蒙古马高负荷运动训练前后转录组与miRNA文库特征及关联分析
发布时间：2019-01-27 14:25
【摘要】：蒙古马是典型的草原马种,经历了漫长的自然选择和人工选择,在草原上以半野生状态生存,所以蒙古马能适应极端恶劣的气候环境及粗放的饲养条件。其基因组中蕴藏着大量的与耐力、抗病、抗寒相关的具有重要利用价值的基因。本实验对6匹蒙古马进行了4个月高负荷运动训练,分别在训练前后采集血液和肌肉样品,检测血液中生理生化指标的变化情况的同时,利用二代测序技术对肌肉样品分别建立转录组和mi RNA文库,对训练前后表达量差异显著的基因进行GO功能富集和KEGG Pathway分析,找到训练前后发生显著变化的代谢通路及运动相关候选基因,并将转录组数据与差异mi RNA靶基因进行关联性分析,绘制蒙古马高负荷训练前后mi RNA与差异表达m RNA的靶向调控网络关系图。然后在体外培养的蒙古马骨骼肌细胞上进行载体构建与细胞转染实验,从而在细胞水平上验证了mi RNA与m RNA的靶向调控关系。经过实验分析本文得到的主要研究结果如下:1.训练后血液中血糖、肌酸激酶、平均血红蛋白含量、平均血红蛋白浓度、血小板计数、血小板压积显著上升;而尿素、血红蛋白、血红细胞、白细胞、淋巴细胞、单核细胞、中性粒细胞、红细胞分布宽度显著下降;血小板平均体积、血细胞比容、红细胞平均容量、乳酸脱氢酶、天冬氨酸转移酶、丙氨酸氨基转移酶训练前后虽然有变化,但不显著。2.转录组数据库筛选到了1102个显著差异基因,其中299个在训练后发生上调,803个下调。这些差异基因同时被注释到3398条GO terms与193条pathway上。其中与运动学紧密相关的生物过程有:肌肉结构发育、心血管系统发育、循环系统发育、肌肉组织发育、肌肉器官发育、肌肉收缩、肌细胞分化、扩张型心肌病、肥厚性心肌病、心肌收缩、钙信号通路、肌动蛋白骨架调节等。3.mi RNA文库共筛选得到248个差异mi RNA,其中54个上调,194个下调。将差异显著的mi RNA进行靶基因预测后进行GO功能富集分析发现,涉及的生物学过程包括:发育过程、解剖结构发育、蛋白质结合、酶结合等。并对531个靶基因进行了KEGG pathway注释,在210条被注释的信号通路中,显著富集并与运动相关的pathway包括:调节肌动蛋白细胞骨架,钙信号通路、心肌收缩等经典信号通路等。4.把mi RNA与转录组文库获得的数据结果进行关联分析,筛选出29个mi RNA对236个基因具有显著靶向调控作用。其中15个上调mi RNA靶向下调68个m RNA;14个下调mi RNA靶向上调168个m RNA。将mi RNA的靶基因结合差异表达基因进行GO功能富集和KEGG Pathway分析,涉及与运动直接相关的通路有:肌动蛋白细胞骨架调节、钙离子信号通路、心肌收缩、肥厚性心肌病,扩张型心肌病等。5.对mi RNA-1及其靶基因IGF1进行载体构建和细胞转染实验,通过RT-PCR检测mi RNA-1、Pre-mir-1及IGF1相对表达量,结果说明Pre-mir-1成功进入骨骼肌细胞,加工为成熟的mi RNA-1,抑制沉默了靶基因IGF1的表达,验证了mi RNA-1与IGF1靶向调控关系及m RNA-mi RNA联合分析结果的正确性。
[Abstract]:The Mongolian horse is a typical grassland horse species, has undergone a long natural selection and a manual selection, and is living in a semi-wild state on the grassland, so the Mongolian horse can adapt to the extreme weather environment and the rough feeding condition. The genome contains a large number of genes with important utilization value related to endurance, disease resistance and cold resistance. A four-month high-load exercise training was carried out on 6 Mongolian horses, blood and muscle samples were collected before and after the training, and the change of the physiological and biochemical indexes in the blood was detected, and the transcriptome and the mi-RNA library were respectively established by the second-generation sequencing technique. carrying out GO function enrichment and KEGG Pathway analysis on a gene with significant difference in pre-and post-training expression quantity, finding a metabolic pathway and a motion-related candidate gene which change significantly before and after training, and carrying out relevance analysis on the transcription group data and the differential mi RNA target gene, The relationship between the mi-RNA and the differentially expressed m-RNA in the high-load training of the Mongolian horse was plotted. and then carrying out vector construction and cell transfection experiments on the Mongolian horse skeletal muscle cells cultured in vitro, so as to verify the targeted regulation and control relation of the mi RNA and the m RNA at the cell level. The main results obtained in this paper are as follows: 1. blood glucose, creatine kinase, average hemoglobin content, mean hemoglobin concentration, platelet count, and platelet pressure in the blood after training are significantly increased; and urea, hemoglobin, red blood cells, white blood cells, lymphocytes, monocytes, neutrophils, The distribution of the red blood cells decreased significantly; the mean volume of platelets, the specific volume of the blood cells, the average capacity of the red blood cells, the lactate dehydrogenase, the aspartate transferase, and the alanine aminotransferase were varied before and after the training, but not significant. The transcriptome database screened 1102 significant difference genes, of which 299 were up-regulated and 803 down-regulated after training. These differential genes were also annotated to 3398 GO terms and 193 patway. in which the biological processes closely related to the kinematics are: muscle structure development, cardiovascular system development, circulatory system development, muscle tissue development, muscle organ development, muscle contraction, muscle cell differentiation, dilated cardiomyopathy, hypertrophic cardiomyopathy, myocardial contraction, calcium signal pathway, A total of 248 differential mi-RNAs were screened by .3.mi-RNA libraries, such as actin cytoskeleton regulation, of which 54 were up-regulated and 194 down-regulated. After the target gene is predicted by the difference significant mi RNA, it is found that the biological process involved includes the development process, the development of the anatomical structure, the protein binding, the enzyme binding, and the like. The KEGG patway annotation was carried out on 531 target genes, and in the signal path annotated with 210, significant enrichment and movement-related patway included the regulation of the actin cytoskeleton, the calcium signal pathway, the classical signal pathway such as the myocardial contraction, and the like. The results of the data obtained from the mi-RNA and the transcription-group library were analyzed, and 29 mi-RNA was selected to have a significant targeted regulatory effect on the 236 genes. Of these, 15 up-regulated mi-RNA was targeted to down-regulate 68 m-RNA, and 14 down-regulated mi-RNA targets up to 168 m-RNA. The target gene of mi-RNA was combined with the differentially expressed genes for GO-function enrichment and the KEGG pathway analysis, which involved the direct-related pathway of actin cytoskeleton regulation, calcium ion signal pathway, myocardial contraction, hypertrophic cardiomyopathy, dilated cardiomyopathy, and the like. The expression of mi-RNA-1, Pre-Mir-1 and IGF1 was detected by RT-PCR, and the expression of the target gene IGF1 was suppressed by the expression of mi-RNA-1, Pre-mir-1 and IGF1 by RT-PCR. The relationship between the targeting and control of mi-RNA-1 and IGF1 and the results of the combined analysis of m-RNA-mi RNA were verified.
【学位授予单位】：内蒙古农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S821.81
本文编号：2416350
[Abstract]:The Mongolian horse is a typical grassland horse species, has undergone a long natural selection and a manual selection, and is living in a semi-wild state on the grassland, so the Mongolian horse can adapt to the extreme weather environment and the rough feeding condition. The genome contains a large number of genes with important utilization value related to endurance, disease resistance and cold resistance. A four-month high-load exercise training was carried out on 6 Mongolian horses, blood and muscle samples were collected before and after the training, and the change of the physiological and biochemical indexes in the blood was detected, and the transcriptome and the mi-RNA library were respectively established by the second-generation sequencing technique. carrying out GO function enrichment and KEGG Pathway analysis on a gene with significant difference in pre-and post-training expression quantity, finding a metabolic pathway and a motion-related candidate gene which change significantly before and after training, and carrying out relevance analysis on the transcription group data and the differential mi RNA target gene, The relationship between the mi-RNA and the differentially expressed m-RNA in the high-load training of the Mongolian horse was plotted. and then carrying out vector construction and cell transfection experiments on the Mongolian horse skeletal muscle cells cultured in vitro, so as to verify the targeted regulation and control relation of the mi RNA and the m RNA at the cell level. The main results obtained in this paper are as follows: 1. blood glucose, creatine kinase, average hemoglobin content, mean hemoglobin concentration, platelet count, and platelet pressure in the blood after training are significantly increased; and urea, hemoglobin, red blood cells, white blood cells, lymphocytes, monocytes, neutrophils, The distribution of the red blood cells decreased significantly; the mean volume of platelets, the specific volume of the blood cells, the average capacity of the red blood cells, the lactate dehydrogenase, the aspartate transferase, and the alanine aminotransferase were varied before and after the training, but not significant. The transcriptome database screened 1102 significant difference genes, of which 299 were up-regulated and 803 down-regulated after training. These differential genes were also annotated to 3398 GO terms and 193 patway. in which the biological processes closely related to the kinematics are: muscle structure development, cardiovascular system development, circulatory system development, muscle tissue development, muscle organ development, muscle contraction, muscle cell differentiation, dilated cardiomyopathy, hypertrophic cardiomyopathy, myocardial contraction, calcium signal pathway, A total of 248 differential mi-RNAs were screened by .3.mi-RNA libraries, such as actin cytoskeleton regulation, of which 54 were up-regulated and 194 down-regulated. After the target gene is predicted by the difference significant mi RNA, it is found that the biological process involved includes the development process, the development of the anatomical structure, the protein binding, the enzyme binding, and the like. The KEGG patway annotation was carried out on 531 target genes, and in the signal path annotated with 210, significant enrichment and movement-related patway included the regulation of the actin cytoskeleton, the calcium signal pathway, the classical signal pathway such as the myocardial contraction, and the like. The results of the data obtained from the mi-RNA and the transcription-group library were analyzed, and 29 mi-RNA was selected to have a significant targeted regulatory effect on the 236 genes. Of these, 15 up-regulated mi-RNA was targeted to down-regulate 68 m-RNA, and 14 down-regulated mi-RNA targets up to 168 m-RNA. The target gene of mi-RNA was combined with the differentially expressed genes for GO-function enrichment and the KEGG pathway analysis, which involved the direct-related pathway of actin cytoskeleton regulation, calcium ion signal pathway, myocardial contraction, hypertrophic cardiomyopathy, dilated cardiomyopathy, and the like. The expression of mi-RNA-1, Pre-Mir-1 and IGF1 was detected by RT-PCR, and the expression of the target gene IGF1 was suppressed by the expression of mi-RNA-1, Pre-mir-1 and IGF1 by RT-PCR. The relationship between the targeting and control of mi-RNA-1 and IGF1 and the results of the combined analysis of m-RNA-mi RNA were verified.
【学位授予单位】：内蒙古农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S821.81
【参考文献】
相关期刊论文 前3条
1 卢鼎厚;;人体运动功能和肌肉损伤[J];体育与科学;2006年05期
2 张海霞,胡扬,田野,许春燕,鲁政,倘绍奎;低氧运动对血清离子浓度的影响及电解质饮料干预的效果[J];中国运动医学杂志;2005年04期
3 李靖,李皓,张蕴琨;耐力训练对力竭运动诱导的大鼠淋巴细胞凋亡的影响[J];中国运动医学杂志;2005年02期
相关博士学位论文 前1条
1 杨丽华;蒙古马运动相关候选基因的分子鉴定[D];内蒙古农业大学;2013年
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