绵羊尾脂的全基因组microRNA表达谱分析及其与性状的关联研究
发布时间：2019-06-15 12:52
【摘要】：畜禽体内脂肪含量的多少决定了其肉质的好坏,而脂肪代谢则是一个由许多因子调控的复杂生物学过程。microRNAs(miRNAs)是一类小分子非编码RNA(small non-coding RNA, sncRNA),长度约为22 nt。miRNA通过与mRNA完全或不完全碱基配对,在转录后水平上参与细胞分化、生长发育和性能表现等生物学过程。了解脂肪代谢相关的miRNAs的调节作用,可以在一定程度上揭示脂肪代谢的分子调控机制。但是,目前关于在绵羊尾脂组织中表达的miRNAs对脂肪代谢的调控研究未见任何报道。为了更好地了解miRNAs对脂肪代谢的作用,本研究构建两个sRNA文库,利用高通量测序鉴定了不同脂尾型绵羊(广灵大尾羊和小尾寒羊)尾脂组织中表达的miRNAs,筛选差异表达的miRNAs,利用生物信息学方法预测差异表达miRNAs的靶基因,并对靶基因进行Gene Ontology分析和KEGG分析。挑选6个miRNAs (miR-10b、miR-29a、 miR-30c、miR-155、miR-192、miR-206),应用荧光定量PCR技术对10月龄个体尾脂中的表达量进行检测,验证测序结果。利用荧光定量PCR的方法对两品种绵羊2、4、6、8、10、12月龄的尾部脂肪组织中6个miRNAs的表达量进行检测。配合一般线性模型分析品种、性别、月龄及二因素互作对miRNAs表达的影响。计算Pearson相关系数,研究miRNAs表达与各性状的关联。得到以下主要结果：1.在广灵大尾羊和小尾寒羊尾脂组织中分别获得了113和131个保守的miRNAs,对应的靶基因数分别为22890和22907。广灵大尾羊与小尾寒羊尾脂中差异表达的miRNAs数为93个,其中58个差异显著。对miRNAs靶基因调控通路进行KEGG分析,在两文库中发现了包括Wnt、MAPK等在内的309个调控通路。用miReap软件筛选两文库中的未知miRNAs,分别获得了208和215个候选序列,对应的靶基因数分别为22927和22929。将这些miRNAs与miRBase21.0中其他物种的miRNAs比对,发现大多miRNAs都可比对到数据库中已有的序列,其中27个为新miRNAs。广灵大尾羊与小尾寒羊尾脂中差异表达的候选新miRNAs数为175个,其中105个差异显著。2.荧光定量PCR结果表明,miR-10b、miR-29a、miR-30c、miR-155、miR-192、 miR-206在广灵大尾羊和小尾寒羊尾脂中的表达量存在显著差异,变化趋势和测序数据一致,推测其可能参与绵羊尾脂中脂肪的代谢。3.基于一般线性模型的方差分析表明,品种对所有6个miRNAs的表达均有显著影响(P0.01或P0.001)。miR-155和miR-192在小尾寒羊中的表达量高于广灵大尾羊,其他4个miRNAs则低于广灵大尾羊。性别只对miR-206的表达影响显著(P0.05)。品种与性别的互作对miR-30c和miR-206的表达量影响显著(P0.01)。品种与月龄的互作对miR-10b、miR-30c和miR-155的表达量影响显著(P0.01或P0.001)。关联分析表明,miR-10b、miR-29a和miR-192与所有性状都呈正相关,其中,与体重、胴体重、绝对尾脂重和相对尾脂重间的相关显著(P0.05或P0.01)。此外,miR-29a与尾长和屠宰率间的相关也达显著水平(P0.05)。miR-30c与所有性状也呈正相关,但只与尾宽的相关显著(P0.05)。miR-155和miR-206与所有性状都呈负相关,miR-206与除屠宰率外的其他6个性状间的负相关显著(P0.05或P0.01)。各miRNAs的表达与性状的关联与其在脂肪代谢中的作用相符。本研究对不同脂尾型绵羊尾脂组织中miRNAs表达谱的分析和关联研究结果为阐明miRNA调节脂肪代谢的机制提供了科学依据,也为进一步研究miRNAs在转录后水平调节脂肪代谢相关基因的功能奠定了基础。
[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.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826;Q78
本文编号：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.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826;Q78
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