【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)學分子機制和調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,內部是遍布神經(jīng)和血管的結締組織和軟骨組織。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,可達到2cm/d。鹿茸軟骨和茸皮組織相互促進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制尚不清楚。microRNAs (miRNAs)是一類長約22nt、內源性的、轉錄后水平調控基因表達的非編碼的單鏈small RNA分子,主要在轉錄后負調控基因表達,通過與靶基因mRNAs 3'非翻譯區(qū)(3’-UTR)的堿基互補配對來降解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。然而關于miRNAs調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?miRNAs在鹿茸不同組織中的表達量如何?研究這些問題可以進一步回答miRNAs與已知的鹿茸再生相關基因之間存在怎樣的相互作用關系?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,然后用生物信息學方法對測序數(shù)據(jù)進行深入挖掘。本文主要研究包括：(1)進行鹿茸軟骨和茸皮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差異表達情況；(4)預測鹿茸miRNAs二級結構和靶基因,并進行靶基因功能注釋,探討miRNAs可能行使的生物學功能；(5)q-PCR驗證測序結果。主要結果如下：1、鹿茸軟骨和茸皮small RNA文庫進行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)過各種質量控制程序處理后,得到9,520,645和3,621,894條高質量可比對序列讀數(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處達到峰值,茸皮組織中序列讀數(shù)在22nt處達到峰值,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、結果顯示,所得單一miRNAs中有大量異構體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個轉換和32個顛換。5、鹿茸軟骨和茸皮文庫miRNAs的表達主要集中在表達量前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表達存在差異,103個miRNAs表達差異顯著。6、首次通過同源比對構建了馬鹿鹿茸的miRNAs表達譜。將鹿茸611個保守miRNAs與miRBase (v18.0)數(shù)據(jù)庫中己注釋的miRNAs序列進行比對,結果顯示鹿茸和牛的保守miRNAs數(shù)目最多,達422個,其次為野豬(42)、小鼠(37)、綿羊(35)和人(27)。7、用mfold軟件預測了鹿茸miRNAs前體二級結構,均可形成典型的發(fā)夾結構。此外,利用TargetScan軟件預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,其功能有待進一步研究。8、通過實時熒光定量PCR (RT-PCR)技術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情況。結果表明,q-PCR的數(shù)據(jù)與測序數(shù)據(jù)基本一致。本次測序結果真實可靠,能夠反映鹿茸軟骨和茸皮miRNAs表達譜數(shù)據(jù)。本研究從miRNA調控鹿茸生長發(fā)育的角度出發(fā),通過高通量測序技術獲得快速生長期鹿茸軟骨和茸皮組織的miRNAs表達譜,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水平,篩選與鹿茸再生相關的miRNAs。馬鹿鹿茸軟骨和茸皮miRNAs的鑒定與功能研究將促進鹿茸再生分子機制的研究。為進一步研究鹿茸miRNAs的功能、基因調控機制和探討miRNAs在鹿茸再生過程中的生物學功能提供重要數(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.
【學位授予單位】：東北林業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q953

【相似文獻】

相關期刊論文 前10條

1 王玫;李思光;羅玉萍;;microRNA識別和鑒定方法[J];細胞生物學雜志;2007年04期

2 王晶;朱榮勝;宋萬坤;張聞博;劉春燕;胡國華;陳慶山;;microRNA的實驗和生物信息學開發(fā)方法[J];生物技術通報;2008年S1期

3 田明;費菁;胡曉湘;李寧;劉娣;孟慶勇;;microRNA-1,-133,-206在鼠肌肉中的表達譜[J];中國畜牧獸醫(yī);2009年08期

4 LEE Younghee;LUSSIER Yves A;;Identification of common microRNA-mRNA regulatory biomodules in human epithelial cancer[J];Chinese Science Bulletin;2010年31期

5 王芳;余佳;張俊武;;小RNA(MicroRNA)研究方法[J];中國生物化學與分子生物學報;2006年10期

6 鄭玉姝;趙樸;趙宏坤;;microRNA及其應用前景[J];生物技術通訊;2007年01期

7 王春梅;李慶章;;microRNA在小鼠乳腺不同發(fā)育時期差異表達譜及作用(英文)[J];遺傳學報;2007年11期

8 鄒文輝;;新奇的調控分子——microRNA[J];安徽農業(yè)科學;2008年34期

9 ;MIRE: A GRAPHICAL R PACKAGE FOR MICRORNARELATED ANALYSIS[J];Chinese Medical Sciences Journal;2008年04期

10 張坤山;李思光;羅玉萍;;調控過氧化物酶體生物合成和增殖的microRNA的計算機分析[J];細胞生物學雜志;2009年02期

相關會議論文 前10條

1 荊清;袁文俊;秦永文;;microRNA的基因調控新功能[A];中國生理學會第五屆全國心血管、呼吸和腎臟生理學學術會議論文摘要匯編[C];2005年

2 靳新;駱志剛;王金華;管乃洋;;microRNA靶標研究進展[A];中國遺傳學會功能基因組學研討會論文集[C];2006年

3 ;Comparasion of Inhibitory Effects on Survivin Gene Expression in Prostate Cancer by Vector-based microRNA and siRNA[A];2008年全國生物化學與分子生物學學術大會論文摘要[C];2008年

4 朱丹霞;徐衛(wèi);繆扣榮;方成;朱華淵;董華潔;王冬梅;范磊;喬純;李建勇;;慢性淋巴細胞白血病microRNA異常表達研究[A];第13屆全國實驗血液學會議論文摘要[C];2011年

5 Yangchao Chen;;Small molecule modulators of microRNA-34a with anti-cancer activities[A];2013醫(yī)學前沿論壇暨第十三屆全國腫瘤藥理與化療學術會議論文集[C];2013年

6 金希;厲有名;;單純性脂變與非酒精性脂肪性肝炎間差異microRNA表達譜研究[A];2009香港-北京-杭州內科論壇暨2009年浙江省內科學學術年會論文匯編[C];2009年

7 賈新正;盧肖男;聶慶華;張細權;;雞部分microRNA的同源預測與克隆驗證[A];中國動物遺傳育種研究進展——第十五次全國動物遺傳育種學術討論會論文集[C];2009年

8 李炯;段德民;鄭克孝;;新型非標記高通量microRNA芯片技術[A];第一屆全國生物物理化學會議暨生物物理化學發(fā)展戰(zhàn)略研討會論文摘要集[C];2010年

9 徐小濤;陸曉;孫婧;束永前;;肺癌側群細胞microRNA表達譜檢測及初步分析[A];2010’全國腫瘤分子標志及應用學術研討會暨第五屆中國中青年腫瘤專家論壇論文匯編[C];2010年

10 王俊峰;李巍;吳小江;阮康成;;大鼠附睪microRNA表達譜的研究[A];第十一次中國生物物理學術大會暨第九屆全國會員代表大會摘要集[C];2009年

相關重要報紙文章 前2條

1 陳英云 喬蕤琳;哈醫(yī)大成功研發(fā)國內首例microRNA轉基因及敲減小鼠模型[N];黑龍江經(jīng)濟報;2010年

2 記者 陳青;2厘米以下肝癌檢出率88%[N];文匯報;2011年

相關博士學位論文 前10條

1 崔洪亮;microRNA的腫瘤表達研究及協(xié)同調控網(wǎng)絡分析[D];中南大學;2014年

2 馬建華;高粱低磷低氮形態(tài)生理特征及低氮響應的microRNA研究[D];山西農業(yè)大學;2014年

3 李虔楨;microRNA相關基因遺傳多態(tài)性與冠心病臨床關聯(lián)及預后研究[D];福建醫(yī)科大學;2015年

4 王耀輝;血清microRNA作為乳腺癌診斷標志物的研究[D];復旦大學;2014年

5 周霽子;環(huán)境因素對心臟畸形胎兒影響的表觀遣傳學機制[D];復旦大學;2013年

6 張麗;microRNA通過調控小膠質細胞炎性反應參與血管性認知障礙發(fā)生發(fā)展[D];復旦大學;2014年

7 查若鵬;肝癌轉移相關microRNA的鑒定及其分子機制研究[D];復旦大學;2013年

8 方硯田;結腸癌干細胞分選、差異microRNA表達譜檢測以及miR-449b-CCND1、E2F3通路在結腸癌干細胞自我更新的機制研究[D];復旦大學;2014年

9 辛成齊;椰棗microRNA鑒定及其在果實發(fā)育過程中的表達譜研究[D];中國科學院北京基因組研究所;2015年

10 李棟;先天性心臟病血漿microRNA表達譜及與GATA4靶序列單核苷酸多態(tài)性的關聯(lián)研究[D];山東大學;2015年

相關碩士學位論文 前10條

1 高智紅;應用多樣性增量方法識別人類基因組microRNA前體序列[D];內蒙古大學;2010年

2 王娜娜;microRNA進化關系及編碼特性研究[D];內蒙古大學;2007年

3 王玫;小麥microRNA的鑒定與分析[D];南昌大學;2007年

4 秦保東;原發(fā)性膽汁性肝硬化microRNA表達譜的檢測及其功能研究[D];第二軍醫(yī)大學;2013年

5 吳曉妍;基于納米復合材料及生物放大技術構建的電化學microRNA傳感器的研究[D];西南大學;2015年

6 周景輝;2型糖尿病microRNA表達譜及其信號通路研究[D];華南理工大學;2015年

7 姜溪;血漿microRNA-486、microRNA-499在肺癌中的表達及臨床價值的研究[D];河北大學;2015年

8 林杉;營養(yǎng)誘導舍飼綿羊非繁殖季節(jié)發(fā)情相關microRNA篩選及其靶基因功能驗證[D];石河子大學;2015年

9 林妹妹;microRNA-192 和 microRNA-21 在 IBD 患者中的表達情況[D];福建醫(yī)科大學;2015年

10 徐嬌陽;循環(huán)microRNA用于低氧性肺動脈高壓早期診斷的實驗研究[D];石河子大學;2015年

，

本文編號：2135266