發(fā)布時間：2018-05-19 02:36

  本文選題：遼寧絨山羊 + 乾華肉用美利奴羊��； 參考：《吉林農業(yè)大學》2016年博士論文

【摘要】：遼寧絨山羊屬絨肉兼用型品種,是我國產(chǎn)絨量最高的優(yōu)良品種,遺傳性能穩(wěn)定,種用價值極高,不但產(chǎn)絨量高,而且絨纖維伸直長度可達10 cm,粗細度適中,纖維直徑平均為15μm左右。乾華肉用美利奴羊是吉林省新選育出來的肉毛兼用型新品種,不但具有良好的產(chǎn)肉性能,而且羊毛纖維直徑也在20.47-21.43μm之間(即品質支數(shù)在66支的范圍內),因此也是優(yōu)質的細毛羊品種。miRNA是一類19-25nt的單鏈非編碼小分子RNA,是大量細胞進程的必需調節(jié)器。毛囊是皮膚重要的附屬可再生器官,控制著毛發(fā)的生長,而且具有自我更新和周期性生長變化的特點。為了明確miRNA對皮膚毛囊發(fā)育的調節(jié)作用并探究可能的作用機制,本研究利用高通量測序技術篩選了皮膚毛囊不同發(fā)育時期的小RNA,并構建了小RNA文庫,同時篩選了皮膚毛囊不同發(fā)育時期差異表達的miRNA;利用生物信息學方法分析預測了差異表達miRNA:miR-1298-5p、miR-1-3p的靶基因,并結合相關文獻確定候選靶基因;利用熒光定量PCR技術從核酸水平水平揭示miRNA及候選靶基因對皮膚毛囊發(fā)育作用的影響;利用Western blot技術揭示候選靶基因對皮膚毛囊發(fā)育作用的影響,從而進一步揭示miRNA及候選靶基因對皮膚毛囊周期性發(fā)育的作用調節(jié)機制。為了進一步揭示miRNA對其候選靶基因的調控作用,本研究采用雙熒光素酶報告系統(tǒng)實驗利用雙熒光素酶活性的變化明確miRNA對其靶基因的靶向調控作用,從而進一步明確miRNA對皮膚毛囊發(fā)育作用的可能調控機制,為進一步揭示毛囊周期性發(fā)育作用機制提供重要的研究依據(jù)。主要研究結果如下:1.構建了遼寧絨山羊和乾華肉用美利奴羊皮膚毛囊生長期、退行期和休止期的小RNA文庫,HiSeq測序結果經(jīng)過過濾獲得純凈reads數(shù)分別為8596054、11039308和10478621,10279515、11712275和10926258,6個文庫小RNA長度分布一致,22 nt小RNA最多,其次是21 nt和23 nt。2.遼寧絨山羊與乾華肉用美利奴羊共獲得了1616個成熟miRNA,其中1397個保守miRNA,219個新mi RNA,大大豐富了羊屬miRNA數(shù)據(jù)庫。遼寧絨山羊和乾華肉用美利奴羊皮膚毛囊生長期、退行期和休止期特有的miRNA數(shù)分別為21個、23個和26個,15個、30個和27個。3.通過熒光定量PCR測定了10個mi RNA在毛囊生長期、退行期和休止期的相對表達量,miRNA相對表達量結果趨勢與高通量測序結果趨勢基本一致,說明高通量測序結果準確。4.利用IDEG6軟件進行差異表達miRNA分析,利用Targetscan v7.0、RNA22 v2.0和MiRanda(2010)三個靶基因預測軟件對miR-1298-5p與mi R-1-3p的靶基因進行了預測分析,結合調控皮膚毛囊發(fā)育的相關信號通路(Wnt,TGF-β,Notch,SHH,MAPK,BMP,Jak-STAT和TNF)進行候選靶基因篩選,并最終確定TGFBR1、TGFBR2和FGF2為miR-1298-5p的候選靶基因,IGF1R和FGF14為mi R-1-3p的候選靶基因。5.熒光定量PCR分析結果:miR-1298-5p在遼寧絨山羊皮膚毛囊生長期低表達,退行期高表達,miR-1-3p生長期低表達,休止期高表達;mi R-1298-5p在乾華肉用美利奴羊皮膚毛囊退行期高表達,休止期低表達,miR-1-3p生長期低表達,休止期高表達;TGFBR1、TGFBR2、FGF2、IGF1R和FGF14五個候選靶基因mRNA在遼寧絨山羊與乾華肉用美利奴羊皮膚組織內均有表達。候選靶基因TGFBR1、TGFBR2和FGF2 mRNA在遼寧絨山羊皮膚毛囊生長期高表達,退行期低表達;TGFBR1、TGFBR2和FGF2 mRNA在乾華肉用美利奴羊皮膚毛囊退行期低表達,候選靶基因TGFBR1和FGF2 mRNA在休止期高表達,候選靶基因TGFBR2 mRNA在生長期高表達;候選靶基因IGF1R mRNA在遼寧絨山羊皮膚毛囊退行期低表達,休止期高表達,候選靶基因FGF14在生長期高表達,休止期低表達;候選靶基因IGF1R和FGF14 mRNA在乾華肉用美利奴羊皮膚毛囊生長期高表達,退行期低表達。6.Western blot分析候選靶基因蛋白相對表達量結果:TGFBR1、TGFBR2、FGF2、IGF1R和FGF14五個候選靶基因蛋白在遼寧絨山羊與乾華肉用美利奴羊皮膚組織內均有表達。候選靶基因TGFBR1、TGFBR2和FGF2蛋白在遼寧絨山羊皮膚毛囊生長期高表達,退行期低表達;TGFBR1、TGFBR2和FGF2蛋白在乾華肉用美利奴羊皮膚毛囊退行期低表達,候選靶基因TGFBR1和FGF2蛋白在休止期高表達,候選靶基因TGFBR2蛋白在生長期高表達;候選靶基因IGF1R蛋白在遼寧絨山羊皮膚毛囊生長期高表達,退行期低表達,候選靶基因FGF14蛋白在生長期高表達,休止期低表達;候選靶基因IGF1R和FGF14蛋白在乾華肉用美利奴羊皮膚毛囊生長期高表達,退行期低表達。7.雙熒光素酶報告基因系統(tǒng)實驗結果表明:對于遼寧絨山羊與乾華肉用美利奴羊,miR-1298-5p都能夠抑制野生型TGFBR1和FGF2,miR-1-3p都能夠抑制野生型IGF1R和FGF14,當靶位點突變后,只有被抑制的FGF2和FGF14熒光素酶活性得到恢復,說明miR-1298-5p可靶向調控FGF2,miR-1-3p可靶向調控FGF14。本研究利用高通量測序技術與熒光定量PCR技術明確了miR-1298-5p和miR-1-3p在遼寧絨山羊與乾華肉用美利奴羊皮膚毛囊組織的表達規(guī)律,利用熒光定量PCR技術和Western blot技術明確了候選靶基因TGFBR1、TGFBR2、FGF2、IGF1R與FGF14在遼寧絨山羊與乾華肉用美利奴羊皮膚毛囊組織內的核酸水平以及蛋白水平的表達規(guī)律,利用雙熒光素酶報告基因系統(tǒng)實驗確定了miR-1298-5p和miR-1-3p的靶基因分別為FGF2和FGF14,而miR-1298-5p和miR-1-3p可能正是通過與其靶基因靶位點的結合引起靶基因mRNA和蛋白的表達量改變,從而誘導毛囊周期性發(fā)生發(fā)育。
[Abstract]:Liaoning cashmere goats are the best cashmere varieties in China, which are the highest quality varieties in China. Their genetic properties are stable and their seed production value is very high, not only has high cashmere production, but also the length of cashmere fiber can reach 10 cm, the coarse fineness is moderate, and the fiber diameter is about 15 mu. The Merino sheep used in Jilin province is a new kind of new type of meat and wool. The variety, not only has good meat production performance, but also the wool fiber diameter is also between 20.47-21.43 mu m (the quality count is within the range of 66 branches), so it is also a fine fine wool sheep variety.MiRNA is a class of 19-25nt single strand non coding small molecule RNA, it is a necessary regulator for a large number of cell processes. The hair follicle is an important accessory renewable skin. The organ, which controls the growth of hair, has the characteristics of self renewal and periodic growth. In order to clarify the regulatory role of miRNA on the development of skin follicles and explore possible mechanisms of action, this study screened small RNA from different developmental stages of skin follicles by high throughput sequencing technology and constructed a small RNA library, which was screened at the same time. MiRNA expressed in different developmental stages of skin hair follicles; using bioinformatics methods to predict the target genes for differentially expressed miRNA:miR-1298-5p and miR-1-3p, and to determine the candidate target genes in combination with relevant literature, and to reveal the effects of miRNA and candidate target genes on the development of skin follicles from the level of nucleic acid at the level of nucleic acid using fluorescence quantitative PCR technology. The effect of candidate target gene on the development of skin hair follicles was revealed by Western blot technique, thus further revealing the regulatory mechanism of miRNA and candidate target genes on the periodic development of skin follicles. In order to further reveal the regulatory role of miRNA on its candidate target genes, this study uses the dual luciferase reporter system for experiment. The changes in the activity of double luciferase clearly define the targeting regulation of miRNA to its target gene, thus further clarifying the possible regulatory mechanism of miRNA on the development of skin follicles, and providing an important research basis for further revealing the mechanism of the periodic development of hair follicles. The main results are as follows: 1. the construction of Liaoning cashmere goats and the use of dry Chinese meat is the main results. The small RNA Library of Merino sheep skin follicles, degenerative and rest periods, HiSeq sequencing results were filtered to obtain the pure reads number of 859605411039308 and 104786211027951511712275 and 10926258,6 small RNA length distribution, 22 NT small RNA, followed by 21 nt and 23 nt.2. Liaoning cashmere goats and the beauty of dry Chinese meat. 1616 mature miRNA were obtained, including 1397 conservative miRNA and 219 new mi RNA, which greatly enriched the miRNA database of the goats. The growth period of the skin follicles of Liaoning cashmere goats and Merino sheep was 21, 23 and 26, 15, 30 and 27.3. measured by fluorescence quantitative PCR. The relative expression of 10 mi RNA in the growth period of hair follicle, degenerative period and repose period, the trend of relative expression of miRNA is basically consistent with the trend of high throughput sequencing results, indicating that high throughput sequencing results are accurate.4. using IDEG6 software for differentially expressed miRNA analysis, and using Targetscan v7.0, RNA22 v2.0 and MiRanda (2010) three target genes. The target gene of miR-1298-5p and MI R-1-3p was predicted and analyzed. The candidate target gene was screened by combining the related signaling pathways regulating the development of skin follicles (Wnt, TGF- beta, Notch, SHH, MAPK, BMP, Jak-STAT and TNF). The results of gene.5. fluorescence quantitative PCR analysis showed that miR-1298-5p was low expression in the growth period of Liaoning cashmere goat hair follicle, high expression in degenerative period, low expression of miR-1-3p at growth stage, high expression in repose period, high expression of MI R-1298-5p in the degenerative period of skin follicle in Merino sheep of dry Chinese meat, low expression of resting stage, low expression of miR-1-3p in growth period, high expression of resting stage, TG The five candidate target genes of FBR1, TGFBR2, FGF2, IGF1R and FGF14 were expressed in the skin tissues of the Liaoning cashmere goat and the Merino sheep of dry Chinese meat. The candidate target gene TGFBR1, TGFBR2 and FGF2 mRNA were high expressed in the growth period of the skin follicles of Liaoning cashmere goats and low expression during the degenerative period; TGFBR1, TGFBR2, and mRNA were in the skin hair of the Merino sheep of dried Chinese meat. The candidate target gene TGFBR1 and FGF2 mRNA were highly expressed in the repose period, and the candidate target gene TGFBR2 mRNA was highly expressed in the growth period, and the candidate target gene IGF1R mRNA was low expressed in the degenerative period of the skin follicle of Liaoning cashmere goats and high expression in the repose period. The candidate target gene FGF14 was expressed in the long term, low expression in the repose period and the candidate target gene IGF1R. And FGF14 mRNA is highly expressed in the growth period of Merino skin hair follicle in dry Chinese meat, and the relative expression of candidate target gene protein of low expression.6.Western blot analysis at degenerative period: the five candidate target gene proteins of TGFBR1, TGFBR2, FGF2, IGF1R and FGF14 are expressed in the skin tissues of Liaoning cashmere goat and dry Chinese Merino sheep. The high expression of TGFBR1, TGFBR2 and FGF2 protein in the growth period of skin follicles in Liaoning cashmere goats, low expression in degenerative period, low expression of TGFBR1, TGFBR2 and FGF2 protein in the degenerative period of Merino skin hair follicle in dry Chinese meat, high expression of candidate target gene TGFBR1 and FGF2 protein in the repose period, high expression of candidate target gene TGFBR2 protein at the growth stage; candidate target base Due to the high expression of IGF1R protein in the growth period of the hair follicle in Liaoning cashmere goat, the degenerative period was low, the candidate target gene FGF14 protein was high expressed in the growth period and low expression in the rest period. The candidate target gene IGF1R and FGF14 protein expressed high in the growth period of the skin hair follicle of the Merino sheep, and the low expression of.7. double luciferase reporter gene system in the retreat period The results showed that for Liaoning cashmere goats and Merino sheep of dry Chinese meat, miR-1298-5p could inhibit wild type TGFBR1 and FGF2, and miR-1-3p could inhibit wild type IGF1R and FGF14. When the target site mutation, only the inhibited activity of FGF2 and FGF14 luciferase was recovered, indicating that miR-1298-5p can be targeted to FGF2, miR-1-3p can be targeted. The FGF14. study made use of high throughput sequencing and fluorescence quantitative PCR to clarify the expression of miR-1298-5p and miR-1-3p in the skin hair follicles of Liaoning cashmere goats and dry Chinese Merino sheep. Using the fluorescence quantitative PCR technology and Western blot technology, the candidate targets were determined by TGFBR1, TGFBR2, FGF2, IGF1R and FGF14 in Liaoning cashmere. The expression of nucleic acid level and protein level in the skin hair follicle of goat and dry Chinese meat Merino sheep. The target genes of miR-1298-5p and miR-1-3p are identified as FGF2 and FGF14 by the double luciferase reporter gene system experiment, and miR-1298-5p and miR-1-3p may be the target of the target gene target. The expression of mRNA and protein changed, thus inducing the development of hair follicle.
【學位授予單位】：吉林農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S827;S826.86

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