【摘要】：鹿茸是哺乳動物中唯一可以周期性表形態(tài)再生的哺乳動物器官,是研究再生醫(yī)學(xué)分子機(jī)制和調(diào)控模式的良好動物模型。鹿茸是公鹿額部生長的尚未骨化的嫩角,外面被有茸毛,內(nèi)部是遍布神經(jīng)和血管的結(jié)締組織和軟骨組織。鹿茸每年周期性地生長、脫落、完全再生,具有明顯的季節(jié)性。晚春和夏季是鹿茸的快速生長期,可達(dá)到2cm/d。鹿茸軟骨和茸皮組織相互促進(jìn)共同完成鹿茸的再生,但是生長期鹿茸軟骨和茸皮相輔相成快速生長的分子機(jī)制尚不清楚。microRNAs (miRNAs)是一類長約22nt、內(nèi)源性的、轉(zhuǎn)錄后水平調(diào)控基因表達(dá)的非編碼的單鏈small RNA分子,主要在轉(zhuǎn)錄后負(fù)調(diào)控基因表達(dá),通過與靶基因mRNAs 3'非翻譯區(qū)(3’-UTR)的堿基互補(bǔ)配對來降解mRNAs或者抑制mRNAs翻譯,從而發(fā)揮沉默特定靶基因的作用。已證實miRNAs廣泛參與調(diào)節(jié)動物、植物和病毒生命過程,其功能幾乎涉及生命活動的各個方面。然而關(guān)于miRNAs調(diào)控鹿茸再生的研究報道極少。我們由此提出如下問題：鹿茸組織中是否有miRNAs的表達(dá)?miRNAs在鹿茸不同組織中的表達(dá)量如何?研究這些問題可以進(jìn)一步回答miRNAs與已知的鹿茸再生相關(guān)基因之間存在怎樣的相互作用關(guān)系?miRNAs是否在鹿茸再生過程中發(fā)揮重要作用?本研究采集了東北馬鹿生長60天時的鹿茸,分離鹿茸軟骨和茸皮組織,進(jìn)行鹿茸軟骨和茸皮small RNA文庫Solexa深度測序,然后用生物信息學(xué)方法對測序數(shù)據(jù)進(jìn)行深入挖掘。本文主要研究包括：(1)進(jìn)行鹿茸軟骨和茸皮small RNA高通量測序,為miRNAs分析提供數(shù)據(jù)；(2)分析鹿茸軟骨和茸皮miRNAs,鑒定鹿茸保守和新型miRNAs;(3)分析鹿茸軟骨和茸皮miRNAs差異表達(dá)情況；(4)預(yù)測鹿茸miRNAs二級結(jié)構(gòu)和靶基因,并進(jìn)行靶基因功能注釋,探討miRNAs可能行使的生物學(xué)功能；(5)q-PCR驗證測序結(jié)果。主要結(jié)果如下：1、鹿茸軟骨和茸皮small RNA文庫進(jìn)行miRNA Solexa測序后,分別獲得13,513,502和5,524,073條原始序列讀數(shù)(raw reads)。經(jīng)過各種質(zhì)量控制程序處理后,得到9,520,645和3,621,894條高質(zhì)量可比對序列讀數(shù)(mappable reads),分別鑒定了389和295個miRNAs。2、miRNAs的序列長度主要分布在18-25nt,其中鹿茸軟骨組織中序列讀數(shù)在18nt處達(dá)到峰值,茸皮組織中序列讀數(shù)在22nt處達(dá)到峰值,在鹿茸軟骨和茸皮文庫中分別占到31.5%、18.0%,說明測序結(jié)果富含潛在miRNAs。3、篩選出7種類型的684個鹿茸miRNAs,其中611個為哺乳動物保守miRNAs,73個為鹿茸新的候選miRNAs。4、結(jié)果顯示,所得單一miRNAs中有大量異構(gòu)體isomiRNAs存在。其中293個保守miRNAs的末端堿基發(fā)生變化,堿基增加的數(shù)目大于減少的數(shù)目,且miRNAs3’端的堿基變化數(shù)目大于5’端；94個miRNAs發(fā)生堿基替換,包括62個轉(zhuǎn)換和32個顛換。5、鹿茸軟骨和茸皮文庫miRNAs的表達(dá)主要集中在表達(dá)量前20位的miRNAs上(均占所有miRNAs總拷貝數(shù)的80%以上),其中miR-21在鹿茸軟骨中拷貝數(shù)最高,miR-127在鹿茸茸皮中拷貝數(shù)最高。兩個組織間共有168個miRNAs表達(dá)存在差異,103個miRNAs表達(dá)差異顯著。6、首次通過同源比對構(gòu)建了馬鹿鹿茸的miRNAs表達(dá)譜。將鹿茸611個保守miRNAs與miRBase (v18.0)數(shù)據(jù)庫中己注釋的miRNAs序列進(jìn)行比對,結(jié)果顯示鹿茸和牛的保守miRNAs數(shù)目最多,達(dá)422個,其次為野豬(42)、小鼠(37)、綿羊(35)和人(27)。7、用mfold軟件預(yù)測了鹿茸miRNAs前體二級結(jié)構(gòu),均可形成典型的發(fā)夾結(jié)構(gòu)。此外,利用TargetScan軟件預(yù)測了鹿茸軟骨和茸皮文庫中保守和新發(fā)現(xiàn)的miRNAs(共39個)的靶基因,其功能有待進(jìn)一步研究。8、通過實時熒光定量PCR (RT-PCR)技術(shù)驗證了鹿茸軟骨和茸皮組織中拷貝數(shù)差異顯著或不顯著的14條保守miRNAs在兩個文庫中的表達(dá)情況。結(jié)果表明,q-PCR的數(shù)據(jù)與測序數(shù)據(jù)基本一致。本次測序結(jié)果真實可靠,能夠反映鹿茸軟骨和茸皮miRNAs表達(dá)譜數(shù)據(jù)。本研究從miRNA調(diào)控鹿茸生長發(fā)育的角度出發(fā),通過高通量測序技術(shù)獲得快速生長期鹿茸軟骨和茸皮組織的miRNAs表達(dá)譜,分析其序列特征,檢測miRNAs在鹿茸快速生長期不同組織中的表達(dá)水平,篩選與鹿茸再生相關(guān)的miRNAs。馬鹿鹿茸軟骨和茸皮miRNAs的鑒定與功能研究將促進(jìn)鹿茸再生分子機(jī)制的研究。為進(jìn)一步研究鹿茸miRNAs的功能、基因調(diào)控機(jī)制和探討miRNAs在鹿茸再生過程中的生物學(xué)功能提供重要數(shù)據(jù)參考。
[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.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q953

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