利用功能分类基因芯片研究不同品种猪脂肪中特定基因的发育性变化

利用Oligo功能分类基因芯片检测了瘦肉型的长白猪和脂肪型的太湖猪在1、2、3、4和5月龄间背部皮下脂肪中脂肪沉积代谢和细胞生长调控相关基因的动态表达变化。差异表达分析结果显示1~5月龄的品种间分别有10、6、11、8和19个基因的表达差异倍数大于2倍, 且长白猪有25个基因在不同月龄间的表达差异达显著水平(P<0.05)。其中血管生成素样蛋白4 (ANGPTL4)、组织蛋白酶K (CTSK) 、异柠檬酸脱氢酶2(NADP+) (IDH2)、脂蛋白脂酶 (LPL)、苹果酸酶1 (ME1)、 硬酯酰辅酶A去饱和酶 (SCD)和解藕联蛋白2 (UCP2)这7个基因不仅在同月龄的品种间和品种内的不同月龄间差异表达, 主成分分析结果也显示其表达模式明显偏离其他基因, 提示受到了特殊的调控。聚类分析结果显示1~5月龄间长白猪中正调控脂肪酸代谢基因的表达量逐渐上调, 太湖猪中参与细胞生长调控基因的表达量平缓波动且变化幅度相对较小。另外, 5个差异表达基因的荧光定量RT-PCR验证结果均与芯片结果呈正相关趋势。结果成功筛选出了对猪胴体和肉质性状可能具有重要影响并值得深入研究的一些候选基因, 初步揭示了相关基因的表达变化规律, 为了解生长发育过程中脂肪酸合成与水解的动态平衡过程提供了基础数据。     

猪背最长肌中肌肉生长和脂肪沉积相关基因的差异表达和主成分分析

骨骼肌细胞和脂肪细胞在分化生长速度上相对竞争的平衡点是猪肉质和胴体性状的决定因素.利用Oligo功能分类芯片检测了瘦肉型的长白猪和脂肪型的太湖猪在初生、1、2、3、4和5月龄间背最长肌中肌肉生长和脂肪沉积相关基因的动态表达变化.差异表达分析结果显示,在初生至5月龄的品种间分别有15、16、11、13、18和20个基因的表达差异倍数大于2倍.品种内的方差分析表明,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<0.01)和显著水平(Ｐ<0.05).主成分分析结果显示,先降后升是两品种内最具代表性的基因表达模式,且长白猪和太湖猪分别有7和6个基因的表达模式明显偏离其他基因,提示其可能受到了重要的调控. 此外,5个差异表达基因的荧光定量RT-PCR验证结果均与芯片结果呈正相关趋势.以上结果筛选出了对于猪肉质和胴体性状可能具有重要影响,值得深入研究的一些候选基因,为深入研究生长发育过程中参与肌纤维生长和脂肪酸合成关键基因的表达变化规律和互作调控机制提供了基础数据.     

长白猪和太湖猪不同生长阶段脂肪中肉质和胴体性状相关基因的表达谱分析

动物个体的皮下脂肪厚度（subcutaneous fat thickness,SFT）和肌内脂肪（intramuscular fat,IMF）含量取决于活体内脂肪酸合成与氧化竞争的动态平衡点．利用包含140个与猪脂肪沉积、代谢和肌肉生长密切相关基因的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1,2,3,4和5月龄间背部皮下脂肪中这些基因的动态表达变化,发现长白猪有25个基因在月龄间的表达差异达显著水平（P〈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〈0．01）．提示两猪种脂肪沉积能力的巨大表型差异可能与这些基因的差异表达规律密切相关．STEM聚类分析显示长白猪和太湖猪各有两个表达模式分别具有极显著（P〈0．01）和显著性意义（P〈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．基于动态贝叶斯模型构建的基因调控网络从一定程度上反映了两猪种在脂肪沉积代谢相关生理生化活动方面存在的明显差异,可从中挖掘相关性状潜在的关键特征基因．此外,对5个差异表达基因的荧光定量RT-PCR验证显示两种实验方法结果的相关系数平均高达0．874±0．071．以上结果筛选出了对于猪脂肪相关性状可能具有重要影响并值得深入研究的一些基因,初步揭示了生长发育过程中脂肪细胞基因表达调控的分子互作机制．     

雌性藏猪不同生长发育阶段肌肉生长和脂肪沉积相关基因的表达模式

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藏猪在2、4、6和8月龄间背最长肌中这些基因的表达量变化,并在2月龄时与脂肪型的太湖猪和瘦肉型的长白猪进行比较.ANOVA分析结果表明:2-8月龄间藏猪分别有10和 7个基因的表达差异达极显著(P<0.01)和显著水平(P<0.05);2月龄时藏猪体重极显著低于长白猪(P<0.01)和显著低于太湖猪(P<0.05),而藏猪肌纤维面积却为最大,但品种间差异未达显著水平(P>0.05);2月龄时3个品种间分别有15和13个基因的表达差异达极显著 (P<0.01)和显著水平(P<0.05).STEM聚类分析结果表明:直线下降和上升是藏猪在2 -8月龄间最具代表性的基因表达模式(P<0.01).另外,5个差异表达基因的荧光定量RT -PC R验证结果与基因芯片结果的Person相关系数平均高达0.856±0.109.提示:藏猪在2-8月龄间骨骼肌生长发育强度较肌内脂肪合成沉积占优势,2月龄时藏猪脂肪酸合成相关基因的表达水平较其他两品种低,而脂肪酸β氧化和肌纤维生长相关基因的表达水平较高,与其在高原独特的自然生态环境和全放牧散养的饲喂方式下长期形成的品种特性相符 [动物学报 54(3):442-452,2008].     

2个猪品种的背最长肌组织中兰尼定受体(RYR1)基因表达的发育性变化

本研究采用实时荧光定量PCR方法检测了长白猪和梅山猪背最长肌组织中RYR1基因mRNA表达量在初生(0月龄)、1月龄、2月龄、3月龄、4月龄和5月龄间的变化及其与体重、肌纤维面积(CSA)和肌内脂肪含量(IMF)之间的相关性。结果表明:长白猪的体重、CSA在多数月龄均高于梅山猪,而梅山猪的IMF在多数月龄均高于长白猪。两品种RYR1基因表达量的发育性表达模式极为相似,均表现为波动起伏中逐渐上升的趋势。相同月龄时,梅山猪RYR1基因的表达量均高于长白猪。本文初步揭示了两猪种生长发育过程中RYR1基因表达的发育性变化模式和品种差异,为进一步研究RYR1基因对猪肉质性状的影响提供了基础数据。     

猪脂肪组织中IGF2和IGFBP3基因表达的发育性变化及其品种差异

采用荧光定量PCR技术分析长白猪和太湖猪背部皮下脂肪组织中胰岛素样生长因子2和胰岛素样生长因子结合蛋白3基因在30、60、90、120和150日龄时表达水平的发育性变化。结果表明: (1)品种内日龄间比较, 长白猪IGF2 mRNA 在30日龄时的表达量极显著高于其他日龄(P<0.01), 之后逐渐下降, 至120日龄降到最低, 150日龄时又明显上升; 太湖猪IGF2 mRNA在30~60日龄的表达量较高, 90日龄降至最低, 120日龄迅速回升, 之后又有所下降。两品种IGFBP3 mRNA表达的发育性变化模式基本相同, 30日龄时表达量最高, 60日龄显著下降(P<0.05), 之后趋于平缓但略有波动。(2)品种间同日龄比较, 120日龄时太湖猪IGF2 mRNA的表达量极显著高于长白猪(P<0.01), 150日龄时太湖猪IGFBP3的表达量极显著低于长白猪(P<0.01), 其余日龄间两品种差异均不显著(P>0.05)。研究结果提示: 猪脂肪组织IGF2和IGFBP3基因表达存在明显的发育性变化和品种差异; IGF2基因表达水平的降低可能与脂肪细胞的增殖有关。     

猪背最长肌中胰岛素样生长因子(IGFs)系统基因的发育表达模式

采用荧光定量PCR技术检测了长白猪和梅山猪的背最长肌组织中胰岛素样生长因子1和2(IGF-1和-2)、胰岛素样生长因子1受体和2受体(IGF-1R和-2R)、胰岛素样生长因子结合蛋白3和5(IGFBP-3和-5)基因mRNA丰度在初生(0月龄)、1、2、3、4和5月龄间的表达变化并分析品种间和不同月龄间基因表达的差异及其对肌肉生长发育的影响。结果表明: 两猪种出生后IGF-1 mRNA表达量均表现为逐渐上调, 而IGF-2则恰好相反, 表现为逐渐下调。这与IGF-2主要在胚胎期发挥作用, 而IGF-1则主要在动物个体出生后才发挥促进细胞增殖和个体发育功能的特点相符。IGFRs mRNA与IGFs mRNA 表达的发育性变化模式并不相似, 提示背最长肌组织中IGFRs mRNA 的表达可能没有受到组织局部产生的IGFs调节。长白猪的IGF-1R、IGF-2R 和IGFBP-3 mRNA表达量均在2月龄时达到最高峰, 提示2月龄可能是长白猪IGFs系统发挥作用最为明显的生长发育阶段。以上结果初步揭示猪生长发育过程中胰岛素样生长因子系统基因表达的发育性变化模式和品种差异, 为深入研究胰岛素样生长因子系统基因的相互调控机制提供了基础数据。     

五指山猪和长白猪背最长肌差异基因的筛选与注释

为了筛选五指山猪和长白猪背最长肌组织差异表达基因,本研究采用RNA-seq技术和生物信息学方法对6月龄、8月龄五指山猪和长白猪的背最长肌进行转录组测序分析,并对差异表达基因进行GO和KEGG Pathway显著性富集分析。通过与猪基因组比对分析后发现,在6月龄和8月龄五指山猪和长白猪均有约44 000 000条Clean reads能够比对到猪基因组上相关基因,每个文库均获得约18 200个表达基因,而转录本的数据为25 000个。在6月龄五指山猪与长白猪中共有30个基因差异表达,在8月龄五指山猪与长白猪中获得29个差异表达的基因。这些差异表达的基因主要富集在与糖酵解代谢、MAPK信号代谢通路和胰岛素信号通路等肌肉发育信号通路,通过筛选获得了与肌肉生长发育有关的候选基因,为探索五指山猪肌肉发育的分子机制提供了理论依据。     

茶树种子发育过程的转录组分析

该研究以‘铁观音’茶树品种的种子为试验材料,采用转录组测序技术分析种子发育的3个时期(幼果期、膨大期、成熟期)的表达差异,探究茶树种子油脂代谢的分子机制。结果表明:(1)经转录组测序、组装后共获得30 940 581个clean reads,经数据合并拼接最终得到36 951条非冗余Unigene序列,其中28 476个Unigene可得到功能注释;在转录本中能够被注释到GO分类的Unigene有11 201条(30.3%),KEGG分析发现共有17 172个基因参与了127个代谢通路。(2)经KEGG通路筛选出14条与脂肪酸代谢相关的通路,且随着茶籽的发育,大部分脂肪酸调控途径相关基因呈下调趋势,其中上调基因数最多的有α-亚麻酸代谢途径和脂肪酸降解途径(有17个基因表达量上调),下调基因数最多的是甘油磷脂代谢途径(有58个基因表达量下调);在茶籽发育幼果期α-亚麻酸代谢途径中表达量上调的基因数超过表达量下调的基因数。(3)研究发现茶籽脂肪酸合成相关的基因涉及14个脂类调控途径,共409条差异基因;随着茶树种子发育到成熟期,上调的差异表达基因数量在减少,下调的差异表达基因数量增加,其中α-亚麻酸途径中的基因PLA2G16、DAD1、pldA、FabF、FabI表达量上调显著,随后表达量下调。(4)qRT-PCR检测结果表明,7个茶树FAD和1个ACP差异表达基因的水平与转录组测序结果基本一致;随着茶籽的发育,基因CsFAD7和Δ6-CsFAD从幼果期、果实膨大期至果实成熟期都为差异下调表达,CsFAD2、CsFAD6和Δ7-CsFAD为差异上调表达,CsFAD8、Δ8-CsFAD和CsACP在幼果期至果实膨大期差异上调表达,在果实膨大期至果实成熟期差异下调表达。     

不同品种猪背最长肌中IGF-1-PI3K/AKT通路基因的生长发育表达模式差异

本文研究了广西巴马小型猪和长白猪背最长肌中IGF-1-PI3K/AKT信号通路相关基因的发育表达模式的差异,为今后研究巴马小型猪的生长发育和肉质性状提供分子理论基础.克隆猪INSR、IRS1、IGF-1、PI3K、PI3KR1、PDK1、PTEN、AKT1、FoxO1、mTOR、4EBP1和β-actin基因,构建荧光定量PCR标准曲线,采用实时PCR法检测上述基因在广西巴马小型猪和长白猪出生后1、30、60、90、180 d背最长肌的表达变化.在同一生长发育阶段,PTEN和4EBP1基因的表达均极显著高于同一品种其他基因的表达,FoxO1、PI3KR1和AKT1基因中度表达,IGF-1、INSR、IRS1、PI3K、PDK1基因低度表达;在不同生长发育阶段基因的表达存在显著差异;两个品种猪在同一生长发育时间的表达也存在差异.本研究结果表明广西巴马小型猪和长白猪IGF-1-PI3K/AKT通路相关基因的表达呈现出明显的品种差异性和时空差异性,PTEN、FoxO1和4EBP1基因表达差异可能是两个品种猪肌肉产量和肉质性状存在差异的原因之一.     

Expression profiling analysis for genes related to meat quality and carcass traits during postnatal development of backfat in two pig breeds

The competitive equilibrium of fatty acid biosynthesis and oxidation in vivo determines porcine sub-cutaneous fat thickness(SFT) and intramuscular fat(IMF) content.Obese and lean-type pig breeds show obvious differences in adipose deposition;however, the molecular mechanism underlying this phenotypic variation remains unclear.We used pathway-focused oligo microarray studies to examine the expression changes of 140 genes associated with meat quality and carcass traits in backfat at five growth stages(1―5 months) of Landrace(a leaner, Western breed) and Taihu pigs(a fatty, indigenous, Chinese breed).Variance analysis(ANOVA) revealed that differences in the expression of 25 genes in Landrace pigs were significant(FDR adjusted permutation, P<0.05) among 5 growth stages.Gene class test(GCT) indicated that a gene-group was very significant between 2 pig breeds across 5 growth stages(PErmineJ<0.01), which consisted of 23 genes encoding enzymes and regulatory proteins associ-ated with lipid and steroid metabolism.These findings suggest that the distinct differences in fat deposition ability between Landrace and Taihu pigs may closely correlate with the expression changes of these genes.Clustering analysis revealed a very high level of significance(FDR adjusted, P<0.01) for 2 gene expression patterns in Landrace pigs and a high level of significance(FDR adjusted, P<0.05) for 2 gene expression patterns in Taihu pigs.Also, expression patterns of genes were more diversified in Taihu pigs than those 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.Based on a dy-namic Bayesian network(DBN) model, gene regulatory networks(GRNs) were reconstructed from time-series data for each pig breed.These two GRNs initially revealed the distinct differences in physiological and biochemical aspects of adipose metabolism between the two pig breeds;from these results, some potential key genes could be identified.Quantitative, real-time RT-PCR(QRT-PCR) was used to verify the microarray data for five modulated genes, and a good correlation between the two measures of expression was observed for both 2 pig breeds at different growth stages(R=0.874±0.071).These results highlight some possible candidate genes for porcine fat characteristics and provide some data on which to base further study of the molecular basis of adipose metabolism.     

Expression profiling analysis for genes related to meat quality and carcass traits during postnatal development of backfat in two pig breeds

The competitive equilibrium of fatty acid biosynthesis and oxidation in vivo determines porcine subcutaneous fat thickness (SFT) and intramuscular fat (IMF) content. Obese and lean-type pig breeds show obvious differences in adipose deposition; however, the molecular mechanism underlying this phenotypic variation remains unclear. We used pathway-focused oligo microarray studies to examine the expression changes of 140 genes associated with meat quality and carcass traits in backfat at five growth stages (1–5 months) of Landrace (a leaner, Western breed) and Taihu pigs (a fatty, indigenous, Chinese breed). Variance analysis (ANOVA) revealed that differences in the expression of 25 genes in Landrace pigs were significant (FDR adjusted permutation, P<0.05) among 5 growth stages. Gene class test (GCT) indicated that a gene-group was very significant between 2 pig breeds across 5 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 between Landrace and Taihu pigs may closely correlate with the expression changes of these genes. Clustering analysis revealed a very high level of significance (FDR adjusted, P<0.01) for 2 gene expression patterns in Landrace pigs and a high level of significance (FDR adjusted, P<0.05) for 2 gene expression patterns in Taihu pigs. Also, expression patterns of genes were more diversified in Taihu pigs than those 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. Based on a dynamic Bayesian network (DBN) model, gene regulatory networks (GRNs) were reconstructed from time-series data for each pig breed. These two GRNs initially revealed the distinct differences in physiological and biochemical aspects of adipose metabolism between the two pig breeds; from these results, some potential key genes could be identified. Quantitative, real-time RT-PCR (QRT-PCR) was used to verify the microarray data for five modulated genes, and a good correlation between the two measures of expression was observed for both 2 pig breeds at different growth stages (R=0.874±0.071). These results highlight some possible candidate genes for porcine fat characteristics and provide some data on which to base further study of the molecular basis of adipose metabolism.     

Identification of extracellular matrix and cell adhesion molecule genes associated with muscle development in pigs

Extracellular matrix (ECM) and cell adhesion molecule (CAM) genes are involved in the regulation of skeletal muscle development; however, their roles in skeletal muscle development in pigs are still poorly understood. 65 days postcopulation (dpc) is a critical time point in pig development. Therefore, we analyzed expression of ECM and CAM genes in the longissimus dorsi muscles at 65?dpc from Landrace (lean-type: L65), Tongcheng (obese-type: T65), and Wuzhishan pigs (miniature-type: W65) using microarray technology. A total of 35 genes were differently expressed between the breeds, and of them, 18, 18, and 20 genes, were observed in the comparisons of L65 versus T65, L65 versus W65, and T65 versus W65 (L65/T65, L65/W65, and T65/W65), respectively. In L65/T65, differently expressed genes were widely distributed, whereas in L65/W65 and T65/W65, they mostly focused on the genes encoding CAMs and ECMs proteins. Moreover, the largest number of up-regulated genes involved in skeletal muscle development was detected in L65, a moderate number in W65, and the smallest number was in T65. Cluster analysis suggested that T65 showed a more similar expression pattern to L65 than W65. In addition, we validated that five genes from microarray data were more highly expressed in the prenatal as compared to postnatal periods in Landrace and Tongcheng pigs and showed a greater range of high-level expression during gestation in Landrace than Tongcheng pigs. Our data indicated that ECM and CAM genes are differently expressed among the three breeds, and more complicated molecular events involving CAMs and ECMs were observed in Wuzhishan pigs. This study advances our knowledge of the molecular basis of phenotypic variation and provides a helpful resource for the identification of candidate genes associated with meat production traits in pigs.     

Molecular characterization of the BTG2 and BTG3 genes in fetal muscle development of pigs

BTG2 and BTG3 are two members of the B-cell translocation gene family with anti-proliferative properties. BTG1 gene in this gene family has been reported to play a key role in muscle growth. In this study, we identified and characterized the porcine BTG2 and BTG3 genes, mapped the two genes to porcine chromosomes, and analyzed their expression differences in the longissimus dorci muscle of 33 dpc (day postconception), 65 dpc and 90 dpc in the lean Landrace and fatty Chinese Tongcheng pig breeds. Expression changes in differentiated C2C12 cells were also investigated with myogenin as internal control. The results showed that the porcine BTG2 and BTG3 genes were mapped on SSC9q21-25 and SSC13q47, respectively. BTG2 gene expressed at high levels in skeletal muscle and heart in both Tongcheng and Landrace pigs whereas BTG3 gene expressed at lower levels in skeletal muscle and heart than in other tissues. Furthermore, BTG3 expressed at higher levels in skeletal muscle of Tonghceng compared with Landrace pig. The expression of BTG2 and BTG3 was significantly different in skeletal muscle among different developmental stages and between the two breeds. Expression analysis in murine myoblast cells showed that both genes were induced in differentiated C2C12 cells, suggesting a role of them in myogenic differentiation. Our study indicated that BTG2 and BTG3, especially BTG3 gene, may be important genes for skeletal muscle growth.