马鹿（Cervus elaphus）鹿茸快速生长期生长点软骨和茸皮组织microRNA表达谱研究

发布时间：2018-07-21 10:43

【摘要】：鹿茸是哺乳动物中唯一可以周期性表形态再生的哺乳动物器官,是研究再生医学分子机制和调控模式的良好动物模型。鹿茸是公鹿额部生长的尚未骨化的嫩角,外面被有茸毛,内部是遍布神经和血管的结缔组织和软骨组织。鹿茸每年周期性地生长、脱落、完全再生,具有明显的季节性。晚春和夏季是鹿茸的快速生长期,可达到2cm/d。鹿茸软骨和茸皮组织相互促进共同完成鹿茸的再生,但是生长期鹿茸软骨和茸皮相辅相成快速生长的分子机制尚不清楚。microRNAs (miRNAs)是一类长约22nt、内源性的、转录后水平调控基因表达的非编码的单链small RNA分子,主要在转录后负调控基因表达,通过与靶基因mRNAs 3'非翻译区(3’-UTR)的碱基互补配对来降解mRNAs或者抑制mRNAs翻译,从而发挥沉默特定靶基因的作用。已证实miRNAs广泛参与调节动物、植物和病毒生命过程,其功能几乎涉及生命活动的各个方面。然而关于miRNAs调控鹿茸再生的研究报道极少。我们由此提出如下问题：鹿茸组织中是否有miRNAs的表达?miRNAs在鹿茸不同组织中的表达量如何?研究这些问题可以进一步回答miRNAs与已知的鹿茸再生相关基因之间存在怎样的相互作用关系?miRNAs是否在鹿茸再生过程中发挥重要作用?本研究采集了东北马鹿生长60天时的鹿茸,分离鹿茸软骨和茸皮组织,进行鹿茸软骨和茸皮small RNA文库Solexa深度测序,然后用生物信息学方法对测序数据进行深入挖掘。本文主要研究包括：(1)进行鹿茸软骨和茸皮small RNA高通量测序,为miRNAs分析提供数据；(2)分析鹿茸软骨和茸皮miRNAs,鉴定鹿茸保守和新型miRNAs;(3)分析鹿茸软骨和茸皮miRNAs差异表达情况；(4)预测鹿茸miRNAs二级结构和靶基因,并进行靶基因功能注释,探讨miRNAs可能行使的生物学功能；(5)q-PCR验证测序结果。主要结果如下：1、鹿茸软骨和茸皮small RNA文库进行miRNA Solexa测序后,分别获得13,513,502和5,524,073条原始序列读数(raw reads)。经过各种质量控制程序处理后,得到9,520,645和3,621,894条高质量可比对序列读数(mappable reads),分别鉴定了389和295个miRNAs。2、miRNAs的序列长度主要分布在18-25nt,其中鹿茸软骨组织中序列读数在18nt处达到峰值,茸皮组织中序列读数在22nt处达到峰值,在鹿茸软骨和茸皮文库中分别占到31.5%、18.0%,说明测序结果富含潜在miRNAs。3、筛选出7种类型的684个鹿茸miRNAs,其中611个为哺乳动物保守miRNAs,73个为鹿茸新的候选miRNAs。4、结果显示,所得单一miRNAs中有大量异构体isomiRNAs存在。其中293个保守miRNAs的末端碱基发生变化,碱基增加的数目大于减少的数目,且miRNAs3’端的碱基变化数目大于5’端；94个miRNAs发生碱基替换,包括62个转换和32个颠换。5、鹿茸软骨和茸皮文库miRNAs的表达主要集中在表达量前20位的miRNAs上(均占所有miRNAs总拷贝数的80%以上),其中miR-21在鹿茸软骨中拷贝数最高,miR-127在鹿茸茸皮中拷贝数最高。两个组织间共有168个miRNAs表达存在差异,103个miRNAs表达差异显著。6、首次通过同源比对构建了马鹿鹿茸的miRNAs表达谱。将鹿茸611个保守miRNAs与miRBase (v18.0)数据库中己注释的miRNAs序列进行比对,结果显示鹿茸和牛的保守miRNAs数目最多,达422个,其次为野猪(42)、小鼠(37)、绵羊(35)和人(27)。7、用mfold软件预测了鹿茸miRNAs前体二级结构,均可形成典型的发夹结构。此外,利用TargetScan软件预测了鹿茸软骨和茸皮文库中保守和新发现的miRNAs(共39个)的靶基因,其功能有待进一步研究。8、通过实时荧光定量PCR (RT-PCR)技术验证了鹿茸软骨和茸皮组织中拷贝数差异显著或不显著的14条保守miRNAs在两个文库中的表达情况。结果表明,q-PCR的数据与测序数据基本一致。本次测序结果真实可靠,能够反映鹿茸软骨和茸皮miRNAs表达谱数据。本研究从miRNA调控鹿茸生长发育的角度出发,通过高通量测序技术获得快速生长期鹿茸软骨和茸皮组织的miRNAs表达谱,分析其序列特征,检测miRNAs在鹿茸快速生长期不同组织中的表达水平,筛选与鹿茸再生相关的miRNAs。马鹿鹿茸软骨和茸皮miRNAs的鉴定与功能研究将促进鹿茸再生分子机制的研究。为进一步研究鹿茸miRNAs的功能、基因调控机制和探讨miRNAs在鹿茸再生过程中的生物学功能提供重要数据参考。
[Abstract]:Antler is the only mammalian organ in mammals that can reproduce periodically in mammals. It is a good animal model to study the molecular mechanism and regulation mode of regenerative medicine. Deer antler is a tender corner of the frontal part of the male deer, with its hair outside and the connective tissue and cartilage tissue all over the nerve and blood vessels. The growth, exfoliation, complete regeneration and obvious seasonal characteristics. Late spring and summer are the rapid growth period of antler antler, which can reach 2cm/d. deer antler cartilage and skin tissue to promote the regeneration of antler. However, the molecular mechanism of.MicroRNAs (miRNAs) is not clear. The non coding single strand small RNA molecules with 22nt, endogenous and post transcriptional levels regulate gene expression, mainly in post transcriptional negative regulatory gene expression, and by complementing the bases of the target gene mRNAs 3'non translation region (3' -UTR) to degrade mRNAs or inhibit mRNAs translation, thus playing the role of silence specific target genes. NAs is widely involved in regulating the life processes of animals, plants and viruses, and their functions are almost involved in all aspects of life activities. However, there are few reports on miRNAs regulation of antler regeneration. We have raised the following questions: is there a miRNAs expression in the deer antler tissue? How does miRNAs Express in different tissues of deer antler? The question can be further answered by the interaction between miRNAs and the known antler regeneration related genes. Is miRNAs playing an important role in the process of antler regeneration? This study collected the antler of 60 days in the growth of northeastern red deer, separated the antler cartilage and the velvet skin, and carried out the pilose antler cartilage and the small RNA Library of the velvet skin Solex A deep sequencing, and then using bioinformatics methods to dig into the sequencing data. The main research includes: (1) high throughput sequencing of antler cartilage and small RNA to provide data for miRNAs analysis; (2) analysis of pilose antler cartilage and miRNAs, identification of antler conservative and new miRNAs; (3) analysis of pilose antler cartilage and velvet skin miRNAs Difference expression; (4) predict the miRNAs two structure and target gene of antler antler, and carry out the target gene function annotation, explore the possible biological function of miRNAs; (5) q-PCR verification sequencing results. The main results are as follows: 1, the antler cartilage and the small RNA Library of the velvet skin were sequenced by miRNA Solexa, and 13513502 and 5524073 were obtained. The initial sequence readings (raw reads). After a variety of quality control programs, 9520645 and 3621894 high quality comparable sequence readings (mappable reads) were obtained, and 389 and 295 miRNAs.2 were identified respectively. The sequence length of miRNAs was mainly distributed in 18-25nt, and the sequence readings in the cartilage tissue of the deer antler reached the peak value at 18nt. The middle sequence readings reached the peak at 22nt, which accounted for 31.5%, 18% in the deer antler cartilage and the velvet skin library respectively, indicating that the sequencing results were rich in potential miRNAs.3, and 7 types of 684 deer antler miRNAs were screened, of which 611 were mammalian conserved miRNAs and 73 were new candidate miRNAs.4 of antler. The results showed that a large number of heterogeneous miRNAs were heterogeneous in the single miRNAs. Body isomiRNAs exists. The terminal base of 293 conservative miRNAs changes, the number of base increase is greater than the number of decrease, and the number of base changes of miRNAs3 'end is greater than 5' end; 94 miRNAs has base substitutions, including 62 conversion and 32 transformation.5. The expression of miRNAs in antler cartilage and skin library mainly concentrates on the expression In the first 20 miRNAs (all 80% of all miRNAs copies), the number of miR-21 in antler cartilage is the highest, and miR-127 has the highest copy number in the pilose antler skin. There are 168 miRNAs expressions in two tissues, and 103 miRNAs expressions with significant difference.6. The miRNAs expression of the deer antler is constructed by homologous comparison for the first time. The comparison was made between the 611 conservative miRNAs and the miRBase (V18.0) database of the deer antler. The results showed that the conservative miRNAs number of the deer antler and the cow was the most, up to 422, followed by the wild boar (42), the mice (37), the sheep (35) and the human (27).7, and the mfold software was used to predict the two structure of the antler miRNAs precursor, which could form a typical hairpin. In addition, TargetScan software was used to predict the conservative and newly discovered miRNAs (39) target genes in the antler cartilage and the velvet skin library. The function needs to be further studied by.8. Through real-time fluorescence quantitative PCR (RT-PCR) technology, the 14 conservative miRNAs, which have significant or not significant difference in the number of torture shells in the pilose antler cartilage and the velvet skin tissue, are verified to be two. The results show that the q-PCR data are basically consistent with the sequencing data. The results are true and reliable, and can reflect the miRNAs expression data of the antler cartilage and the velvet skin. From the perspective of the growth and development of the deer antler growth and development, this study obtained the rapid growth stage of the antler cartilage and the skin tissue by high throughput sequencing technology. MiRNAs expression profile, analysis of its sequence characteristics, detection of the expression level of miRNAs in different tissues in the rapid growth period of antler antler, the identification and function study of miRNAs. deer antler cartilage and skin miRNAs related to antler regeneration will promote the study of the molecular mechanism of antler regeneration. In order to further study the function of antler miRNAs, gene regulation and regulation will be further studied. The mechanism and discussion of miRNAs provide important data for reference in the biological function of velvet antler regeneration.
【学位授予单位】：东北林业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q953

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