【作者】 李明洲；

【导师】 李学伟；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 骨骼肌细胞和脂肪细胞在生长发育过程中受到的微效多基因间的互作调控机制是决定猪胴体和肉质等相关数量性状的分子基础。本研究利用包含140个与猪肌肉生长和脂肪沉积密切相关基因的Oligo功能分类基因芯片,检测了脂肪型的太湖猪和瘦肉型的长白猪在出生后不同生长发育阶段,背最长肌（初生,1,2,3,4和5月龄）和背部皮下脂肪（1,2,3,4和5月龄）中相关基因表达谱的变化。①方差分析（ANOVA）和基因分组检验（GCT）结果显示:背最长肌表达谱中,长白猪分别有18和22个基因,太湖猪分别有3和7个基因在月龄间的表达差异达极显著(P_ANOVA＜0.01)和显著水平(P_ANOVA＜0.05),且长白猪中包含10个基因的“参与脂肪酸生物合成的酶”基因分组具有极显著意义(P_ErmineJ＜0.01)。皮下脂肪表达谱中,长白猪有25个基因在月龄间的表达差异达显著水平(P_ANOVA＜0.05),且包含23个基因的“参与脂肪或类固醇代谢的酶和调节蛋白”基因分组在品种间具有极显著意义(P_ErmineJ＜0.01)。提示两猪种骨骼肌生长速度和脂肪沉积能力表型的巨大差异可能与这些基因的差异表达规律相关。②短时间序列表达聚类（STEM）结果显示:背最长肌表达谱中,先降后升、逐渐上升和逐渐下降、逐渐上升分别是长白猪和太湖猪在初生至5月龄间最具代表性的基因表达模式(P_STEM＜0.01),其中正调控肌纤维生长和脂肪酸合成的基因主要表现为逐渐上调,而抑制细胞增殖和正调控脂肪酸β氧化的基因则主要表现为逐渐下降。皮下脂肪表达谱中,长白猪和太湖猪各有两个表达模式分别具有极显著(P_STEM＜0.01)和显著性意义(P_STEM＜0.05),但太湖猪基因表达谱比较分散,占主导优势的表达模式没有长白猪明显,提示太湖猪脂肪细胞内参与相关代谢活动的基因间的调控关系较长白猪复杂。③主成分分析（PCA）结果显示:长白猪和太湖猪的背最长肌表达谱中分别有7和6个基因,皮下脂肪表达谱中分别有16和13个基因的时序表达模式明显偏离其他基因,提示可能受到了特殊的调控。④ANOVA和二维层级聚类的PCA映射结果显示:长白猪分别有42和27个基因,太湖猪分别有51和20个基因在背最长肌和皮下脂肪间的表达差异达极显著(P_ANOVA＜0.01)和显著水平(P_ANOVA＜0.05)。53个与脂肪沉积相关基因在两组织内的时序表达模式大致可分为3类,可按照不同标准筛选存在组织特异性表达模式的基因,为探讨IMF和皮下脂肪沉积的分子机制积累了基础资料。⑤基于动态贝叶斯模型（DBN）构建的基因调控网络（GRNs）从一定程度上揭示了两品种在肌肉生长和脂肪沉积等生理生化代谢活动方面的明显差异。网络中基因的调控地位与已知功能信息大致相符,具有一定的准确性和可信度,可从中挖掘相关性状潜在的关键特征基因。⑥Pathway映射结果显示:两个代表脂肪沉积动态平衡的通路——脂肪酸代谢和脂肪酸生物合成,两个代表脂肪沉积调控的通路——PPAR信号转导和脂肪细胞因子信号转导,所涉及的生物学功能在两猪种间和生长发育过程中可能发生了变化。从一定程度上揭示了不同生产类型的两猪种在脂肪沉积性状上巨大表型差异的分子基础。⑦通过基于表达模式识别（STEM）结果的文献网络子网挖掘,在等级分值较高的子网中寻找到了大量已有相关研究支撑、对宏观表型具有重要影响和研究价值的种子基因,建议可作为影响猪肌肉和脂肪性状的关键特征基因进行深入研究。⑧利用物种人的同源基因预测了本研究背最长肌表达谱中差异表达基因间的转录调控网络（TRNs）,发现26个表达差异达显著水平的基因(P_AVOVA＜0.05和0.01)受到E2F转录因子（E2F1～8）、上游结合蛋白1（LBP-1/UBP1）、激活增强子结合蛋白2γ（AP2-gamma）和PHD锌指转录因子蛋白（BPTF）等27个转录因子（TFs）具有显著性意义的调控(P_TF＜0.05)。提示这些TFs通过调节靶基因的表达,可能对猪肌肉和脂肪性状的最终表型产生重要影响。⑨对片内和片间重复性的评价结果显示:4个片内重复的平均变异系数（?）和3个重复检测芯片间的平均相关系数（?）在皮下脂肪和背最长肌中分别平均为5.95%±3.15%、5.19%±2.87%和0.894±0.038、0.917±0.041,均高于（?）＜15%和r＞0.700的成功实验标准。对两组织内各5个差异表达基因的QRT-PCR验证显示:两种实验方法检测结果均为正相关,相关系数（r）在皮下脂肪和背最长肌表达谱中分别平均为0.874±0.071和0.876±0.095。表明本研究结果准确可靠,检测数据能够真实反映基因的表达变化规律。本研究筛选出了对于猪胴体和肉质性状可能具有重要影响和较大研究价值的基因,初步揭示了生长发育过程中骨骼肌和脂肪细胞基因表达调控的局部分子互作机制,以及造成猪肌肉和脂肪宏观性状表型差异的分子网络基础。更多还原

【Abstract】 During the growth and development of skeletal muscle cells and adipose cells,the regulatory mechanism of micro-effect polygenes determines porcine meat quality,carcass characteristics and other relative quantitative traits.Obese and lean type pig breeds show obvious differences in muscle growth and adipose deposition;however,the molecular mechanism underlying this phenotypic variation remains unknown.We used pathway-focused oligo microarray studies to examine the expression changes of 140 genes associated with muscle growth and adipose deposition in longissimus dorsi muscle at six growth stages（birth,1,2,3,4 and 5 months）and in backfat at five growth stages（1,2,3,4 and 5 months）of Landrace（a leaner,Western breed）and Taihu pigs（a fatty,indigenous,Chinese breed）.（ⅰ）Variance analysis（ANOVA）revealed that differences in the expression of 18 genes in Landrace pigs and 3 genes in Taihu pigs were very significant(P_ANOVA＜0.01)and differences for 22 genes in Landrace pigs and 7 genes in Taihu pigs were significant(P_ANOVA＜0.05)in longissimus dorsi muscle among six growth stages.The differences in the expression of 25 genes in Landrace pigs were significant(P_ANOVA＜0.05)in backfat among five growth stages.Gene class test（GCT）indicated that a gene-group was very significant in longissimus dorsi muscle of Landrace pigs across six growth stages(P_ErmineJ＜0.01),which consisted of 10 genes for enzymes related to fatty acid biosynthesis.A gene-group was very significant in backfat between two pig breeds across five growth stages(P_ErmineJ＜0.01),which consisted of 23 genes encoding enzymes and regulatory proteins associated with lipid and steroid metabolism.These findings suggest that the distinct differences in fat-deposition ability,muscle-fiber number and growth rate between Landrace and Taihu pigs may closely correlate with the expression changes of these genes.（ⅱ）STEM（Short Time-series Expression Miner）clustering analysis revealed a very high level of significance(P_STEM＜0.01)for 4 gene expression patterns in longissimus dorsi muscle,in which genes that showed strong up-regulation were mainly associated with the positive regulation of myofiber formation and fatty acid biogenesis and genes that showed strong down-regulation were mainly associated with the inhibition of cell proliferation and positive regulation of fatty acidβ-oxidation.A very high level of significance(P_STEM＜0.01)for 2 gene expression patterns in Landrace pigs and a high level of significance(P_STEM＜0.05)for 2 gene expression patterns in Taihu pigs for backfat.Also,expression patterns of genes were more diversified in Taihu pigs than in Landrace pigs,which suggests that the regulatory mechanism of micro-effect polygenes in adipocytes may be more complex in Taihu pigs than in Landrace pigs.（ⅲ）Principal component analysis（PCA）revealed that 7 and 16 genes of Landrace,6 and 13 genes of Taihu pigs in longissimus dorsi muscle and backfat displayed distinctive expression pattern from other genes,which suggested that these genes maybe came under special regulation during the growth and development of skeletal muscle cells and adipose cells.（ⅳ）ANOVA revealed that differences in the expression of 42 genes in Landrace pigs and 51 genes in Taihu pigs were very significant(P_ANOVA＜0.01)and differences for 27 genes in Landrace pigs and 20 genes in Taihu pigs were significant(P_ANOVA＜0.05)between longissimus dorsi muscle and backfat across five growth stages.The resluts of two-way hierarchical clustering could be segmented to three clusters,and mapped to the two- and three- dimensional map of PCA.We can highlight some genes which have tissue specific expression patterns on which to base further study of the differences in molecular mechanism between the depostation of subcutaneous fat and intramuscular fat（IMF）.（ⅴ）Based on a dynamic Bayesian network（DBN）model,gene regulatory networks（GRNs） were reconstructed from time-series data for each pig breed and tissue.These four GRNs initially revealed the distinct differences in physiological and biochemical aspects of muscle growth and adipose deposition between the two pig breeds;from these results,some potential key genes could been identified.（ⅵ）Pathway mapping revealed that the biological functions of four pathways maybe changed during the process of growth and development between two pig breeds,in which the pathway of fat acid metabolism and biosynthesis involved the competitive equilibrium of fat depostation,and the pathway of PPAR（peroxisome proliferator-activated receptor）signaling and adipocytokine signaling involved the rugulation of fat depostation.These results could be explained the phenotypic variation of fat traits between obese and lean type pig breeds to some extent.（ⅶ）Based on a natural language processing（NLP）approach,literature co-citation networks of genes that belong to predominant temporal expression patterns using a unique STEM algorithm were reconstructed.We can choose seed genes from sub-networks that have higher rank scores as potential key genes for porcine meat quality and carcass traits.（ⅷ）Based on human homologous genes,transcriptional regulatory networks（TRNs）of differentially expressed genes(P_ANOVA＜0.05 and 0.01)in longissimus dorsi muscle were inferred. We found 27 transcription factors（TFs）have significantly overabundant binding sites in the promoters of 26 genes(P_TF＜0.05).These findings suggest that these TFs maybe indirectly influence muscle and fat traits via directly stimulate target genes.（ⅸ）Repeatability analysis indicated that average coefficient of variation（（?））within-arrays and average correlation coefficient（（?））between-arrays were 5.95%±3.15%and 0.894±0.038 for expression profiling of backfat,and 5.19%±2.87%and 0.917±0.041 for expression profiling of longissimus dorsi muscle.Quantitative,real-time RT-PCR（QRT-PCR）was used to verify the microarray data for five modulated genes within each tissue,and a good correlation between the two measures of expression was observed for both two pig breeds at different growth stages （backfat:r=0.874±0.071 and longissimus dorsi muscle:r=0.876±0.095）.These resultes demonstrate that the microarray technique used in this study is accurate and reproducible.These results highlight some possible candidate genes for porcine meat quality and carcass traits and provide some data on which to base further study of the molecular mechanism of muscle growth and fat deposition.更多还原