胞骨架调控,细胞增殖和分化有关的通路(P<0.01)等5个通路(P<0.05)。miR-181a的靶基因KEGG分析结果表明,靶基因富集于神经营养因子通路、长时程增强效应通路、II型糖尿病通路、MAPK信号通路、醛固酮调节钠的重吸收、T细胞受体信号通路和卵母细胞减数分裂等与代谢有关的7个通路(P<0.05)。miR-92的靶基因主要富集在长时程增强效应通路、II型糖尿病通路和MAPK信号通路等3个通路(P<0.05)。实验三、构建鸡下丘脑mRNA数字表达谱及初步分析采用solexa技术对1日龄和36周龄鸡下丘脑mRNA进行高通量测序测序,并进行生物信息分析。结果表明在1日龄和36周龄鸡下丘脑中干净有效的tag序列的拷贝数在两个文库中分布规律相似,即随着转录本拷贝数的增多,其所包含的tag的总数和种类越来越少。拷贝数超过100的转录本,在两个文库中分别为4.81%,5.18%。超过超过80%的转录本的拷贝数在2-50个,40%的转录本的拷贝数在2-5个。以1日龄鸡下丘脑组织为对照,在36周龄中共筛差异基因有1143个,其中上调基因413个,下调基因730个,1日龄特异表达的50个,36周龄特异表达的有17个。差异基因KEGG分析表明基因富集的通路大部分与代谢和疾病有关。GO分析表明大部分靶基因与细胞代谢,和调控细胞代谢有关,相关的结果和miRNA靶基因富集的通路相似。实验四、鸡PMCH基因的克隆及生物信息学分析采用RT-PCR, 3’-RACE and 5’-RACE扩增获得PMCH基因cDNA全长803bp(登陆号:HM853641),生物信息学分析表明鸡PMCH有完整的ORF,其长度为492bp,编码163氨基酸,起始密码子ATG位于69bp和终止密码子位于558-560 bp。起始密码子前的5’非翻译区包含68bp,终止密码子后有243bp的3,非翻译区。在3,末端的polyA尾上游20bp处有加尾信号AATAAA,包括3个外显子和2个内含子。与人、小鼠、牛、黑猩猩,和大鼠的核酸相应序列同源性分别为65.95%、62.69%、65.19%、66.54%和59.62%。氨基酸序列的同源性分别为52.07%、50.30%、52.07%、50.89%和50.30%。鸡PMCH基因编码的蛋白为可溶性蛋白。编码蛋白的氨基酸序列存在pro-MCH、CSP,IL7、XPGI和low complexity sequence等5种结构域,含有8个磷酸化位点;同时在鸡PMCH的3’非翻译区预测到3个microRNA靶位点。综上所述,本研究采用新一代测序技术对鸡下丘脑不同发育阶段进行miRNA和mRNA表达谱研究,应用系统生物学的方法分析二者的关系,拓展了鸡已知miRNA的数量;同时构建了miR -9、miR -92和miR -181a的组织表达谱;克隆了鸡PMCH基因并对其的生物学功能进行生物信息分析。更多还原

【Abstract】 MicroRNAs （miRNAs） are small non-coding regulatory RNAs with 19–24 nucleotides （nt）, which play an important role in the post-transcriptional regulation of gene expression during development, and in other biological processes. Most of these miRNAs have been identified from whole embryos, somites and primary fibroblast cells. However, relatively little work has been done to study miRNA expression and miRNA function in the later stages of development in the chicken. miRNA operate highly complex regulatory networks. Current estimates indicate that each miRNA potentially could have hundreds of target genes, and each gene can combine with numerous miRNAs. Former studies mainly focus on the miRNA expression profiling,then predict the target genes, which may not completely illustrate the whole gene regulation network.Therefore, identification of new miRNAs in different organisms and phases is a critical step in the study of the biological functions of miRNAs.In this subject, expression profile of miRNA and mRNA in hypothalamus in different developmental stages chicken are to be studied by solexa sequencing technology. The expermental results are to be verified by real-time quantitative PCR, and the corresponding binding sites of miRNA target genes are to be analyzed through bioinformatics.we explore the gene and miRNAs expression profile in hypothalamus,to illustrate the mechanism of miRNAs and genes involved in hypothalamus.Simultaneously,we try to use the comparative genomics and systems biology to find out the mechanism of the possible interaction between miRNAs and genes, to clarify the regulation mechanism of miRNA in animal growth and development and meat conformation, to find out new target genes for gene engineering breeding in meat quality, to offer new idea and useful clue for molecular improvement in meat quality.1、Differentially expressed miRNAs in two phases of chicken by Deep sequencing In this study, we used Deep sequencing technologies to identify the chicken miRNAs, and we compared the characteristics and expression patterns of miRNA in the hypothalamus of 1-day-old and 36-weeks chickens, and KEGG （Kyoto Encyclopedia of Genes and Genomes） pathway analysis of predicted target genes to determine the global biological functions of different miRNA.The number of RNAs with high-quality reads was different in the two RNA libraries. For example, in the library from 1-day-old chickens, almost half of the high-quality reads （44.35%） were 22nt in length, followed by 23nt （26.37%）; however, in the library from 36-weeks chickens, the length distribution peaked at 23nt （23.06%）, followed by 22nt （20.24%）. The proportions of other small RNA types in the two libraries were significantly different. We found 179 miRNAs that had significant differences in expression between the two libraries （174 miRNA that had more than one-fold difference, p<0.01; 5 miRNA, 0.01<p<0.05）; 157 of them appeared to be down-regulated from the 1-day-old to the 36-weeks chicken, while 22 seemed to be up-regulated in the same order.In this study, we identified 363 potential novel miRNAs that corresponded collectively to 363 independent genomic loci. In total, 85 miRNAs were present in the two libraries, white 112 and 166 miRNAs were expressed specifically in the 1-day-old and 36-weeks hypothalamus, respectively.The 4362 target genes were predicted by RNAhyrid which are involved with KEGG pathway and GO overrepresentation. Target genes were mostly existed in the pathway of the cell and renal cell metabolic and regulation cell metabolic using KEGG functiongal annotations. The gene ontology category indicated that these target genes mainly participate in cellular process,biological regulation, regulation of cellular process, regulation of biological process and metabolic process.2、Expressions of miRNA gene in different developmental stages and tissues The expression profiles of miR-9, miR-181a and miR-92 in 11 normal tissues of two stage chicken were studied by real-time RT-PCR using stem-loop RT-primers. TargetScan 5.1 and PicTar were used to predict the target genes of miR-9, miR-181a and miR-92 and the intersection of the results as gene set was analyzed by KEGG analysis.To confirmation of differentially expressed miRNAs. The real-time RT-PCR were used to confirm the expression pattern of differentially expressed miRNAs in chicken hypothalamus. The change directions of differentially expressed miRNAs between SYBR Green qPCR and deep sequence analysis in four miRNAs was general consistency, although the expressed miRNAs level was different between SYBR Green qPCR and deep sequence analysis. The results of SYBR Green PCR showed that the expressions of miR-9、miR-181a and miR-92 was significantly different in different tissues and different stage.The 160,137,157 target genes were predicted by algorithms PicTar and TargetScan which are involved with KEGG pathway. Target genes of miR-9 were mostly existed in the pathway of the regulation of cytoskeleton and Renal cell carcinoma etc using KEGG functiongal annotations. miR-181a target genes were mainly involved in neurotrophin signaling pathway, long-term potentiation, Aldosterone-regulated sodium reabsorption, T cell receptor signaling pathway and Oocyte meiosis. miR-92 target genes were mainly involved in Long-term potentiation, Type II diabetes mellitus, MAPK signaling pathway, Amino sugar and nucleotide sugar metabolism These experimental results would provide a beneficial reference for further study of miRNA in brain on the regulation in animal metabolic and development.3、Transcriptome profiling of developing chicken by deep-sequencingThe result that the distribution of total and distinct tag counts over different tag abundance categories showed very similar tendencies for two libraries. Among the distinct tags, 4.81% and 5.18% copy number were expressed specifically in the 1-day-old and 36-weeks hypothalamus, respectively. White as 80% of the tags were present between 2 and 50 copies, and more than 40% of the transcripts were 2–5 copies.The differentially expressed mRNA in the hypothalamus of 1-day-old and 36-weeks chickens were screened by deep sequencing. The results showed 1143 mRNAs were down-regulated and 413 mRNAs were up-regulated in 36-weeks hypothalamus. 50 and 17 were expressed specifically in the 1-day-old and 36-weeks hypothalamus, respectively.Different genes were mostly existed in the pathway of metabolic and regulation cell metabolic using KEGG functiongal annotations and ontology category.4、Clone and bioinformatics analysis of chicken PMCH gene In this study, The full-length cDNA was amplified by RT-PCR, 3’-RACE and 5’-RACE from chicken hypothalamus. A cDNA of 803 bp sequence was obtained, and was submitted on GenBank （accession number: HM853641）. The exon 1 of chicken PMCH gene contained the translation start site. The full-length cDNA contained an ORF of 492 bp, which coded for a protein of 163 amino acids. The 5’-untranslated region （-UTR） was 68 bp and was followed by an ATG initiation codon. The stop codon TGA was at the position 558-560 bp. The putative polyadenylation signal AATAAA was found 20 bp upstream from the 14-nucleotide poly （A） tail, which suggested a 243 bp long of 3’-UTR in the chicken PMCH full-length mRNA. The introns and exons were obtained by contig analysis and GT-AG rule. Chicken PMCH gene is composed of 3 exons, which is consistent with those of human and bovine. Its sequence was shorter than the predicted PMCH （XM₄16324.1） at the 5’end. This was due to the mistaken prediction of one of exon1. A nucleotide BLAST search showed that full-length cDNA was homologous to mammalian PMCH genes. It was 65.95%, 62.69%, 65.19%, 66.54% and 59.62% identical to the cDNA sequences of human,mouse,cattle,Pan troglodytes and rat PMCH, respectively. Otherwise, the chicken shared 52.07%, 50.30%, 52.07%, 50.89% and 50.30% homology with human,mouse,cattle,Pan troglodytes and rat in the amino acid sequence of PMHC, respectively.The PMCH protein sequence is predicted to have several functional domains, including pro-MCH,CSP,IL7,XPGI and some low complexity sequence. It has 8 phosphorylation sites and no signal peptide sequence. There is a hydrophobic region in 140-160aa.Ten function motifs has be found in the amino acid sequence. Three predicted microRNA targeting site was found in the 3′untranslated region of chicken PMCH mRNA.In a word, our work study the expression profile of miRNA and mRNA in chicken hypothalamus of different developmental stages by deep sequencing technology, firstly. And we provide a thorough account of the miRNA transcriptome in chicken hypothalamus tissue. Imultaneously, we use the systems biology to study interaction between miRNAs and genes. Meanwhile, our data also provide new insights into the biological role of miR-9, miR-181a and miR-92.We first study to clone and analysized of chicken PMCH gene.更多还原