綿羊尾脂的全基因組microRNA表達(dá)譜分析及其與性狀的關(guān)聯(lián)研究
發(fā)布時間：2019-06-15 12:52
【摘要】：畜禽體內(nèi)脂肪含量的多少決定了其肉質(zhì)的好壞,而脂肪代謝則是一個由許多因子調(diào)控的復(fù)雜生物學(xué)過程。microRNAs(miRNAs)是一類小分子非編碼RNA(small non-coding RNA, sncRNA),長度約為22 nt。miRNA通過與mRNA完全或不完全堿基配對,在轉(zhuǎn)錄后水平上參與細(xì)胞分化、生長發(fā)育和性能表現(xiàn)等生物學(xué)過程。了解脂肪代謝相關(guān)的miRNAs的調(diào)節(jié)作用,可以在一定程度上揭示脂肪代謝的分子調(diào)控機(jī)制。但是,目前關(guān)于在綿羊尾脂組織中表達(dá)的miRNAs對脂肪代謝的調(diào)控研究未見任何報道。為了更好地了解miRNAs對脂肪代謝的作用,本研究構(gòu)建兩個sRNA文庫,利用高通量測序鑒定了不同脂尾型綿羊(廣靈大尾羊和小尾寒羊)尾脂組織中表達(dá)的miRNAs,篩選差異表達(dá)的miRNAs,利用生物信息學(xué)方法預(yù)測差異表達(dá)miRNAs的靶基因,并對靶基因進(jìn)行Gene Ontology分析和KEGG分析。挑選6個miRNAs (miR-10b、miR-29a、 miR-30c、miR-155、miR-192、miR-206),應(yīng)用熒光定量PCR技術(shù)對10月齡個體尾脂中的表達(dá)量進(jìn)行檢測,驗(yàn)證測序結(jié)果。利用熒光定量PCR的方法對兩品種綿羊2、4、6、8、10、12月齡的尾部脂肪組織中6個miRNAs的表達(dá)量進(jìn)行檢測。配合一般線性模型分析品種、性別、月齡及二因素互作對miRNAs表達(dá)的影響。計算Pearson相關(guān)系數(shù),研究miRNAs表達(dá)與各性狀的關(guān)聯(lián)。得到以下主要結(jié)果：1.在廣靈大尾羊和小尾寒羊尾脂組織中分別獲得了113和131個保守的miRNAs,對應(yīng)的靶基因數(shù)分別為22890和22907。廣靈大尾羊與小尾寒羊尾脂中差異表達(dá)的miRNAs數(shù)為93個,其中58個差異顯著。對miRNAs靶基因調(diào)控通路進(jìn)行KEGG分析,在兩文庫中發(fā)現(xiàn)了包括Wnt、MAPK等在內(nèi)的309個調(diào)控通路。用miReap軟件篩選兩文庫中的未知miRNAs,分別獲得了208和215個候選序列,對應(yīng)的靶基因數(shù)分別為22927和22929。將這些miRNAs與miRBase21.0中其他物種的miRNAs比對,發(fā)現(xiàn)大多miRNAs都可比對到數(shù)據(jù)庫中已有的序列,其中27個為新miRNAs。廣靈大尾羊與小尾寒羊尾脂中差異表達(dá)的候選新miRNAs數(shù)為175個,其中105個差異顯著。2.熒光定量PCR結(jié)果表明,miR-10b、miR-29a、miR-30c、miR-155、miR-192、 miR-206在廣靈大尾羊和小尾寒羊尾脂中的表達(dá)量存在顯著差異,變化趨勢和測序數(shù)據(jù)一致,推測其可能參與綿羊尾脂中脂肪的代謝。3.基于一般線性模型的方差分析表明,品種對所有6個miRNAs的表達(dá)均有顯著影響(P0.01或P0.001)。miR-155和miR-192在小尾寒羊中的表達(dá)量高于廣靈大尾羊,其他4個miRNAs則低于廣靈大尾羊。性別只對miR-206的表達(dá)影響顯著(P0.05)。品種與性別的互作對miR-30c和miR-206的表達(dá)量影響顯著(P0.01)。品種與月齡的互作對miR-10b、miR-30c和miR-155的表達(dá)量影響顯著(P0.01或P0.001)。關(guān)聯(lián)分析表明,miR-10b、miR-29a和miR-192與所有性狀都呈正相關(guān),其中,與體重、胴體重、絕對尾脂重和相對尾脂重間的相關(guān)顯著(P0.05或P0.01)。此外,miR-29a與尾長和屠宰率間的相關(guān)也達(dá)顯著水平(P0.05)。miR-30c與所有性狀也呈正相關(guān),但只與尾寬的相關(guān)顯著(P0.05)。miR-155和miR-206與所有性狀都呈負(fù)相關(guān),miR-206與除屠宰率外的其他6個性狀間的負(fù)相關(guān)顯著(P0.05或P0.01)。各miRNAs的表達(dá)與性狀的關(guān)聯(lián)與其在脂肪代謝中的作用相符。本研究對不同脂尾型綿羊尾脂組織中miRNAs表達(dá)譜的分析和關(guān)聯(lián)研究結(jié)果為闡明miRNA調(diào)節(jié)脂肪代謝的機(jī)制提供了科學(xué)依據(jù),也為進(jìn)一步研究miRNAs在轉(zhuǎn)錄后水平調(diào)節(jié)脂肪代謝相關(guān)基因的功能奠定了基礎(chǔ)。
[Abstract]:The amount of fat in the body of the animal determines the quality of its meat, and the fat metabolism is a complex biological process that is regulated by many factors. The microRNAs (miRNAs) are a kind of small non-coding RNA (sncRNA), and the length is about 22nt. The miRNAs are paired with the mRNA in a complete or incomplete manner, and are involved in the biological processes such as cell differentiation, growth and performance in the post-transcriptional level. To understand the regulation of the miRNAs associated with the metabolism of fat, it is possible to reveal the molecular regulation mechanism of the fat metabolism to a certain extent. However, no reports have been reported on the regulation of fat metabolism by miRNAs expressed in sheep tail-fat tissue. In order to better understand the effect of miRNAs on the metabolism of fat, two sRNA libraries were constructed, and the miRNAs expressed in the tail-fat tissues of different fat-tail-type sheep (wide-tailed sheep and small-tailed Han sheep) were identified by high-throughput sequencing, and the miRNAs of the differentially expressed miRNAs were screened. The target gene of miRNAs was expressed by bioinformatics method, and the target gene was analyzed by Gene Oncology and KEGG. Six miRNAs (miR-10b, miR-29a, miR-30c, miR-155, miR-192, miR-206) were selected, and the expression amount of the 10-month-old individual tail fat was detected by the fluorescence quantitative PCR technique to verify the sequencing result. The expression of 6 miRNAs in 2,4,6,8,10 and 12 months of tail adipose tissue of two breeds of sheep was detected by the method of fluorescence quantitative PCR. The effect of two factors on the expression of miRNAs was analyzed by the general linear model. Pearson correlation coefficient was calculated to study the correlation between the expression of miRNAs and the characters. The following main results were obtained:1. 113 and 131 conservative miRNAs were obtained in the tail-fat tissue of the large-tailed sheep and the tail-tail of the small-tailed Han sheep, and the corresponding number of target genes was 22890 and 22907, respectively. The number of miRNAs expressed in the tail fat of the large-tailed sheep and the tail fat of the small-tailed Han sheep was 93, and 58 of them were significant. In the two libraries,309 regulatory pathways, including Wnt, MAPK, and the like, were identified by the KEGG analysis of the miRNAs target gene regulatory pathway. The unknown miRNAs in the two libraries were screened by miReap software, and 208 and 215 candidate sequences were obtained. The corresponding number of target genes was 22927 and 22929, respectively. The miRNAs of these miRNAs were compared with the miRNAs of other species in miRBase21.0, and most miRNAs were found to be comparable to those already present in the database, of which 27 were new miRNAs. The number of new miRNAs expressed in the difference between the large-tailed sheep and the tail fat of the small-tailed Han sheep was 175, of which 105 were significant. The fluorescence quantitative PCR results show that the expression of miR-10b, miR-29a, miR-30c, miR-155, miR-192 and miR-206 in the tail fat of the large-tailed sheep and the tail fat of the small-tailed Han sheep is significantly different, and the variation trend and the sequencing data are consistent, and it is presumed that the miR-10b, miR-29a, miR-30c, miR-155, miR-192 and miR-206 may be involved in the metabolism of fat in the tail fat of the sheep. The analysis of variance based on the general linear model showed that the variety had a significant effect on the expression of all 6 miRNAs (P0.01 or P0.01). The expression of miR-155 and miR-192 in the small-tailed Han sheep is higher than that of the large-tailed sheep, and the other four miRNAs are lower than the wide-tailed sheep. The effect of gender on the expression of miR-206 was significant (P0.05). There was a significant effect on the expression of miR-30c and miR-206 (P0.01). The expression of miR-10b, miR-30c and miR-155 in miR-10b, miR-30c and miR-155 was significant (P0.01 or P0.01). The correlation analysis showed that miR-10b, miR-29a and miR-192 were positively correlated with all characters, among which, the correlation with body weight, carcass weight, absolute tail fat weight and relative tail fat weight was significant (P0.05 or P0.01). In addition, the correlation between miR-29a and the length of the tail and the slaughter rate reached a significant level (P0.05). The miR-30c was positively related to all characters, but only correlated with the width of the tail (P0.05). The miR-155 and miR-206 were negatively correlated with all characters, and the negative correlation between the miR-206 and the other 6 characters except the slaughter rate was significant (P0.05 or P0.01). The correlation of the expression and the character of miRNAs in fat metabolism. The results of the study on miRNAs expression profile in the tail-fat tissue of different fat-tail-type sheep provide a scientific basis for elucidating the mechanism of miRNAs to regulate the fat metabolism, and also lays a foundation for further study of the function of miRNAs in regulating the fat metabolism related genes at the post-transcriptional level.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S826;Q78
本文編號：2500220
[Abstract]:The amount of fat in the body of the animal determines the quality of its meat, and the fat metabolism is a complex biological process that is regulated by many factors. The microRNAs (miRNAs) are a kind of small non-coding RNA (sncRNA), and the length is about 22nt. The miRNAs are paired with the mRNA in a complete or incomplete manner, and are involved in the biological processes such as cell differentiation, growth and performance in the post-transcriptional level. To understand the regulation of the miRNAs associated with the metabolism of fat, it is possible to reveal the molecular regulation mechanism of the fat metabolism to a certain extent. However, no reports have been reported on the regulation of fat metabolism by miRNAs expressed in sheep tail-fat tissue. In order to better understand the effect of miRNAs on the metabolism of fat, two sRNA libraries were constructed, and the miRNAs expressed in the tail-fat tissues of different fat-tail-type sheep (wide-tailed sheep and small-tailed Han sheep) were identified by high-throughput sequencing, and the miRNAs of the differentially expressed miRNAs were screened. The target gene of miRNAs was expressed by bioinformatics method, and the target gene was analyzed by Gene Oncology and KEGG. Six miRNAs (miR-10b, miR-29a, miR-30c, miR-155, miR-192, miR-206) were selected, and the expression amount of the 10-month-old individual tail fat was detected by the fluorescence quantitative PCR technique to verify the sequencing result. The expression of 6 miRNAs in 2,4,6,8,10 and 12 months of tail adipose tissue of two breeds of sheep was detected by the method of fluorescence quantitative PCR. The effect of two factors on the expression of miRNAs was analyzed by the general linear model. Pearson correlation coefficient was calculated to study the correlation between the expression of miRNAs and the characters. The following main results were obtained:1. 113 and 131 conservative miRNAs were obtained in the tail-fat tissue of the large-tailed sheep and the tail-tail of the small-tailed Han sheep, and the corresponding number of target genes was 22890 and 22907, respectively. The number of miRNAs expressed in the tail fat of the large-tailed sheep and the tail fat of the small-tailed Han sheep was 93, and 58 of them were significant. In the two libraries,309 regulatory pathways, including Wnt, MAPK, and the like, were identified by the KEGG analysis of the miRNAs target gene regulatory pathway. The unknown miRNAs in the two libraries were screened by miReap software, and 208 and 215 candidate sequences were obtained. The corresponding number of target genes was 22927 and 22929, respectively. The miRNAs of these miRNAs were compared with the miRNAs of other species in miRBase21.0, and most miRNAs were found to be comparable to those already present in the database, of which 27 were new miRNAs. The number of new miRNAs expressed in the difference between the large-tailed sheep and the tail fat of the small-tailed Han sheep was 175, of which 105 were significant. The fluorescence quantitative PCR results show that the expression of miR-10b, miR-29a, miR-30c, miR-155, miR-192 and miR-206 in the tail fat of the large-tailed sheep and the tail fat of the small-tailed Han sheep is significantly different, and the variation trend and the sequencing data are consistent, and it is presumed that the miR-10b, miR-29a, miR-30c, miR-155, miR-192 and miR-206 may be involved in the metabolism of fat in the tail fat of the sheep. The analysis of variance based on the general linear model showed that the variety had a significant effect on the expression of all 6 miRNAs (P0.01 or P0.01). The expression of miR-155 and miR-192 in the small-tailed Han sheep is higher than that of the large-tailed sheep, and the other four miRNAs are lower than the wide-tailed sheep. The effect of gender on the expression of miR-206 was significant (P0.05). There was a significant effect on the expression of miR-30c and miR-206 (P0.01). The expression of miR-10b, miR-30c and miR-155 in miR-10b, miR-30c and miR-155 was significant (P0.01 or P0.01). The correlation analysis showed that miR-10b, miR-29a and miR-192 were positively correlated with all characters, among which, the correlation with body weight, carcass weight, absolute tail fat weight and relative tail fat weight was significant (P0.05 or P0.01). In addition, the correlation between miR-29a and the length of the tail and the slaughter rate reached a significant level (P0.05). The miR-30c was positively related to all characters, but only correlated with the width of the tail (P0.05). The miR-155 and miR-206 were negatively correlated with all characters, and the negative correlation between the miR-206 and the other 6 characters except the slaughter rate was significant (P0.05 or P0.01). The correlation of the expression and the character of miRNAs in fat metabolism. The results of the study on miRNAs expression profile in the tail-fat tissue of different fat-tail-type sheep provide a scientific basis for elucidating the mechanism of miRNAs to regulate the fat metabolism, and also lays a foundation for further study of the function of miRNAs in regulating the fat metabolism related genes at the post-transcriptional level.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S826;Q78
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