本文選題：北京鴨 + 胸肌發(fā)育�。� 參考：《西北農(nóng)林科技大學(xué)》2014年博士論文

【摘要】：北京鴨是世界著名的肉鴨品種，其中” Z型北京鴨瘦肉型配套系”具有飼料轉(zhuǎn)化效率高、瘦肉率高的特點，是研究禽類骨骼肌發(fā)育的極好模型，但其骨骼肌發(fā)育研究尚不多見。但是，骨骼肌形成與再生的分子機制遠(yuǎn)非單個調(diào)節(jié)因子作用的加減，而是所有調(diào)節(jié)因子相互作用的網(wǎng)絡(luò)。因此，以大規(guī)模的數(shù)據(jù)分析為基礎(chǔ)尋找不同生長階段北京鴨胸肌發(fā)育的差異表達(dá)基因及差異表達(dá)基因與北京鴨胸肌的關(guān)系研究是深度挖掘骨骼肌發(fā)育機制的基礎(chǔ)。 本研究應(yīng)用抑制消減雜交（SSH）和RNA-seq技術(shù)分離鑒定了不同生長階段北京鴨胸肌發(fā)育差異表達(dá)基因。分析了經(jīng)典的骨骼肌發(fā)育調(diào)控基因（MSTN、IGF1和FOXO3）分子特征、它們在北京鴨胸肌發(fā)育過程中的表達(dá)變化規(guī)律及其與胸肌發(fā)育之間的關(guān)系。同時，開展了新發(fā)現(xiàn)的MUSTN1基因分子特征、時空表達(dá)模式及其與北京鴨胸腿肌發(fā)育的關(guān)系以及北京鴨胸肌內(nèi)活體轉(zhuǎn)染pcDNA3.1（+）-MUSTN1真核表達(dá)載體以探索MUSTN1基因?qū)Ρ本喰丶“l(fā)育的作用。試驗方法及結(jié)果如下： 1、應(yīng)用SSH技術(shù)分離鑒定不同生長階段北京鴨胸肌發(fā)育差異表達(dá)基因 應(yīng)用SSH技術(shù)從2周齡和6周齡北京鴨胸肌SSH文庫中分離得到257個差異表達(dá)基因（165個已知基因和92個未知基因）。多數(shù)差異表達(dá)基因在6周齡北京鴨胸肌內(nèi)高表達(dá)且與機體的能量代謝和蛋白合成有關(guān)。利用qRT-PCR分析了23個差異表達(dá)基因在北京鴨2、4、6、8周齡胸肌中的表達(dá)模式，通過聚類分析可以將這23個基因分為表達(dá)量持續(xù)下降、表達(dá)量先升高后下降和表達(dá)量持續(xù)上升等三種類別。 2、應(yīng)用RNA-seq技術(shù)分離鑒定不同生長階段北京鴨胸肌發(fā)育的差異表達(dá)基因 對2、4、6周齡北京鴨胸肌和皮脂組織進(jìn)行cDNA文庫構(gòu)建及RNA-seq測序與分析，共得到21800萬個高質(zhì)量序列，占原始序列的95.76%。生物信息學(xué)分析發(fā)現(xiàn)這些高質(zhì)量序列在鴨子參考基因上隨機分布，覆蓋了鴨子已注釋基因的89.4%，序列的RPKM值與熒光定量值整體趨勢一致。相同時期組織間及同一組織不同生長階段間的差異表達(dá)基因數(shù)量從幾百個到數(shù)千個，其中組織間差異表達(dá)基因遠(yuǎn)多于生長階段間，而皮脂間差異表達(dá)基因多于胸肌間。GO和KEGG分析發(fā)現(xiàn)，差異表達(dá)基因被富集到不同GO條目中，而大量差異表達(dá)基因（如IGF1R、IGF1、MSTN、TGFβ3、TGFβ1、TGIF1、FOXO6、FOXO3等）被顯著富集到肌肉發(fā)育及脂肪沉積的信號通路中，僅MAPK通路就富集了214個差異表達(dá)基因。 3、肌肉抑制素基因（MSTN）分子特征及其多態(tài)性與北京鴨胸肌性狀的關(guān)聯(lián)分析 應(yīng)用qRT-PCR技術(shù)檢測MSTN基因在第1d和2、4、6、8周齡北京鴨胸肌中的表達(dá)量發(fā)現(xiàn)：從1d到4周齡該基因表達(dá)量逐漸升高，但6周齡的表達(dá)量顯著低于4周齡和8周齡的表達(dá)量。分子克隆及生物信息分析發(fā)現(xiàn)，，北京鴨MSTN基因的CDS區(qū)長度為1128bp，其編碼蛋白質(zhì)與多數(shù)脊椎動物同源蛋白質(zhì)具有很高的相似性。MSTN基因具有2個可變剪切體，分別編碼375個氨基酸序列和251個氨基酸序列。通過對368只6周齡北京鴨該基因的克隆、SNP掃描、分型及關(guān)聯(lián)分析發(fā)現(xiàn)：該基因含有3個SNP位點，其中T129C位點BB基因型鴨子的胸肌厚顯著大于AA基因型鴨子的胸肌厚，T952C位點AB基因型鴨子的龍骨長顯著長于BB基因型鴨子的龍骨長。 4、IGF1和FOXO3表達(dá)模式及出雛后北京鴨胸腿肌發(fā)育關(guān)系研究 北京鴨胸腿肌在第2d和2、4、6周齡石蠟切片表明，北京鴨胸肌發(fā)育落后于腿肌，其中胸肌在4~6周齡生長最快，而腿肌在2~4周齡生長速度最大。應(yīng)用qRT-PCR技術(shù)檢測IGF1和FOXO3在2d和2、4、6周齡胸腿肌中表達(dá)情況表明，IGF1分別在第6周和第2周胸肌和腿肌中的表達(dá)量最大，而FOXO3分別在第6周和第2周胸肌內(nèi)表達(dá)量最小。 5、MUSTN1基因分子特征及其與北京鴨胸肌發(fā)育關(guān)系研究 經(jīng)過對北京鴨MUSTN1基因cDNA和DNA序列克隆、拼接與分析發(fā)現(xiàn)，該基因編碼一個含有78個氨基酸的蛋白質(zhì)，由3個外顯子和2個內(nèi)含子組成，其啟動子區(qū)包含許多轉(zhuǎn)錄因子結(jié)合位點。MUSTN1基因在北京鴨胸肌、腿肌中的表達(dá)量分別在5周齡和3周齡達(dá)到峰值�；铙w轉(zhuǎn)染pcDNA3.1+MUSTN1表達(dá)載體到北京鴨胸肌后，該重組載體能夠在北京鴨胸肌中高效表達(dá)，同時引起一些骨骼肌發(fā)育調(diào)控基因表達(dá)量的明顯變化。 綜上所述，本研究分離鑒定了北京鴨胸肌發(fā)育不同階段的差異表達(dá)基因。對部分經(jīng)典的骨骼肌發(fā)育調(diào)控基因及新發(fā)現(xiàn)的MUSTN1基因進(jìn)行了分子特征及其與北京鴨胸肌發(fā)育的關(guān)系研究。本研究結(jié)果將對深入探索北京鴨骨骼肌發(fā)育機制提供基礎(chǔ)。
[Abstract]:Beijing duck is a famous breed of meat ducks in the world. The "Z type Beijing duck lean type matching line" has the characteristics of high feed conversion efficiency and high lean meat rate. It is an excellent model for studying the development of skeletal muscle in poultry. However, the study of skeletal muscle development is not very common. However, the molecular mechanism of skeletal muscle formation and regeneration is far from a single regulatory factor. It is the network of all regulating factors interacting with each other. Therefore, on the basis of large-scale data analysis, the study on the relationship between differentially expressed genes in the development of Beijing ducks at different growth stages and the relationship between differentially expressed genes and the breast muscle of Beijing duck is the basis for deep excavation of skeletal muscle development machine.
In this study, the differential expression genes of the breast muscle development of Beijing ducks at different growth stages were isolated and identified by subtractive subtractive hybridization (SSH) and RNA-seq technology. The characteristics of the classical skeletal muscle development regulation genes (MSTN, IGF1 and FOXO3) were analyzed, and their expression changes in the development of Beijing duck breast muscle and their relationship with the development of the chest muscle were also analyzed. At the same time, the molecular characteristics of the MUSTN1 gene, the spatio-temporal expression pattern and the relationship with the development of the Beijing duck breast and leg muscles, and the transfection of the pcDNA3.1 (+) -MUSTN1 eukaryotic expression vector in the living body of the Beijing duck breast muscle were carried out to explore the effect of the MUSTN1 gene on the development of the breast muscle of Beijing duck. The test method and results were as follows:
1, isolation and identification of differentially expressed genes of Beijing duck pectoral muscle at different growth stages by SSH technology.
257 differentially expressed genes (165 known genes and 92 unknown genes) were isolated from the 2 week and 6 week old Beijing duck breast muscle SSH library by SSH technology. Most of the differentially expressed genes were highly expressed in the 6 week old Beijing duck breast muscle and related to the body's energy metabolism and protein synthesis. 23 differentially expressed genes were analyzed by qRT-PCR. The expression patterns in the 2,4,6,8 week old breast muscle of Beijing duck can be divided into three categories by clustering analysis. These 23 genes can be divided into three categories: the expression quantity continues to decline, the expression level increases first and the expression amount continues to rise.
2, isolation and identification of differentially expressed genes of Beijing duck pectoral muscle at different growth stages by RNA-seq technology.
A total of 218 million high quality sequences were obtained from the construction of the cDNA Library of the 2,4,6 week old Beijing duck breast and sebum tissues. A total of 218 million high quality sequences were obtained. The 95.76%. bioinformatics analysis of the original sequence found that these high quality sequences were randomly distributed on duck reference genes, covering 89.4% of the annotated duck genes, the RPKM value of the sequence and the fluorescence of the sequence. The overall trend of light quantitative values is consistent. The number of differentially expressed genes between intertissue and the same tissue in the same period from several hundred to thousands, of which the intertissue differentially expressed genes are far more than between the growth stages, and the differentially expressed genes in the sebum are more than the.GO and KEGG analysis between the pectoral muscles. In the same GO entry, a large number of differentially expressed genes (such as IGF1R, IGF1, MSTN, TGF beta 3, TGF beta 1, TGIF1, FOXO6, FOXO3, etc.) were significantly enriched in the signaling pathways of muscle development and fat deposition, and only 214 differentially expressed genes were enriched by MAPK pathway.
3, molecular characterization and polymorphism of myostatin gene (MSTN) and its association with pectoral muscle traits in Beijing ducks
The expression of MSTN gene in Beijing duck breast muscles at the age of 1D and 2,4,6,8 weeks was detected by qRT-PCR technique. The expression of the gene expression was gradually increased from 1D to 4 weeks old, but the expression amount at the age of 6 weeks was significantly lower than that of 4 weeks and 8 weeks of age. Molecular cloning and bioinformatics analysis found that the length of the CDS region of the Beijing duck MSTN gene was 1128bp. The code protein has a high similarity with most vertebrate homologous proteins with 2 variable shear bodies, which encode 375 amino acid sequences and 251 amino acid sequences. Through the cloning of the 368 6 week old Beijing duck gene, SNP scanning, typing and correlation analysis found that the gene contains 3 SNP loci, of which T129C The breast muscle thickness of the duck with the locus BB genotype was significantly greater than that of the AA genotype duck, and the length of the T952C locus AB duck was longer than that of the BB genotypic duck.
4, the expression pattern of IGF1 and FOXO3 and the relationship between the development of the pectoral muscles of Beijing ducks after hatching
The paraffin sections of Beijing duck chest and leg muscles at the age of 2D and 2,4,6 show that the development of the breast muscle in Beijing ducks is lagging behind the leg muscles, of which the growth of the chest muscles is fastest at the age of 4~6 weeks and the leg muscle grows at the age of 2~4. The expression of IGF1 and FOXO3 in the chest and leg muscles at the age of 2D and 2,4,6 weeks by qRT-PCR technique shows that IGF1 is in the chest muscles of sixth and second weeks, respectively. The expression level of leg muscle was the largest, while FOXO3 was the smallest in sixth weeks and second weeks.
5, the molecular characteristics of MUSTN1 gene and its relationship with the development of pectoral muscles in Beijing ducks
After cloned the cDNA and DNA sequences of the Beijing duck MUSTN1 gene, it was found that the gene encodes a protein containing 78 amino acids, consisting of 3 exons and 2 introns. The promoter region contains a number of transcription factor binding site.MUSTN1 genes in the breast muscle of Beijing duck, and the expression of the leg muscles at 5 weeks and 3 weeks, respectively. When the pcDNA3.1+MUSTN1 expression vector was transfected into the Beijing duck breast muscle, the recombinant vector could be expressed efficiently in the breast muscle of Beijing duck, and the expression of some skeletal muscle development regulation genes was obviously changed.
To sum up, this study isolated and identified the differentially expressed genes in the different stages of the development of the Beijing duck breast muscle. The molecular characteristics of some classic skeletal muscle development regulation genes and the newly discovered MUSTN1 genes and their relationship with the development of the Beijing duck breast muscle were studied. The results of this study will explore the mechanism of skeletal muscle development in Beijing duck. For the foundation.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：S834

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