鸡肌肉生长相关基因的表达与肌苷酸关键酶基因网络调控的构建
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摘要
本文选取中国地方鸡种如皋黄鸡和外来鸡种隐性白羽鸡种作为研究对象,利用Agilent表达谱芯片技术,分别对不同生长阶段的如皋黄鸡和隐性白羽鸡肌肉组织中相关基因转录特性进行研究,着重分析了2、12周龄两鸡种肌肉组织的差异表达基因,并结合GO分析进行相关差异基因的生物功能分类,探讨不同鸡种肌肉组织中相关基因表达谱的差异及其联系与规律,以期了解不同鸡种肌肉组织的生长发育差异,为进一步探明影响鸡肌肉生长发育的分子机制提供基础。
肌苷酸(Inosine Monophosphate,IMP)是肉品中重要的风味物质,其在体内的合成代谢过程十分复杂,肌苷酸关键酶基因包括：磷酸焦磷酸酰胺转移酶(PPAT),磷酸核糖氨基咪唑碳酸酵素(PAICS),甘氨酰胺核苷酸合成酶(GARS),5-氨基咪唑核苷酸合成酶(AIRS),甘氨酰胺核苷酸转甲基酶(GART),5-氨基咪唑-4-甲酰氨核糖核苷酸甲酰转移酶(ATIC),腺苷琥珀酸裂解酶(ADSL),腺苷单磷酸脱氨酶(AMPD)。本文在如皋黄鸡基因芯片数据的基础上,运用Perl语言和皮尔森系数方程求解相关基因间线性关系,结合Cytoscape和GO将影响肌肉组织中肌苷酸关键酶相关基因的网络调控图进行了分析,构建了影响IMP合成途径中关键酶基因网络调控图,以期探明肌肉组织中IMP合成途径中关键酶之间的相互作用机理,主要研究结果如下：
1、同一鸡种不同生长阶段差异表达基因的比较(1)利用基因芯片技术,分析了不同生长阶段下(2-12周龄)如皋黄鸡和隐性白羽鸡肌肉组织基因的表达情况,获得了13379个基因的差异表达动态图谱。在如皋黄鸡中,与2周龄相比(P<0.001),4周龄时有114个基因上调表达,127个基因下调表达；6周龄时有116个基因上调表达,101个基因下调表达；8周龄时有130个基因上调表达,110个基因下调表达；10周龄时有136个基因上调表达,118个基因下调表达；12周龄时有94个基因上调表达,114个基因下调表达。4周龄、12周龄时上调表达基因数量少于下调表达基因的数量,而6周龄、8周龄、10周龄时上调表达基因数量明显多于下调表达基因的数量；在隐性白羽鸡中,与2周龄相比(P<0.001),6周龄时有72个基因上调表达,51个基因下调表达；10周龄时有42个基因上调表达,30个基因下调表达；12周龄时有44个基因上调表达,34个基因下调表达。3个生长阶段,上调表达的基因数量均多于下调表达的基因数量。
(2)将表达趋势一致的基因归为一类,所有基因可分为12类,相关系数大都集中在0-1之间,如皋黄鸡第6类与第7类相关系数为1,第3类与第7类,第4类与第8类之间相关系数为0；隐性白羽鸡第1类与第2类,第6类与第3、7、11类相关系数均是0,而第6类与第5、10类,第8类与第12类相关系数均是1,可得到：生长发育过程中,隐性白羽鸡表达量发生变化的基因之间的相关系数普遍高于如皋黄鸡。
2、不同时期(2周龄、12周龄)生长相关差异基因的表达
(1)应用表达谱芯片,对如皋黄鸡和隐性白羽鸡不同时期肌肉组织抽提及纯化的cRNA进行芯片杂交,并对基因表达谱进行分析,如皋黄鸡在不同生长时期(2周龄和12周龄相比)共筛选出差异表达基因208条(P<0.001),其中已知功能基因108条,表达上调94条,表达下调114条；隐性白羽鸡在不同生长时期(2周龄和12周龄相比)共筛选出差异表达基因78条(P<0.001),其中已知功能基因39条,表达上调44条,表达下调34条,经GO分类,两鸡种筛选的基因主要涉及生长发育、分子代谢、免疫应答、生物合成、细胞通信以及蛋白质合成与分解等相关基因,同时经荧光定量PCR技术与蛋白质组技术验证,表达谱芯片结果可靠。在差异基因中包括一些未知的新基因,但具体的功能以及在鸡生长发育、能量代谢等的过程所起到的作用需进一步验证。
(2)在差异基因中,如皋黄鸡中共筛选出6条与生长发育相关差异基因,隐性白羽鸡共筛选出1条差异基因。α-烯醇化酶基因(NM205120)均参与如皋黄鸡和隐性白羽鸡生长发育过程,该基因在如皋黄鸡和隐性白羽鸡中表达模式存在差异,其在如皋黄鸡中4周龄时表达量最低,2周龄和12周龄表达量并无变化；而在隐性白羽鸡中从2周龄到12周龄表达持续下降,推测α-烯醇化酶基因可能是影响鸡早期生长性状的候选功能基因；如皋黄鸡中另外5个生长相关差异基因表达情况是：HOP同源体(HOPX)高度表达：adducin1(ADD1)、受体酪氨酸激酶2(DDR2)和细胞周期因子B2(CCNB2)基因表达趋势保持平缓；胰岛素样生长因子1(IGF1)基因低度表达,表达水平持续下降。
3、肌苷酸相关基因网络调控图的构建
运用Perl语言和皮尔森系数方程求解相关基因的作用关系,结合Cytoscape软件生成肌苷酸相关基因的网络图,结合如皋黄鸡芯片数据构建肌苷酸相关基因网络调控图,运用Cytoscape和GO将肌苷酸关键酶相关基因的网络调控图进行了功能分类。
(1)通过相关系数模型分析肌苷酸相关基因之间的关联程度,相关系数在0.75以上时,肌苷酸相关基因包括：PPAT, PAICS, ADSL, AMPD, ATIC, GART, AIRS, GARS;相关系数在0.80以上时,肌苷酸相关基因包括：PPAT, ATIC, AMPD, GART, AIRS, GARS;相关系数大于等于0.85时,肌苷酸相关基因包括PPAT, AMPD, GART, AIRS, GARS;相关系数在0.90以上时,肌苷酸相关基因包括：AMPD, GART, AIRS, GARS,可得到：AMPD与GARS-AIRS-GART复合酶基因相关性最高,PPAT次之。
(2)对如皋黄鸡肌苷酸8个关键酶基因表达情况的分析：在如皋黄鸡肌苷酸含量的调控中,起较主要作用的可能是AMPD和ADSL基因。GART的表达趋势与GARS, AIRS和AMPD的表达趋势相反,但是由相关系数模型可得到GART, GARS, AIRS和AMPD的相关系数最高,表明8个关键酶之间存在正相关和负相关。
(3)在6、10周龄肌苷酸含量最高时,此时AMPD, AIRS和GARS的表达量呈上升趋势,对肌苷酸调控可能呈现正相关关系；而PPAT, ADSL和GART表达量呈下降趋势,对肌苷酸调控可能呈现负相关关系。
(4)将由芯片数据所得到的网络调控图与基于KEGG的网络调控图进行比较,可得到：KEGG中GART, PAICS, ADSL, ATIC, AMPD这5个关键酶在如皋黄鸡肌苷酸网络调控中也起着主要作用,而其余三个基因,PPAT, AIRS, GARS在如皋黄鸡肌肉中属于低度表达,这可能与不同鸡种的遗传背景有关。
Chinese local chicken breeds Rugao Yellow chicken and exotic breeds Recessive White chicken were selected as the object of study, in order to study genes expression changes of Rugao Yellow chicken and Recessive white chicken from the overall level at different growth times and the growth and development mechanism, which is of great significance for understanding the molecular mechanisms of chicken muscle growth and development.In this study, gene expression profiles of2,4,6,8,10,12weeks of age Rugao Yellow chicken and2,6,10,12weeks of age Recessive White chicken muscle tissues were studied respectively by cDNA microarrays and differentially expressed genes of2,12weeks of age of two kinds of chickens were screened to investigate the intrinsic link of different expression genes and their possible molecular biology and laws of the muscle tissues between two experimental groups, differential genes biological functions were classified by GO, in order to provide a theoretical basis further study.
Meat flavor is one of the most important aspects of the meat quality in chicken, many evidences indicated that Inosine Monophosphate (IMP) is one of the key components for meat flavor. The process of the synthesis and metabolism for IMP is very complicated, on the basis of Rugao Yellow chicken microarray data, the Perl language and the Pearson coefficient equation were used for solving the linear correlation of related genes, Cytoscape software was combined to generate genes network diagram of IMP, regulation network of IMP genes and its related genes was built based on the KEGG database at last, the genes functions of this regulation network were classified by Cytoscape and GO. IMP genes include PPAT, PAICS, GARS, AIRS, GART, ATIC, ADSL and AMPD. The purpose of building a regulation network is that the model was screened based on RNA levels on the basis of molecular hybridization, the IMP genes expression difference and the mechanism of interaction were revealed at the transcriptional level in the IMP synthesis pathway; the regulation network model of IMP was built in different spatial and temporal expression with RNA-mediated in order to understand function of cells, tissues from the molecular level and clarify molecular mechanism of different spatial and temporal expression for our local chicken flavor substance-IMP and provide a theoretical basis for industrial development. The main results were as follows:
1. Comparison at different growth stages of same chicken breeds
(1) The gene expressions of Rugao Yellow chicken and Recessive White chicken muscle were analyzed in different periods (2w-12w) by GeneChip. The expression profiles of13,379differentially expressed genes were obtained, Rugao Yellow chicken, compared to2weeks^P<0.001),114genes were up-regulated,127genes down-regulated, when it was4weeks old,116genes were up-regulated,101genes down-regulated, when it was6weeks old,130genes were up-regulated,110genes down-regulated, when it was8weeks old,136genes were up-regulated,118genes down-regulated, when it was10weeks old,94genes were up-regulated,114genes down-regulated, when it was12weeks old, the data analysis showed that at6weeks8and10weeks, the number of up-regulated genes were significantly more than the number of down-regulated genes, at4and12weeks, the number of up-regulated genes were significantly less than down-regulated genes; Recessive white chickens, compared to2weeks(P<0.001),72genes were up-regulated,51genes down-regulated, when it was4weeks old,42genes were up-regulated,30genes down-regulated, when it was10weeks old,44genes were up-regulated,34genes down-regulated, when it was4weeks old, the data analysis showed that at the three time points, the number of up-regulated genes were significantly more than the number of down-regulated genes.
(2) Different genes were divided into12categories, the correlation coefficients were concentrated in the between0and1, Rugao Yellow chicken, the correlation coefficient between Class6and Class7was1, the correlation coefficient between Class3and Class7, Class4and Class8were0; Recessive white chicken, the correlation coefficient between Class1and class2, Class6and3,7,11were0, the correlation coefficient between Class6and class5,10. Class8and12were1, the correlation coefficient of different genes in Recessive white chicken were generally higher than that in Rugao Yellow chicken in growth and development process
2. Gene expression in different periods (2w,12w)
(1) GeneChip was used to construct gene expression profile in order to screen differentially expressed genes of muscle tissue in Rugao Yellow chicken and Recessive white chicken and investigate the molecular mechanism related with muscle tissue traits between the two groups, respectively.208differentially expressed genes (P<0.001) were screened out, of which108are known genes,94up-regulated,114down-regulated for Rugao Yellow chicken;78differentially expressed genes (P<0.001) were screened out, of which39were known genes,44up-regulated,34down-regulated for Recessive white chicken, which were involved in growth, molecular mechanism, immune system process, biosynthetic process, cell communication, protein synthesis and degradation were screened out. Meanwhile, some genes among all these differentially expressed genes that had no annotation in GenBank were screened out; they were presumed to be unknown new genes. At the same time, the results were verified by real-time quantitative PCR and proteomic technology, indicating that microarray results were reliable. The roles that they may play in chicken growth and development, energy metabolism needed to be clarified later.
(2)6differentially expressed genes were screened out in the process of growth and development of Rugao Yellow chickens,1differentially expressed gene was screened out in the process of growth and development of Recessive white chickens, RCJMB04_24e12(NM_205120) was involved in the process of growth and development in Rugao Yellow chicken and Recessive white chicken, the expression of which was the lowest at4weeks old in Rugao Yellow chicken, while continued to decline in Recessive white chicken, suggesting that it may be a candidate marker of early growth traits in chicken, another five genes related growth-related differences in Rugao Yellow chicken were:HOPX, CCNB2, DDR2, ADD1and IGF1had a certain impact on the growth and development, HOPX was highly expressed, gene expression tendency of to CCNB2, DDR2, ADD1remained gentle, IGF1was lowly expressed.
3、Construction of IMP genes regulatory networks
The Perl language and the Pearson coefficient equation were used for solving the linear correlation of related genes, Cytoscape software was combined to generate genes network diagram of IMP, regulation network of IMP genes and its related genes was built based on the KEGG database at last, the genes functions of this regulation network were classified by Cytoscape and GO.
(1)Correlation between IMP related genes were analyzed, when correlation coefficients were greater than or equal to0.75, genes included:PPAT, PAICS, ADSL, AMPD, ATIC, GART, AIRS and GARS, when correlation coefficient greater than or equal to0.80, genes included: PPAT, ATIC, AMPD, GART, AIRS, GARS, when correlation coefficient greater than or equal to0.85, genes included:PPAT, AMPD, GART, AIRS, GARS, when correlation coefficient greater than or equal to0.90, genes included:AMPD, GART, AIRS, GARS, conclusion between AMPD and GARS-AIRS-GART composite gene was the highest, the PPAT followed.
(2) Expressions of8key IMP genes were analyzed in Rugao Yellow chicken:AMPD and ADSL genes may play major roles and AMPD was a positive regulatory role. Expression trend between GART and GARS, AIRS, AMPD was the opposite, but it could be obtained by the correlation coefficient model that conclusion between AMPD and GARS-AIRS-GART composite gene was the highest, it can be seen that8key IMP genes may have a positive or a negative correlation.
(3) Expression of AMPD of AIRS and GARS were upward trend while IMP contents were highest at6and10weeks, which may showed a positive correlation; expression of PPAT, ADSL and GART were downward trend that may be negatively correlated.
(4) Comparison of network regulation based on GeneChip and KEGG:GART, PAICS, ADSL, ATIC and AMPD played roles in IMP network regulation of Rugao Yellow chicken, while regulatory role of PPAT, AIRS and GARS were lowly expressed, this reason may be related to the genetic background of the different breeds.
肌苷酸(Inosine Monophosphate,IMP)是肉品中重要的风味物质,其在体内的合成代谢过程十分复杂,肌苷酸关键酶基因包括：磷酸焦磷酸酰胺转移酶(PPAT),磷酸核糖氨基咪唑碳酸酵素(PAICS),甘氨酰胺核苷酸合成酶(GARS),5-氨基咪唑核苷酸合成酶(AIRS),甘氨酰胺核苷酸转甲基酶(GART),5-氨基咪唑-4-甲酰氨核糖核苷酸甲酰转移酶(ATIC),腺苷琥珀酸裂解酶(ADSL),腺苷单磷酸脱氨酶(AMPD)。本文在如皋黄鸡基因芯片数据的基础上,运用Perl语言和皮尔森系数方程求解相关基因间线性关系,结合Cytoscape和GO将影响肌肉组织中肌苷酸关键酶相关基因的网络调控图进行了分析,构建了影响IMP合成途径中关键酶基因网络调控图,以期探明肌肉组织中IMP合成途径中关键酶之间的相互作用机理,主要研究结果如下：
1、同一鸡种不同生长阶段差异表达基因的比较(1)利用基因芯片技术,分析了不同生长阶段下(2-12周龄)如皋黄鸡和隐性白羽鸡肌肉组织基因的表达情况,获得了13379个基因的差异表达动态图谱。在如皋黄鸡中,与2周龄相比(P<0.001),4周龄时有114个基因上调表达,127个基因下调表达；6周龄时有116个基因上调表达,101个基因下调表达；8周龄时有130个基因上调表达,110个基因下调表达；10周龄时有136个基因上调表达,118个基因下调表达；12周龄时有94个基因上调表达,114个基因下调表达。4周龄、12周龄时上调表达基因数量少于下调表达基因的数量,而6周龄、8周龄、10周龄时上调表达基因数量明显多于下调表达基因的数量；在隐性白羽鸡中,与2周龄相比(P<0.001),6周龄时有72个基因上调表达,51个基因下调表达；10周龄时有42个基因上调表达,30个基因下调表达；12周龄时有44个基因上调表达,34个基因下调表达。3个生长阶段,上调表达的基因数量均多于下调表达的基因数量。
(2)将表达趋势一致的基因归为一类,所有基因可分为12类,相关系数大都集中在0-1之间,如皋黄鸡第6类与第7类相关系数为1,第3类与第7类,第4类与第8类之间相关系数为0；隐性白羽鸡第1类与第2类,第6类与第3、7、11类相关系数均是0,而第6类与第5、10类,第8类与第12类相关系数均是1,可得到：生长发育过程中,隐性白羽鸡表达量发生变化的基因之间的相关系数普遍高于如皋黄鸡。
2、不同时期(2周龄、12周龄)生长相关差异基因的表达
(1)应用表达谱芯片,对如皋黄鸡和隐性白羽鸡不同时期肌肉组织抽提及纯化的cRNA进行芯片杂交,并对基因表达谱进行分析,如皋黄鸡在不同生长时期(2周龄和12周龄相比)共筛选出差异表达基因208条(P<0.001),其中已知功能基因108条,表达上调94条,表达下调114条；隐性白羽鸡在不同生长时期(2周龄和12周龄相比)共筛选出差异表达基因78条(P<0.001),其中已知功能基因39条,表达上调44条,表达下调34条,经GO分类,两鸡种筛选的基因主要涉及生长发育、分子代谢、免疫应答、生物合成、细胞通信以及蛋白质合成与分解等相关基因,同时经荧光定量PCR技术与蛋白质组技术验证,表达谱芯片结果可靠。在差异基因中包括一些未知的新基因,但具体的功能以及在鸡生长发育、能量代谢等的过程所起到的作用需进一步验证。
(2)在差异基因中,如皋黄鸡中共筛选出6条与生长发育相关差异基因,隐性白羽鸡共筛选出1条差异基因。α-烯醇化酶基因(NM205120)均参与如皋黄鸡和隐性白羽鸡生长发育过程,该基因在如皋黄鸡和隐性白羽鸡中表达模式存在差异,其在如皋黄鸡中4周龄时表达量最低,2周龄和12周龄表达量并无变化；而在隐性白羽鸡中从2周龄到12周龄表达持续下降,推测α-烯醇化酶基因可能是影响鸡早期生长性状的候选功能基因；如皋黄鸡中另外5个生长相关差异基因表达情况是：HOP同源体(HOPX)高度表达：adducin1(ADD1)、受体酪氨酸激酶2(DDR2)和细胞周期因子B2(CCNB2)基因表达趋势保持平缓；胰岛素样生长因子1(IGF1)基因低度表达,表达水平持续下降。
3、肌苷酸相关基因网络调控图的构建
运用Perl语言和皮尔森系数方程求解相关基因的作用关系,结合Cytoscape软件生成肌苷酸相关基因的网络图,结合如皋黄鸡芯片数据构建肌苷酸相关基因网络调控图,运用Cytoscape和GO将肌苷酸关键酶相关基因的网络调控图进行了功能分类。
(1)通过相关系数模型分析肌苷酸相关基因之间的关联程度,相关系数在0.75以上时,肌苷酸相关基因包括：PPAT, PAICS, ADSL, AMPD, ATIC, GART, AIRS, GARS;相关系数在0.80以上时,肌苷酸相关基因包括：PPAT, ATIC, AMPD, GART, AIRS, GARS;相关系数大于等于0.85时,肌苷酸相关基因包括PPAT, AMPD, GART, AIRS, GARS;相关系数在0.90以上时,肌苷酸相关基因包括：AMPD, GART, AIRS, GARS,可得到：AMPD与GARS-AIRS-GART复合酶基因相关性最高,PPAT次之。
(2)对如皋黄鸡肌苷酸8个关键酶基因表达情况的分析：在如皋黄鸡肌苷酸含量的调控中,起较主要作用的可能是AMPD和ADSL基因。GART的表达趋势与GARS, AIRS和AMPD的表达趋势相反,但是由相关系数模型可得到GART, GARS, AIRS和AMPD的相关系数最高,表明8个关键酶之间存在正相关和负相关。
(3)在6、10周龄肌苷酸含量最高时,此时AMPD, AIRS和GARS的表达量呈上升趋势,对肌苷酸调控可能呈现正相关关系；而PPAT, ADSL和GART表达量呈下降趋势,对肌苷酸调控可能呈现负相关关系。
(4)将由芯片数据所得到的网络调控图与基于KEGG的网络调控图进行比较,可得到：KEGG中GART, PAICS, ADSL, ATIC, AMPD这5个关键酶在如皋黄鸡肌苷酸网络调控中也起着主要作用,而其余三个基因,PPAT, AIRS, GARS在如皋黄鸡肌肉中属于低度表达,这可能与不同鸡种的遗传背景有关。
Chinese local chicken breeds Rugao Yellow chicken and exotic breeds Recessive White chicken were selected as the object of study, in order to study genes expression changes of Rugao Yellow chicken and Recessive white chicken from the overall level at different growth times and the growth and development mechanism, which is of great significance for understanding the molecular mechanisms of chicken muscle growth and development.In this study, gene expression profiles of2,4,6,8,10,12weeks of age Rugao Yellow chicken and2,6,10,12weeks of age Recessive White chicken muscle tissues were studied respectively by cDNA microarrays and differentially expressed genes of2,12weeks of age of two kinds of chickens were screened to investigate the intrinsic link of different expression genes and their possible molecular biology and laws of the muscle tissues between two experimental groups, differential genes biological functions were classified by GO, in order to provide a theoretical basis further study.
Meat flavor is one of the most important aspects of the meat quality in chicken, many evidences indicated that Inosine Monophosphate (IMP) is one of the key components for meat flavor. The process of the synthesis and metabolism for IMP is very complicated, on the basis of Rugao Yellow chicken microarray data, the Perl language and the Pearson coefficient equation were used for solving the linear correlation of related genes, Cytoscape software was combined to generate genes network diagram of IMP, regulation network of IMP genes and its related genes was built based on the KEGG database at last, the genes functions of this regulation network were classified by Cytoscape and GO. IMP genes include PPAT, PAICS, GARS, AIRS, GART, ATIC, ADSL and AMPD. The purpose of building a regulation network is that the model was screened based on RNA levels on the basis of molecular hybridization, the IMP genes expression difference and the mechanism of interaction were revealed at the transcriptional level in the IMP synthesis pathway; the regulation network model of IMP was built in different spatial and temporal expression with RNA-mediated in order to understand function of cells, tissues from the molecular level and clarify molecular mechanism of different spatial and temporal expression for our local chicken flavor substance-IMP and provide a theoretical basis for industrial development. The main results were as follows:
1. Comparison at different growth stages of same chicken breeds
(1) The gene expressions of Rugao Yellow chicken and Recessive White chicken muscle were analyzed in different periods (2w-12w) by GeneChip. The expression profiles of13,379differentially expressed genes were obtained, Rugao Yellow chicken, compared to2weeks^P<0.001),114genes were up-regulated,127genes down-regulated, when it was4weeks old,116genes were up-regulated,101genes down-regulated, when it was6weeks old,130genes were up-regulated,110genes down-regulated, when it was8weeks old,136genes were up-regulated,118genes down-regulated, when it was10weeks old,94genes were up-regulated,114genes down-regulated, when it was12weeks old, the data analysis showed that at6weeks8and10weeks, the number of up-regulated genes were significantly more than the number of down-regulated genes, at4and12weeks, the number of up-regulated genes were significantly less than down-regulated genes; Recessive white chickens, compared to2weeks(P<0.001),72genes were up-regulated,51genes down-regulated, when it was4weeks old,42genes were up-regulated,30genes down-regulated, when it was10weeks old,44genes were up-regulated,34genes down-regulated, when it was4weeks old, the data analysis showed that at the three time points, the number of up-regulated genes were significantly more than the number of down-regulated genes.
(2) Different genes were divided into12categories, the correlation coefficients were concentrated in the between0and1, Rugao Yellow chicken, the correlation coefficient between Class6and Class7was1, the correlation coefficient between Class3and Class7, Class4and Class8were0; Recessive white chicken, the correlation coefficient between Class1and class2, Class6and3,7,11were0, the correlation coefficient between Class6and class5,10. Class8and12were1, the correlation coefficient of different genes in Recessive white chicken were generally higher than that in Rugao Yellow chicken in growth and development process
2. Gene expression in different periods (2w,12w)
(1) GeneChip was used to construct gene expression profile in order to screen differentially expressed genes of muscle tissue in Rugao Yellow chicken and Recessive white chicken and investigate the molecular mechanism related with muscle tissue traits between the two groups, respectively.208differentially expressed genes (P<0.001) were screened out, of which108are known genes,94up-regulated,114down-regulated for Rugao Yellow chicken;78differentially expressed genes (P<0.001) were screened out, of which39were known genes,44up-regulated,34down-regulated for Recessive white chicken, which were involved in growth, molecular mechanism, immune system process, biosynthetic process, cell communication, protein synthesis and degradation were screened out. Meanwhile, some genes among all these differentially expressed genes that had no annotation in GenBank were screened out; they were presumed to be unknown new genes. At the same time, the results were verified by real-time quantitative PCR and proteomic technology, indicating that microarray results were reliable. The roles that they may play in chicken growth and development, energy metabolism needed to be clarified later.
(2)6differentially expressed genes were screened out in the process of growth and development of Rugao Yellow chickens,1differentially expressed gene was screened out in the process of growth and development of Recessive white chickens, RCJMB04_24e12(NM_205120) was involved in the process of growth and development in Rugao Yellow chicken and Recessive white chicken, the expression of which was the lowest at4weeks old in Rugao Yellow chicken, while continued to decline in Recessive white chicken, suggesting that it may be a candidate marker of early growth traits in chicken, another five genes related growth-related differences in Rugao Yellow chicken were:HOPX, CCNB2, DDR2, ADD1and IGF1had a certain impact on the growth and development, HOPX was highly expressed, gene expression tendency of to CCNB2, DDR2, ADD1remained gentle, IGF1was lowly expressed.
3、Construction of IMP genes regulatory networks
The Perl language and the Pearson coefficient equation were used for solving the linear correlation of related genes, Cytoscape software was combined to generate genes network diagram of IMP, regulation network of IMP genes and its related genes was built based on the KEGG database at last, the genes functions of this regulation network were classified by Cytoscape and GO.
(1)Correlation between IMP related genes were analyzed, when correlation coefficients were greater than or equal to0.75, genes included:PPAT, PAICS, ADSL, AMPD, ATIC, GART, AIRS and GARS, when correlation coefficient greater than or equal to0.80, genes included: PPAT, ATIC, AMPD, GART, AIRS, GARS, when correlation coefficient greater than or equal to0.85, genes included:PPAT, AMPD, GART, AIRS, GARS, when correlation coefficient greater than or equal to0.90, genes included:AMPD, GART, AIRS, GARS, conclusion between AMPD and GARS-AIRS-GART composite gene was the highest, the PPAT followed.
(2) Expressions of8key IMP genes were analyzed in Rugao Yellow chicken:AMPD and ADSL genes may play major roles and AMPD was a positive regulatory role. Expression trend between GART and GARS, AIRS, AMPD was the opposite, but it could be obtained by the correlation coefficient model that conclusion between AMPD and GARS-AIRS-GART composite gene was the highest, it can be seen that8key IMP genes may have a positive or a negative correlation.
(3) Expression of AMPD of AIRS and GARS were upward trend while IMP contents were highest at6and10weeks, which may showed a positive correlation; expression of PPAT, ADSL and GART were downward trend that may be negatively correlated.
(4) Comparison of network regulation based on GeneChip and KEGG:GART, PAICS, ADSL, ATIC and AMPD played roles in IMP network regulation of Rugao Yellow chicken, while regulatory role of PPAT, AIRS and GARS were lowly expressed, this reason may be related to the genetic background of the different breeds.
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