蒙古馬高負荷運動訓(xùn)練前后轉(zhuǎn)錄組與miRNA文庫特征及關(guān)聯(lián)分析
發(fā)布時間：2019-01-27 14:25
【摘要】：蒙古馬是典型的草原馬種,經(jīng)歷了漫長的自然選擇和人工選擇,在草原上以半野生狀態(tài)生存,所以蒙古馬能適應(yīng)極端惡劣的氣候環(huán)境及粗放的飼養(yǎng)條件。其基因組中蘊藏著大量的與耐力、抗病、抗寒相關(guān)的具有重要利用價值的基因。本實驗對6匹蒙古馬進行了4個月高負荷運動訓(xùn)練,分別在訓(xùn)練前后采集血液和肌肉樣品,檢測血液中生理生化指標的變化情況的同時,利用二代測序技術(shù)對肌肉樣品分別建立轉(zhuǎn)錄組和mi RNA文庫,對訓(xùn)練前后表達量差異顯著的基因進行GO功能富集和KEGG Pathway分析,找到訓(xùn)練前后發(fā)生顯著變化的代謝通路及運動相關(guān)候選基因,并將轉(zhuǎn)錄組數(shù)據(jù)與差異mi RNA靶基因進行關(guān)聯(lián)性分析,繪制蒙古馬高負荷訓(xùn)練前后mi RNA與差異表達m RNA的靶向調(diào)控網(wǎng)絡(luò)關(guān)系圖。然后在體外培養(yǎng)的蒙古馬骨骼肌細胞上進行載體構(gòu)建與細胞轉(zhuǎn)染實驗,從而在細胞水平上驗證了mi RNA與m RNA的靶向調(diào)控關(guān)系。經(jīng)過實驗分析本文得到的主要研究結(jié)果如下:1.訓(xùn)練后血液中血糖、肌酸激酶、平均血紅蛋白含量、平均血紅蛋白濃度、血小板計數(shù)、血小板壓積顯著上升;而尿素、血紅蛋白、血紅細胞、白細胞、淋巴細胞、單核細胞、中性粒細胞、紅細胞分布寬度顯著下降;血小板平均體積、血細胞比容、紅細胞平均容量、乳酸脫氫酶、天冬氨酸轉(zhuǎn)移酶、丙氨酸氨基轉(zhuǎn)移酶訓(xùn)練前后雖然有變化,但不顯著。2.轉(zhuǎn)錄組數(shù)據(jù)庫篩選到了1102個顯著差異基因,其中299個在訓(xùn)練后發(fā)生上調(diào),803個下調(diào)。這些差異基因同時被注釋到3398條GO terms與193條pathway上。其中與運動學(xué)緊密相關(guān)的生物過程有:肌肉結(jié)構(gòu)發(fā)育、心血管系統(tǒng)發(fā)育、循環(huán)系統(tǒng)發(fā)育、肌肉組織發(fā)育、肌肉器官發(fā)育、肌肉收縮、肌細胞分化、擴張型心肌病、肥厚性心肌病、心肌收縮、鈣信號通路、肌動蛋白骨架調(diào)節(jié)等。3.mi RNA文庫共篩選得到248個差異mi RNA,其中54個上調(diào),194個下調(diào)。將差異顯著的mi RNA進行靶基因預(yù)測后進行GO功能富集分析發(fā)現(xiàn),涉及的生物學(xué)過程包括:發(fā)育過程、解剖結(jié)構(gòu)發(fā)育、蛋白質(zhì)結(jié)合、酶結(jié)合等。并對531個靶基因進行了KEGG pathway注釋,在210條被注釋的信號通路中,顯著富集并與運動相關(guān)的pathway包括:調(diào)節(jié)肌動蛋白細胞骨架,鈣信號通路、心肌收縮等經(jīng)典信號通路等。4.把mi RNA與轉(zhuǎn)錄組文庫獲得的數(shù)據(jù)結(jié)果進行關(guān)聯(lián)分析,篩選出29個mi RNA對236個基因具有顯著靶向調(diào)控作用。其中15個上調(diào)mi RNA靶向下調(diào)68個m RNA;14個下調(diào)mi RNA靶向上調(diào)168個m RNA。將mi RNA的靶基因結(jié)合差異表達基因進行GO功能富集和KEGG Pathway分析,涉及與運動直接相關(guān)的通路有:肌動蛋白細胞骨架調(diào)節(jié)、鈣離子信號通路、心肌收縮、肥厚性心肌病,擴張型心肌病等。5.對mi RNA-1及其靶基因IGF1進行載體構(gòu)建和細胞轉(zhuǎn)染實驗,通過RT-PCR檢測mi RNA-1、Pre-mir-1及IGF1相對表達量,結(jié)果說明Pre-mir-1成功進入骨骼肌細胞,加工為成熟的mi RNA-1,抑制沉默了靶基因IGF1的表達,驗證了mi RNA-1與IGF1靶向調(diào)控關(guān)系及m RNA-mi RNA聯(lián)合分析結(jié)果的正確性。
[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.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：S821.81
本文編號：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.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：S821.81
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