北京鸭胸肌发育差异表达基因分离鉴定及其与胸肌发育的关系研究

发布时间：2018-07-12 15:59

本文选题：北京鸭 + 胸肌发育　；参考：《西北农林科技大学》2014年博士论文

【摘要】：北京鸭是世界著名的肉鸭品种，其中” Z型北京鸭瘦肉型配套系”具有饲料转化效率高、瘦肉率高的特点，是研究禽类骨骼肌发育的极好模型，但其骨骼肌发育研究尚不多见。但是，骨骼肌形成与再生的分子机制远非单个调节因子作用的加减，而是所有调节因子相互作用的网络。因此，以大规模的数据分析为基础寻找不同生长阶段北京鸭胸肌发育的差异表达基因及差异表达基因与北京鸭胸肌的关系研究是深度挖掘骨骼肌发育机制的基础。本研究应用抑制消减杂交（SSH）和RNA-seq技术分离鉴定了不同生长阶段北京鸭胸肌发育差异表达基因。分析了经典的骨骼肌发育调控基因（MSTN、IGF1和FOXO3）分子特征、它们在北京鸭胸肌发育过程中的表达变化规律及其与胸肌发育之间的关系。同时，开展了新发现的MUSTN1基因分子特征、时空表达模式及其与北京鸭胸腿肌发育的关系以及北京鸭胸肌内活体转染pcDNA3.1（+）-MUSTN1真核表达载体以探索MUSTN1基因对北京鸭胸肌发育的作用。试验方法及结果如下： 1、应用SSH技术分离鉴定不同生长阶段北京鸭胸肌发育差异表达基因应用SSH技术从2周龄和6周龄北京鸭胸肌SSH文库中分离得到257个差异表达基因（165个已知基因和92个未知基因）。多数差异表达基因在6周龄北京鸭胸肌内高表达且与机体的能量代谢和蛋白合成有关。利用qRT-PCR分析了23个差异表达基因在北京鸭2、4、6、8周龄胸肌中的表达模式，通过聚类分析可以将这23个基因分为表达量持续下降、表达量先升高后下降和表达量持续上升等三种类别。 2、应用RNA-seq技术分离鉴定不同生长阶段北京鸭胸肌发育的差异表达基因对2、4、6周龄北京鸭胸肌和皮脂组织进行cDNA文库构建及RNA-seq测序与分析，共得到21800万个高质量序列，占原始序列的95.76%。生物信息学分析发现这些高质量序列在鸭子参考基因上随机分布，覆盖了鸭子已注释基因的89.4%，序列的RPKM值与荧光定量值整体趋势一致。相同时期组织间及同一组织不同生长阶段间的差异表达基因数量从几百个到数千个，其中组织间差异表达基因远多于生长阶段间，而皮脂间差异表达基因多于胸肌间。GO和KEGG分析发现，差异表达基因被富集到不同GO条目中，而大量差异表达基因（如IGF1R、IGF1、MSTN、TGFβ3、TGFβ1、TGIF1、FOXO6、FOXO3等）被显著富集到肌肉发育及脂肪沉积的信号通路中，仅MAPK通路就富集了214个差异表达基因。 3、肌肉抑制素基因（MSTN）分子特征及其多态性与北京鸭胸肌性状的关联分析应用qRT-PCR技术检测MSTN基因在第1d和2、4、6、8周龄北京鸭胸肌中的表达量发现：从1d到4周龄该基因表达量逐渐升高，但6周龄的表达量显著低于4周龄和8周龄的表达量。分子克隆及生物信息分析发现，，北京鸭MSTN基因的CDS区长度为1128bp，其编码蛋白质与多数脊椎动物同源蛋白质具有很高的相似性。MSTN基因具有2个可变剪切体，分别编码375个氨基酸序列和251个氨基酸序列。通过对368只6周龄北京鸭该基因的克隆、SNP扫描、分型及关联分析发现：该基因含有3个SNP位点，其中T129C位点BB基因型鸭子的胸肌厚显著大于AA基因型鸭子的胸肌厚，T952C位点AB基因型鸭子的龙骨长显著长于BB基因型鸭子的龙骨长。 4、IGF1和FOXO3表达模式及出雏后北京鸭胸腿肌发育关系研究北京鸭胸腿肌在第2d和2、4、6周龄石蜡切片表明，北京鸭胸肌发育落后于腿肌，其中胸肌在4~6周龄生长最快，而腿肌在2~4周龄生长速度最大。应用qRT-PCR技术检测IGF1和FOXO3在2d和2、4、6周龄胸腿肌中表达情况表明，IGF1分别在第6周和第2周胸肌和腿肌中的表达量最大，而FOXO3分别在第6周和第2周胸肌内表达量最小。 5、MUSTN1基因分子特征及其与北京鸭胸肌发育关系研究经过对北京鸭MUSTN1基因cDNA和DNA序列克隆、拼接与分析发现，该基因编码一个含有78个氨基酸的蛋白质，由3个外显子和2个内含子组成，其启动子区包含许多转录因子结合位点。MUSTN1基因在北京鸭胸肌、腿肌中的表达量分别在5周龄和3周龄达到峰值。活体转染pcDNA3.1+MUSTN1表达载体到北京鸭胸肌后，该重组载体能够在北京鸭胸肌中高效表达，同时引起一些骨骼肌发育调控基因表达量的明显变化。综上所述，本研究分离鉴定了北京鸭胸肌发育不同阶段的差异表达基因。对部分经典的骨骼肌发育调控基因及新发现的MUSTN1基因进行了分子特征及其与北京鸭胸肌发育的关系研究。本研究结果将对深入探索北京鸭骨骼肌发育机制提供基础。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：S834

【参考文献】