【作者】 徐永杰；

【导师】 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 猪肉是肉类消费的重要组成部分,是人类获得蛋白质营养物质的主要途径之一,在国人饮食结构中占据绝对的比重；而猪不仅可作为肉产品的重要来源,还可作为模式器官动物。因此,深入探讨猪骨骼肌生长发育的机制不仅对畜牧生产有重要意义,同时也为人类医学研究提供重要补充。本研究通过建立的以双向电泳和质谱为基础的蛋白质组学分析平台,开展猪肌肉组织蛋白质组学相关研究,系统分析了国内外典型品种(梅山猪与大白猪)及梅山猪不同发育时期骨骼肌的蛋白质组学差异,并就部分差异蛋白在基因水平做了进一步的研究,以期为阐明猪肌肉品质形成的分子机制奠定了分子基础。取得的主要研究结果如下：1.从不同的样品处理、不同上样量、不同pH IPG胶条的选择、不同二向胶浓度、不同染色方法等方面对猪肌肉组织蛋白双向电泳分离条件进行优化,并就一些常见问题进行了详细分析,从而建立了猪肌肉组织蛋白双向电泳分离技术。结果显示,对于猪肌肉组织样品,采用液氮研磨+超声破碎的策略比单纯液氮研磨更合适,蛋白质溶解性更好,核酸污染少,图谱比较均匀清晰；18cm pH4-7的胶条比pH3-10NL分离效果好；对于银染18cm pH4-7的胶条150μg上样量比较适宜。与前人相比,采用本方法获得图谱的分辨率及重复性都有所提高。2.采用基于双向电泳和质谱技术为基础的蛋白质组学方法对纯种大白猪和梅山猪背最长肌进行蛋白质组学分析。共发现25个差异表达蛋白点,梅山猪中高表达的10个,而大白猪中高表达的15个,其中20个蛋白点经MALDI-TOF-MS方法成功鉴定,得到14个不同的蛋白,这些蛋白按功能可以分为4类：代谢相关蛋白,防御与应激相关蛋白,肌纤维结构相关蛋白,其他未归类蛋白。此外,我们采用荧光定量PCR方法对其中7个差异表达蛋白做了mRNA水平上的表达分析,结果表明蛋白质水平和mRNA水平存在较理想的相关性。同时我们还分析了大白猪和梅山猪背最长肌肌球蛋白重链4种亚型(Ⅰ、ⅡA、ⅠB和ⅡX)在mRNA水平上的表达情况,结果显示Ⅰ型纤维在两品种间没显著差异(P>0.05),ⅡA和ⅡX在梅山猪中显著高表达(P<0.05),而ⅡB在大白猪中显著高表达(P<0.05)。3.利用18cm pH4-7的胶条,第二向12.5%的SDS-PAGE对梅山猪背最长肌总蛋白进行2-DE分离,共得到36块2-DE胶(4*3*3),采用软件对图像进行分析,经one-way ANOVA分析(P<0.01),共发现不同时期间差异表达蛋白点(>1.5倍)共99个,其中胚胎65天与出生后3天相比,有78个(下调27个,上调51个)；出生后3天与60天相比,有70个(下调21个,上调49个)；出生后60天与120天相比,有40个(下调22个,上调18个)。经MALDI-TOF/MS质谱成功鉴定90个蛋白点,得到66个不同的蛋白,按功能分为8类：代谢相关蛋白为最大一类,占25%,包括糖酵解代谢相关酶、线粒体氧化代谢相关酶及其他代谢相关酶类,糖酵解代谢相关酶随着肌肉的生长发育呈上调表达趋势,而线粒体氧化代谢相关酶呈相反趋势；第二大类为肌纤维调节相关蛋白,占19%,包括MLC1sa、MLC1sb、MyBP-H、ACTN3、CapZ-β等；细胞骨架蛋白占11%,包括desmin、cofilin-1、FSCN1、gelsolin等7个,这些蛋白随着肌肉的生长发育均呈下调表达趋势；细胞防御相关蛋白占14%,包括HSP27、Hsp60、Hsp90、DJ1、TCP1等；信号转导相关蛋白占6%；抗氧化与解毒相关蛋白8%；运输蛋白6%；其他未归类蛋白占11%。这些蛋白按细胞组成可分为6类,有43%的属于胞浆蛋白,26%的属于细胞骨架蛋白,6%的属于膜蛋白,11%的为线粒体蛋白,核蛋白占9%。用蛋白质组学数据与梅山猪背最长肌不同发育时期转录表达谱数据比较分析大部分基因在转录水平与翻译水平存在较好的一致性,只是在翻译水平由于存在翻译后修饰,蛋白水平的更为复杂。此外,我们通过设计特意引物对梅山猪背最长肌不同发育时期的肌动蛋白重链的四种亚型进行荧光定量分析,发现在胚胎65天ⅡX和ⅡB型纤维已开始表达,但占比例很少,主要为Ⅰ型纤维(81.5%),而就整个发育过程Ⅰ型纤维由胚胎65天的81.5%递减到出生后120天的15.5%,Ⅱb型纤维却由2.8%递增到48.7%,四种肌纤维在猪肌肉发育过程中处于Ⅰ(?)ⅠA(?)ⅡX(?)ⅡB相互转换的动态平衡状态。另外,我们采用Western Blot方法,选取AFP、vimentin、desmin和HSP90等4个蛋白进行验证分析,发现结果与蛋白质组学数据较一致,进一步说明了我们2-DE和质谱结果的可靠性。4.选取ATP5B、ATP5A、14-3-3ζ等三个差异表达蛋白对应的基因,作了进一步的分析。组织表达谱显示：ATP5B和ATP5A在睾丸、子宫、脂肪等组织相对高表达,心、睾丸、卵巢、小肠等组织次之,肌肉和肺中的表达相对较低,而在各组织中ATP5B的表达量均比ATP5A高；发现14-3-3ζ基因转录本1在脂肪中表达量最高,心脏、小肠和肌肉次之,肝脏、大脑、睾丸、卵巢中等表达,在肾脏和胃中表达量最低,转录本2相对于转录子1在各组织中表达丰度都很低,从两个转录本在不同类型肌肉表达分布来看,14-3-3ζ基因的肌纤维特异性似乎不明显。不同时期发育表达谱显示：ATP5B和ATP5A在3天和胚胎期高表达,生长后期表达量较低,而梅山中在大多数时期都比大白高；14-3-3ζ基因的两个转录子在胚胎65天特异的高表达,这与差异蛋白质组学分析中的该基因蛋白水平表达是一致的,而不同时期品种间相比,两个转录本在大白中的表达均高于梅山。多态性分析：ATP5B基因第八外显子75处有—A/G突变,利用梅山猪和大白猪杂交组建的F2代资源家系对该位点进行关联分析,发现与肥肉率、瘦肥肉比例、内脂率、肌内脂肪含量、肌内水分含量等肉质性状存在显著相关(P<0.05)；14-3-3ζ基因第五外显子77bp处有—T/C突变,该位点只与皮率和瘦肉率两个性状存在显著相关(P<0.05),TC基因型比TT基因型具有更高的瘦肉率更低的皮率,与其他的性状关联不显著。更多还原

【Abstract】 Skeletal muscle represents the most abundant tissue in the adult mammalian body and plays a central role in motility and the control of whole-body metabolism. So understanding of growth and development of skeletal muscle is one of the most important goals in meat production science and is also related to particular aspects of human medicine. In this study, the platform for porcine skeletal muscle proteomics research was established using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The skeletal muscle proteome of different pig breeds and different development stages was analyzed. Screening and molecular biological analysis of differentially expressed proteins were carried. The aim is to understand the molecular mechanism of meat quality formation. The results are as follws:1. The experimental conditions of two-dimensional gel electrophoresis in porcine skeletal muscle was optimized by different experimental conditions, including different sample processing, different loading quantities, different pH IPG strips, different concentration in gels, different methods for gel staining, et al. The results show that the method of grinding in liquid nitrogen and ultrasonication is more appropriate than the method of grinding in liquid nitrogen for porcine skeletal muscle. The better map of two-dimensional gel electrophoresis can be obtained; low nucleic acid contamination and more protein spots can be separated from the method of grinding in liquid nitrogen and ultrasonication. Compared with previous studies, the 2-DE maps from this work have better resolution and repeatability.2. Western and indigenous Chinese pig breeds show obvious differences in muscle growth and meat quality, however, the underlying molecular mechanism remains unclear. In this study, proteome analysis of longissimus dorsi muscle between purebred Meishan and Large White pigs was performed by two-dimensional gel electrophoresis and mass spectrometry. A total of 25 protein spots were differentially expressed in the two breeds. The 14 identified proteins could be divided into 4 groups:energy metabolism, defense and stress, myofibrillar filaments, and other unclassified proteins. Quantitative real-time PCR was used to analyze the partly differentially expressed proteins in mRNA level, which revealed a positive correlation between the content of the proteins and their mRNA levels. We also analyzed the mRNA levels of myosin heavy chain isoforms using quantitative real-time PCR. The results indicated that IIA and IIX fibers were elevated in Meishan pigs, whereas the IIB fiber was more highly expressed in Large White pigs. To the best of our knowledge, this was the first proteomics-based investigation of total skeletal muscle protein in different pig breeds, and these results may provide valuable information for understanding the molecular mechanism responsible for breed specific differences in growth performance and meat quality.3. We used 2-DE method in combination with mass spectrometric.2-DE gels were generated in triplicate at four stages from embryo to mature. Using Image Master 2D Platinum software Version 6.0, protein spots were quantified on every gel at stages 65 dpc, 3d,60d and 120d. Only spots present in all three animals per group and under at least 2 of 4 conditions were taken into consideration. Statistical analysis (one-way ANOVA, P<0.01) revealed 78 proteins significantly altered in expression in 65 dpc compared to 3d (51 up-and 27 down-regulated); 70 altered proteins in 3d compared to 60d (49 up- and 21 down-regulated); and 40 alterations in 60d compared to 120d (18 up- and 22 down-regulated). In total,99 protein spots were altered in expression during development, which was consistently found between neighbouring stages points investigated. Therefore, most differential spots characterized 65dpc animals, whereas 60-day and 120-day adults exhibited similar patterns of expression. For subsequent MALDI-TOF-MS analysis, a total of 90 spots were successfully identified, and matched to 66 different proteins. To compare the functions of differential proteins, we collected the functional annotations (biological process and molecular function) of all identified proteins. It is evident that a large portion (25%) of the proteins has roles in metabolism. The second largest group of proteins (19%) was identified as myofibrillar regulatory protein, whereas 11% could not be classified on the basis of available information. Other major categories comprised of proteins involved in celluar defense and stress (14%), cytoskeleton (11%), signal transduction (6%), antioxidant and detoxification (8%) and transport (6%). The 66 differential proteins in control term placenta localized to the cytoplasm (43%), cytoskeleton (26%), membrane (6%), mitochondria (11%), nucleus (9%), and other (5%). Four proteins (desmin, AFP, vimentin and HSP90) were confirmed by immunoblotting. It is highly likely that these proteins are closely related with the 2-DE and MS results. Comparing with the 2-DE experiments, the results obtained with microarrays were significantly more comprehensive. To demonstrate whether protein expression levels changed during skeletal muscle development can be correlated with their mRNA expression levels, we assessed the differential expression at the RNA level of those proteins that were identified as relatively abundant in either stage. The expression patterns of most differential proteins showed a positive correlation with their gene expression at the transcript level during skeletal muscle development.4. Three differentially expressed proteins, ATP5B, ATP5A and 14-3-3ζwere further analyzed in mRNA expression levels of spatio-temporal, polymorphism detection and association analysis with meat quality traits.更多还原