辽宁绒山羊与乾华肉用美利奴羊皮肤毛囊miRNA筛选及靶基因验证

发布时间：2018-05-19 02:36

本文选题：辽宁绒山羊 + 乾华肉用美利奴羊　；参考：《吉林农业大学》2016年博士论文

【摘要】：辽宁绒山羊属绒肉兼用型品种,是我国产绒量最高的优良品种,遗传性能稳定,种用价值极高,不但产绒量高,而且绒纤维伸直长度可达10 cm,粗细度适中,纤维直径平均为15μm左右。乾华肉用美利奴羊是吉林省新选育出来的肉毛兼用型新品种,不但具有良好的产肉性能,而且羊毛纤维直径也在20.47-21.43μm之间(即品质支数在66支的范围内),因此也是优质的细毛羊品种。miRNA是一类19-25nt的单链非编码小分子RNA,是大量细胞进程的必需调节器。毛囊是皮肤重要的附属可再生器官,控制着毛发的生长,而且具有自我更新和周期性生长变化的特点。为了明确miRNA对皮肤毛囊发育的调节作用并探究可能的作用机制,本研究利用高通量测序技术筛选了皮肤毛囊不同发育时期的小RNA,并构建了小RNA文库,同时筛选了皮肤毛囊不同发育时期差异表达的miRNA;利用生物信息学方法分析预测了差异表达miRNA:miR-1298-5p、miR-1-3p的靶基因,并结合相关文献确定候选靶基因;利用荧光定量PCR技术从核酸水平水平揭示miRNA及候选靶基因对皮肤毛囊发育作用的影响;利用Western blot技术揭示候选靶基因对皮肤毛囊发育作用的影响,从而进一步揭示miRNA及候选靶基因对皮肤毛囊周期性发育的作用调节机制。为了进一步揭示miRNA对其候选靶基因的调控作用,本研究采用双荧光素酶报告系统实验利用双荧光素酶活性的变化明确miRNA对其靶基因的靶向调控作用,从而进一步明确miRNA对皮肤毛囊发育作用的可能调控机制,为进一步揭示毛囊周期性发育作用机制提供重要的研究依据。主要研究结果如下:1.构建了辽宁绒山羊和乾华肉用美利奴羊皮肤毛囊生长期、退行期和休止期的小RNA文库,HiSeq测序结果经过过滤获得纯净reads数分别为8596054、11039308和10478621,10279515、11712275和10926258,6个文库小RNA长度分布一致,22 nt小RNA最多,其次是21 nt和23 nt。2.辽宁绒山羊与乾华肉用美利奴羊共获得了1616个成熟miRNA,其中1397个保守miRNA,219个新mi RNA,大大丰富了羊属miRNA数据库。辽宁绒山羊和乾华肉用美利奴羊皮肤毛囊生长期、退行期和休止期特有的miRNA数分别为21个、23个和26个,15个、30个和27个。3.通过荧光定量PCR测定了10个mi RNA在毛囊生长期、退行期和休止期的相对表达量,miRNA相对表达量结果趋势与高通量测序结果趋势基本一致,说明高通量测序结果准确。4.利用IDEG6软件进行差异表达miRNA分析,利用Targetscan v7.0、RNA22 v2.0和MiRanda(2010)三个靶基因预测软件对miR-1298-5p与mi R-1-3p的靶基因进行了预测分析,结合调控皮肤毛囊发育的相关信号通路(Wnt,TGF-β,Notch,SHH,MAPK,BMP,Jak-STAT和TNF)进行候选靶基因筛选,并最终确定TGFBR1、TGFBR2和FGF2为miR-1298-5p的候选靶基因,IGF1R和FGF14为mi R-1-3p的候选靶基因。5.荧光定量PCR分析结果:miR-1298-5p在辽宁绒山羊皮肤毛囊生长期低表达,退行期高表达,miR-1-3p生长期低表达,休止期高表达;mi R-1298-5p在乾华肉用美利奴羊皮肤毛囊退行期高表达,休止期低表达,miR-1-3p生长期低表达,休止期高表达;TGFBR1、TGFBR2、FGF2、IGF1R和FGF14五个候选靶基因mRNA在辽宁绒山羊与乾华肉用美利奴羊皮肤组织内均有表达。候选靶基因TGFBR1、TGFBR2和FGF2 mRNA在辽宁绒山羊皮肤毛囊生长期高表达,退行期低表达;TGFBR1、TGFBR2和FGF2 mRNA在乾华肉用美利奴羊皮肤毛囊退行期低表达,候选靶基因TGFBR1和FGF2 mRNA在休止期高表达,候选靶基因TGFBR2 mRNA在生长期高表达;候选靶基因IGF1R mRNA在辽宁绒山羊皮肤毛囊退行期低表达,休止期高表达,候选靶基因FGF14在生长期高表达,休止期低表达;候选靶基因IGF1R和FGF14 mRNA在乾华肉用美利奴羊皮肤毛囊生长期高表达,退行期低表达。6.Western blot分析候选靶基因蛋白相对表达量结果:TGFBR1、TGFBR2、FGF2、IGF1R和FGF14五个候选靶基因蛋白在辽宁绒山羊与乾华肉用美利奴羊皮肤组织内均有表达。候选靶基因TGFBR1、TGFBR2和FGF2蛋白在辽宁绒山羊皮肤毛囊生长期高表达,退行期低表达;TGFBR1、TGFBR2和FGF2蛋白在乾华肉用美利奴羊皮肤毛囊退行期低表达,候选靶基因TGFBR1和FGF2蛋白在休止期高表达,候选靶基因TGFBR2蛋白在生长期高表达;候选靶基因IGF1R蛋白在辽宁绒山羊皮肤毛囊生长期高表达,退行期低表达,候选靶基因FGF14蛋白在生长期高表达,休止期低表达;候选靶基因IGF1R和FGF14蛋白在乾华肉用美利奴羊皮肤毛囊生长期高表达,退行期低表达。7.双荧光素酶报告基因系统实验结果表明:对于辽宁绒山羊与乾华肉用美利奴羊,miR-1298-5p都能够抑制野生型TGFBR1和FGF2,miR-1-3p都能够抑制野生型IGF1R和FGF14,当靶位点突变后,只有被抑制的FGF2和FGF14荧光素酶活性得到恢复,说明miR-1298-5p可靶向调控FGF2,miR-1-3p可靶向调控FGF14。本研究利用高通量测序技术与荧光定量PCR技术明确了miR-1298-5p和miR-1-3p在辽宁绒山羊与乾华肉用美利奴羊皮肤毛囊组织的表达规律,利用荧光定量PCR技术和Western blot技术明确了候选靶基因TGFBR1、TGFBR2、FGF2、IGF1R与FGF14在辽宁绒山羊与乾华肉用美利奴羊皮肤毛囊组织内的核酸水平以及蛋白水平的表达规律,利用双荧光素酶报告基因系统实验确定了miR-1298-5p和miR-1-3p的靶基因分别为FGF2和FGF14,而miR-1298-5p和miR-1-3p可能正是通过与其靶基因靶位点的结合引起靶基因mRNA和蛋白的表达量改变,从而诱导毛囊周期性发生发育。
[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.
【学位授予单位】：吉林农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S827;S826.86

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