猪不同肌纤维类型骨骼肌的基因组表达谱差异
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摘要
本研究选用中国肉质优良的脂肪型地方品种荣昌猪和肉质相对较差的瘦肉型国外引入品种长白猪为动物模型,采用Agilent商业化的全基因组芯片检测了在相同饲养条件下(7月龄)背最长肌和腰大肌中相关基因的差异表达,探讨在猪生长发育后期过程中肌纤维类型和相关基因的表达差异及品种特点。
①MyHC家族基因的差异表达与肉质性状的关联性分析结果显示：长白猪的MyHCIIb mRNA表达量在混合性别效应的背最长肌和腰大肌中均极显著的高于荣昌猪(P<0.01)。同时MyHCIIb mRNA表达量与pH2之间呈极显著负相关(P<0.01),与贮存损失成极显著正相关(P<0.01),显示出肉质较差的特性。
②差异表达,聚类和主成份分析结果显示：在品种内性别内组织间,品种内组织内性别间和性别内组织内品种间平均存在1,180,833和1,798个差异基因,表明品种>组织>性别差异。对8类组织中的差异基因聚类发现基因可以聚为11大类,而组织聚为4大类。主成份分析发现第一主成份的差异表达基因的模式为主要表达模式,即8类组织之间的差异基因存在着相似的表达模式。
③T检验得到差异基因的GO分析结果显示：在品种内性别内组织间,品种内组织内性别间和性别内组织内品种间以及在混合性别的组织效应和品种效应中,共发现了34,12,15,15和7个与组织、品种、性别类型存在差异的GO,主要集中在糖酵解,骨骼肌系统形态学发生,ATP代谢过程,电子传递链等方面；表明大量的GO存在于组织>品种>性别之间,也可以看出背最长肌和腰大肌作为两种代谢类型的极端代表之间存在着生物学过程差异。
④T检验得到差异基因的GSEA分析结果显示：在品种内性别内组织间,品种内组织内性别间和性别内组织内品种间以及在混合性别的组织效应和品种效应中分别发现了18,13和2,13和1个显著的Pathway途径,主要集中在氧化磷酸化和与免疫学相关以及信号传导等途径,表明大量的Pathway存在于组织>性别>品种之间,可以看出两种肌肉组织在参与能量代谢的同时,在参与体内免疫学相关的通路中也同样具有很活跃的功能。
⑤GSEA筛选出来的差异基因分析结果显示：在品种内性别内组织间,品种内组织内性别间和性别内组织内品种间的交集中,分别发现了5(PIK3R5,PLA2Gl B, SLA-DQA1,CD86和CAMK2D),2(PLA2G1B和ABAT)和1(CTH)个差异基因,表明这些基因在生物学通路中的重要作用。
⑥染色体富集分析结果显示：在品种内性别内组织间的交集中,共发现差异表达倍数比较高(千倍以上)的基因主要集中在6,7,9,13,14号染色体上；在品种内组织内性别间的交集中,主要集中在1,2,9,14,15号染色体上,性别内组织内品种间的交集中,主要集中在5,13,14号染色体上。
⑦芯片数据可靠性验证的结果显示：对芯片内部变异程度的评价分析表明,变异系数范围为3.40%-10.01%,低于C.V 15%的标准,同时qRT-PCR技术对芯片数据结果的验证显示,两种技术之间的相关系数为0.718±0.140,具有较高的相关性,说明本次实验所检测的基因能够真实的反映出它们的表达变化规律,数据真实可信。
In this study, we choose Rongchang Pigs of Chinese domestic breed with better meat quality,stronger fat deposition and Landrace of introduced breed with worse meat quality,less fat deposition as the animal model, used Genome transcript microarray of Agilent company studies to example the expression changes of genome transcript profiling in longissimus dorsi muscle and psoas major muscle at 210 d feed with the same condition and discuss the expression changes associated with different fibertypes and breeds characters during post growth development.
①The result about difference expression of MyHC gene family and the association with meat qulity showed that:the MyHCⅡb mRNA level in Landrace were higher than Rongchang pigs at very significant level(P<0.01),when mixed gender effect. Meanwhile the expression of MyHCⅡb mRNA was negative with pH2 at very significant level(P<0.01), and positive with drip loss at very significant level(P<0.01),presented the worse meat quality characters.
②Difference expression, clustering and principal component analysis showed that: between the same breed, same gender and the differet tissues, between the same breed, same tissue and the different gengers, between the same genger, same tissue and different breeds, we found 1,180,833 and 1,798 respectively, suggested that the difference between the breeds> tissues> genders.While the 8 diffetent tissues were classified 11 clusters,while different tissues were classifed 4 clusters.The futher principal component analysis found that the first principal component was the chief expression profile, the genes of 8 diffetent tissues existed the similar expression profiles.
③The results of GO analysis selected by T test showed that:between the same breed, same gender and the differet tissues, between the same breed, same tissue and the different gengers, between the same genger, same tissue and different breeds and mixed gender, considering the tissues effects and breeds effects, we found 34,12,15,15 and 7 GO respectively, primary focusing on glucolysis, Skeletal muscular system morphology, ATP metablism process, electron transport chain and so on; the lots of GO existed in the tissues > breeds> genders, suggested that different biology process between longissimus dorsi muscle and psoas major muscle which presented the two extremity metablism types.
④The results of GSEA analysis selected by T test showed that:between the same breed, same gender and the differet tissues, between the same breed, same tissue and the different gengers, between the same genger, same tissue and different breeds and mixed gender, considering the tissues effects and breeds effects, we found 18,13,2,13 and 1 Pathway significantly, The Pathway primary focusing on oxidation phosphorylation, immunology and signal transduction, the lots of GO existed in the tissues> genders> breeds, we also saw that these two muscular tissues play an important role in participation the Pathway related to immunology, when they played a part in energy metabolism.
⑤The different genes selected by GSEA analysis showed that:analyzing the high frequency genes emerged in different Pathway by leading edge analysis showed thay: between the same breed, same gender and the differet tissues, between the same breed, same tissue and the different gengers and between the same genger, same tissue and different breeds we found 5 (PIK3R5, PLA2G1B, SLA-DQA1, CD86 and CAMK2D),2 (PLA2G1B and ABAT) and 1 (CTH) difference expression genes respectively, suggested these genes may play an impotrant role in Pathway.
⑥The results of chromosome co-localization analysis showed that:intersection set existed among the same breed, same gender and the differet tissues,we found the expression genes (thousandfold and more) were mostly focused on the chrosome of 6,7, 9,13,14; intersection set existed among the same breed, same tissue and the differet genders, we found the expression genes (thousandfold and more) were mostly focused on the chrosome of 1,2,9,14,15; intersection set existed among the same gender, same tissue and the differet breeds,we found the expression genes (thousandfold and more) were mostly focused on the chrosome of 5,13,14.
⑦Reliability evaluation from microarray data showed thaty:the C.V range from 3.4%-10.01% within-arrays, lower the standard of within C.V 15%, meanwhile qRT-PCR was used to verify the microarray data, the results showed that the coefficient correlation between these two technology was:0.718±0.140, These resultes demonstrate that the microarray technique used in this study is accurate and reproducible.
①MyHC家族基因的差异表达与肉质性状的关联性分析结果显示：长白猪的MyHCIIb mRNA表达量在混合性别效应的背最长肌和腰大肌中均极显著的高于荣昌猪(P<0.01)。同时MyHCIIb mRNA表达量与pH2之间呈极显著负相关(P<0.01),与贮存损失成极显著正相关(P<0.01),显示出肉质较差的特性。
②差异表达,聚类和主成份分析结果显示：在品种内性别内组织间,品种内组织内性别间和性别内组织内品种间平均存在1,180,833和1,798个差异基因,表明品种>组织>性别差异。对8类组织中的差异基因聚类发现基因可以聚为11大类,而组织聚为4大类。主成份分析发现第一主成份的差异表达基因的模式为主要表达模式,即8类组织之间的差异基因存在着相似的表达模式。
③T检验得到差异基因的GO分析结果显示：在品种内性别内组织间,品种内组织内性别间和性别内组织内品种间以及在混合性别的组织效应和品种效应中,共发现了34,12,15,15和7个与组织、品种、性别类型存在差异的GO,主要集中在糖酵解,骨骼肌系统形态学发生,ATP代谢过程,电子传递链等方面；表明大量的GO存在于组织>品种>性别之间,也可以看出背最长肌和腰大肌作为两种代谢类型的极端代表之间存在着生物学过程差异。
④T检验得到差异基因的GSEA分析结果显示：在品种内性别内组织间,品种内组织内性别间和性别内组织内品种间以及在混合性别的组织效应和品种效应中分别发现了18,13和2,13和1个显著的Pathway途径,主要集中在氧化磷酸化和与免疫学相关以及信号传导等途径,表明大量的Pathway存在于组织>性别>品种之间,可以看出两种肌肉组织在参与能量代谢的同时,在参与体内免疫学相关的通路中也同样具有很活跃的功能。
⑤GSEA筛选出来的差异基因分析结果显示：在品种内性别内组织间,品种内组织内性别间和性别内组织内品种间的交集中,分别发现了5(PIK3R5,PLA2Gl B, SLA-DQA1,CD86和CAMK2D),2(PLA2G1B和ABAT)和1(CTH)个差异基因,表明这些基因在生物学通路中的重要作用。
⑥染色体富集分析结果显示：在品种内性别内组织间的交集中,共发现差异表达倍数比较高(千倍以上)的基因主要集中在6,7,9,13,14号染色体上；在品种内组织内性别间的交集中,主要集中在1,2,9,14,15号染色体上,性别内组织内品种间的交集中,主要集中在5,13,14号染色体上。
⑦芯片数据可靠性验证的结果显示：对芯片内部变异程度的评价分析表明,变异系数范围为3.40%-10.01%,低于C.V 15%的标准,同时qRT-PCR技术对芯片数据结果的验证显示,两种技术之间的相关系数为0.718±0.140,具有较高的相关性,说明本次实验所检测的基因能够真实的反映出它们的表达变化规律,数据真实可信。
In this study, we choose Rongchang Pigs of Chinese domestic breed with better meat quality,stronger fat deposition and Landrace of introduced breed with worse meat quality,less fat deposition as the animal model, used Genome transcript microarray of Agilent company studies to example the expression changes of genome transcript profiling in longissimus dorsi muscle and psoas major muscle at 210 d feed with the same condition and discuss the expression changes associated with different fibertypes and breeds characters during post growth development.
①The result about difference expression of MyHC gene family and the association with meat qulity showed that:the MyHCⅡb mRNA level in Landrace were higher than Rongchang pigs at very significant level(P<0.01),when mixed gender effect. Meanwhile the expression of MyHCⅡb mRNA was negative with pH2 at very significant level(P<0.01), and positive with drip loss at very significant level(P<0.01),presented the worse meat quality characters.
②Difference expression, clustering and principal component analysis showed that: between the same breed, same gender and the differet tissues, between the same breed, same tissue and the different gengers, between the same genger, same tissue and different breeds, we found 1,180,833 and 1,798 respectively, suggested that the difference between the breeds> tissues> genders.While the 8 diffetent tissues were classified 11 clusters,while different tissues were classifed 4 clusters.The futher principal component analysis found that the first principal component was the chief expression profile, the genes of 8 diffetent tissues existed the similar expression profiles.
③The results of GO analysis selected by T test showed that:between the same breed, same gender and the differet tissues, between the same breed, same tissue and the different gengers, between the same genger, same tissue and different breeds and mixed gender, considering the tissues effects and breeds effects, we found 34,12,15,15 and 7 GO respectively, primary focusing on glucolysis, Skeletal muscular system morphology, ATP metablism process, electron transport chain and so on; the lots of GO existed in the tissues > breeds> genders, suggested that different biology process between longissimus dorsi muscle and psoas major muscle which presented the two extremity metablism types.
④The results of GSEA analysis selected by T test showed that:between the same breed, same gender and the differet tissues, between the same breed, same tissue and the different gengers, between the same genger, same tissue and different breeds and mixed gender, considering the tissues effects and breeds effects, we found 18,13,2,13 and 1 Pathway significantly, The Pathway primary focusing on oxidation phosphorylation, immunology and signal transduction, the lots of GO existed in the tissues> genders> breeds, we also saw that these two muscular tissues play an important role in participation the Pathway related to immunology, when they played a part in energy metabolism.
⑤The different genes selected by GSEA analysis showed that:analyzing the high frequency genes emerged in different Pathway by leading edge analysis showed thay: between the same breed, same gender and the differet tissues, between the same breed, same tissue and the different gengers and between the same genger, same tissue and different breeds we found 5 (PIK3R5, PLA2G1B, SLA-DQA1, CD86 and CAMK2D),2 (PLA2G1B and ABAT) and 1 (CTH) difference expression genes respectively, suggested these genes may play an impotrant role in Pathway.
⑥The results of chromosome co-localization analysis showed that:intersection set existed among the same breed, same gender and the differet tissues,we found the expression genes (thousandfold and more) were mostly focused on the chrosome of 6,7, 9,13,14; intersection set existed among the same breed, same tissue and the differet genders, we found the expression genes (thousandfold and more) were mostly focused on the chrosome of 1,2,9,14,15; intersection set existed among the same gender, same tissue and the differet breeds,we found the expression genes (thousandfold and more) were mostly focused on the chrosome of 5,13,14.
⑦Reliability evaluation from microarray data showed thaty:the C.V range from 3.4%-10.01% within-arrays, lower the standard of within C.V 15%, meanwhile qRT-PCR was used to verify the microarray data, the results showed that the coefficient correlation between these two technology was:0.718±0.140, These resultes demonstrate that the microarray technique used in this study is accurate and reproducible.
引文
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