【作者】 朱正茂；

【导师】 李奎； 赵书红；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2004， 博士

【摘要】 猪是为人类提供动物蛋白的最主要畜种之一，提高产肉性状一直是养猪业追求的目标。猪瘦肉产量和质量在遗传因素上主要取决于胚胎发育期肌纤维数量的增生和生后期(postnatal)肌纤维体积的膨大，但骨骼肌的生长发育过程十分复杂，涉及大批基因的表达及网络式调控，从而增加了应用标记辅助选择等现代育种方法改良这类性状的困难。大批量基因在骨骼肌不同发育阶段表达谱的建立，以及对猪基因的结构、功能及与性状表型关系等的充分了解，则将是应用分子育种技术对猪产肉性状进行遗传改良的基础。 本论文利用cDNA宏阵列(cDNA Macroarray)技术分析了来源于猪肌肉组织cDNA文库的327个EST克隆在胚胎发育期差异表达的基因，并对部分差异表达基因进行了比较深入的探讨，取得了如下结果： 1．对cDNA Macroarray技术的相关技术条件进行了优化，在本研究室建立了大规模阵列表达数据的统计分析方法。应用杜洛克猪和通城猪胚胎发育33天到75天的眼肌RNA为材料，在杜洛克中经分析发现98条EST序列在不同发育期有差异表达，代表43个已知基因和27条无明显匹配的EST。通城猪中发现有138条EST序列在不同发育期有差异表达，代表58个已知基因和52条无明显匹配的EST。在这些差异表达基因中，有11个已知基因表现为上调表达模式，有25个基因表现为下调表达模式，有16个已知基因表现为波浪式表达模式。 2．首次发现血红蛋白相关基因的表达随着骨骼肌细胞分化的完成表达水平呈现显著的差异下调表达。血红素beta链(Hemoglobin beta chain)(最大2.6倍变化)，血红素alpha链(Hemoglobin alpha chain)(最大2.9倍变化)，Beta-1金属结合血红蛋白(Beta-1-metal binding globulin)(最大2.4倍变化)，转铁蛋白(transferrin)(最大2.45倍变化)。 3．进一步证实了绝大多数核糖体组成因子在细胞分化前后呈现下调表达的规律，本研究结果发现有12个核糖体组成因子的表达水平发生了改变。他们分别为Sus scrofa ribosomal protein L3在通城猪中先上调后下调，而在杜洛克中表现为下调，ribosomal protein S10表现为先上调后下调的表达模式，从33天到55天表达水平上升，从55天到75天表达量下降，而且这两者在通城猪中的表达量高于杜洛克品种。mitochondrial ribosomal protein L42，T09551 ribosomal protein L4，ribosomal protein L29／cell surface heparin binding protein HIP在这段时间内上调表达。40S ribosomal protein S7，40S应用cDNA宫阵列技术欲砚翻奋茶健冬产肉性状的候选基因ribosomal Protein 520，605 aeidie ribosomal Protein PO，605 ribosomal Protein L37a，405dbosomal protein s3A，605 aeidic ribosomalprotein’ pl一致表现为下调表达。 4.层次聚类法聚类结果表明来源于同一基因的EST聚在一起，如UniGene Cluster (Ssc.7157)中的17个EST中，仅E3一aae一b一06没有聚在一类:功能相近的基因被聚类在一起，如与核糖体相关基因的EST聚在一起。表明这种聚类关系可以对基因功能的研究提供比较有效的信息。同时发现在两个品种中胚胎发育第33天与第45天左右的基因表达谱相近:而通城猪65天到75天的表达谱相近，杜洛克猪变化较大，而通城猪变化比较平缓。 5.采用。DNA末端快速扩增方法获得了差异表达EST M165，M63的cDNA序列，部分获得了EST MZ18的3‘端序列，并获得了EST M165同源基因cavcohn一3的全长cDNA克隆。M63的cDNA序列推导的氨基酸序列与人未知功能基因PNAS一4有86%的同源。EST MZ18的3‘端序列在人与鼠的NCBI各数据库中均未发现同源基因或片段，性状关联分析结果表明该基因对产肉性状和肉质性状有明显的影响。 6，表达谱分析结果显示cavcohn一3是一个骨骼肌特异表达因子，仅在骨骼肌组织和心脏器官中表达。在通城猪和杜洛克猪胎儿骨骼肌发育期的表达规律一致，从33天到75天表达量逐渐上升，在第75天左右达到最大，在此之后表达量逐渐下降。统计结果表明在通城猪品种中最高表达量与最低表达量相差1 .75倍，在杜洛克猪中的比值高达1 .9倍，通城猪的最高表达量是杜洛克猪最低表达量的2.6倍。猪CGI一146基因在各组织中均有表达，在心，肝，脾脏中表达量较高，在胎儿骨骼肌发育阶段表现为差异表达，在杜洛克猪中，仅在第33天左右检测到较高的表达，在通城猪中，在第45天左右检测到较高的表达，在第55天和65天有较微弱的表达，而在其它阶段则未检测到表达信息。从统计学角度差异的标准判断，EST M218在胎儿骨骼肌发育阶段也表现出差异表达。 7.用猪x啮齿类体细胞杂种板将三个新分离的猪差异表达基因进行染色体区域定位，用猪辐射杂种板进行染色体精细定位。EST M 165(cavcohn一3)的区域定位结果为ssC 1 3C q23一(l/2 q41)，染色体精细定位结果为M165(eaveolin一3)与Sw96o紧密连锁，Lon值为3.47:EsT MZls的区域定位结果可能为SSCo7o(q26)或者SSC7E(qlZ一q23)，染色体精细定位结果为MZ18与5 SC 12B09紧密连锁，LOD值为2.99，综合分析表明EsT MZ 18的区域定位结果只能为SSCo7G(q26):M63(PNAS一4)的区域定位结果为SSC 1 OA(pll一pl6)染?更多还原

【Abstract】 Pig serves as an important source of protein for human consumption as one of the major meat animals. Swine industries have been pursuing higher lean growth rate and better meat quality in pigs as two of their highest priority goals for profitability for many years. Muscle fiber characteristics are critical for meat quality, pigs with greater muscle fiber numbers and moderate fiber size can produce better pork. The number of muscle fibers is determined at the prenatal stage, while the fiber sizes are determined in a postnatal development process. In order to make the genetic improvement in growth rate and meat quality traits of pigs by using the molecular breeding strategy, the useful approaches to identify candidate genes related to rapid muscle growth and better meat production in pigs is to understand the variation of gene expression patterns in different developmental stages together with the understanding of gene structure and it’s function.The objective of this thesis was to monitor-the gene activities in different developmental stages of porcine fetal’ skeletal muscle and to isolate, characterize some of those differential expression genes. The main results are as follows:1. The condition of related techniques of cDNA Macroarray was improved by trial and error; linear mixed models were used to mine the useful information of the porcine cDNA macroarry data set. 98 ESTs were revealed differential expression among the five swine developmental stages in Duroc within a total of 327 expressed-sequence tags (ESTs) in the analyses, which represents 43 known genes and 27 ESTs with no good matchs. 138 ESTs were revealed differential expression among the five swine developmental stages in Tongcheng pig within a total of 327 expressed-sequence tags (ESTs) in the analyses, which represents 58 known genes and 52 ESTs with no good macths. Browse those genes in the OMIM database, it was found that NUDT3 , ACTN2 , cavec4in-M , transferring , ribosomal protein L3, ribosomal protein S7, 60S acidic ribosomal protein PI, ribosomal protein s20, ribosomal protein, large PO, and vitamin binding protein etc. genes had differential expression in the other researcher’s result.2. The fators related to the Hemoglobin function include Hemoglobin beta chain, Hemoglobin alpha chain, Beta-1-metal binding globulin, Transferrin show a significant downregulation expression during the fetal’s stage from the 33thday to the 75thday.3. RP factors were found differential expression during the stage from the 33thday to the 75thday. The downregulated genes include 40S ribosomal protein S7, 40S ribosomal proteinS20, 60S acidic ribosomal protein PO, 60S ribosomal protein L37a, 40S ribosomal protein S3 A, 60S acidic ribosomal protein PI; the upregulated genes included mitochondrial ribosomal protein L42, T09551 ribosomal protein L4 and ribosomal protein L29/cell surface heparin binding protein HIP. Sus scrofa ribosomal protein L3 was first upregulated in the Tongcheng pig from the 33thd to the 55thd and then down regulated from the 55th d to the 75thd, but in Duroc, it was a downregulated gene; the gene ribosomal protein S10 shows upregulated from the 33thd to the 55thd and then downregulated from the 55.thd to the 75thd.4. Hierarchical Clustering results displayed that the EST came from Ae same gene were in a cluster, for example, 16 ESTs from UniGene Cluster (Ssc.7157) which represented the gene Chain B of Carbonmonoxy-Haemoglobin were in a cluster except one EST did not in the cluster. And the genes that had the similar function or joined the same process were most likely clustered together, for example, EST (E6 acc-d-07 (ribosomal protein L3), E2-ab-g-07(ribosomal protein S7), E2-aa-h-ll (60S acidic ribosomal protein PI), E2-ab-f-ll (ribosomal protein s20), E3-aaf-a-09(ribosomal.protein,large P0))and so on were clustered closely. This could give some clue for the understanding -the function of new genes. The results also indicated that development stages with similar gene expression action mode were clustered together. The 33thd and the更多还原