利用基因芯片技术构建中国草原红牛公牛与阉牛差异基因表达谱
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摘要
20世纪70年代以后,随着基因组学、系统生物学、生物信息学等新兴学科的迅猛发展及人们消费观念的改变,动物育种目标由单一的常规育种向分子与常规联合育种转变,肉用性能相关基因的筛选成为研究热点。基因芯片及荧光定量PCR技术的出现为肉牛肉质性状的系统研究提供了强有利的手段。
研究以草原红牛为研究对象,在生产性能评定的基础上,采用AffymetrixBovine GeneChip进行表达谱分析,通过表达谱的构建选出与肉牛生长、代谢相关的功能基因。同时采用实时荧光定量PCR的方法比较了公牛与阉牛不同组织中IGF1、IGF2、IGF1R、IGFBP3、IGFBP5的表达差异,研究结果为探索牛肉质性状的主效基因奠定了分子生物学基础,也为肉牛的育种改良提供了依据。
参照国家肉牛屠宰标准及肉质分析方法,分析了草原红牛公牛与阉牛的屠宰性能、肉常规营养组成、肉质性状、氨基酸含量、脂肪酸含量进行了分析及比较发现:屠宰性状中,宰前活重、净肉重、日增重、胴体重、胴体产肉率、屠宰率、净肉率显著差异;高档肉块分割上,牛柳、西冷、大米龙、小黄瓜条肉差异显著;肉质性状上失水率差异极显著;剪切力值和肌纤维直径差异显著:肉常规营养成分分析中,干物质含量及粗蛋白质含量、初水分含量差异显著;氨基酸组成上公牛的谷氨酸、丝氨酸、组氨酸、缬氨酸、蛋氨酸、苯丙氨酸、赖氨酸、肉豆蔻酸、棕榈油酸、油酸、亚油酸、亚麻酸、棕榈酸、硬脂酸以及氨基酸总和差异显著。
利用Affymetrix牛基因组芯片对成年公牛和阉牛背最长肌组织进行生长发育、脂肪代谢等性状的差异表达基因进行筛选,共有529个探针在公牛和阉牛中差异表达倍数在1.5倍以上:上调基因435个,下调基因94个。探针中403个基因与已知功能基因具有较高的氨基酸相似性。对差异表达基因GO分析发现大部分基因均能找到注释:生物过程294个,细胞组成121个,分子功能243个,未分类126个。通过基因注解结合关键词筛选出与生长、发育、脂肪代谢相关的基因25个。通过KEGG pathway通路分析,在脂肪酸生物合成、胆汁酸生物合成、脂肪酸代谢、过氧化物酶体增生物激活受体信号通路、肌动蛋白细胞骨架调控5条pathway中筛选到ADH5、OXSM、FABP3、ARPC3、BDKRB2基因分别与脂肪酸代谢、肌肉发育、生长调节等相关。
对草原红牛公牛、阉牛不同组织(心、肝、脾、肺、肾、瘤胃、十二指肠、背最长肌)中IGF1、IGF2、IGF1R、IGFBP3、IGFBP5 mRNA表达量进行了比较,结果发现部分组织中的表达量存在一定的差异。
Meat quality and carcass traits are comprehensive, including a series of evaluation index. There are so many factors affecting the meat quality and carcass traits and we know little on meat quality, flavor, and taste. For thoroughly understanding the factors on genetic mechanisms of meat quality, it requires rapid and efficient methods to find major specific expression genes in bovine muscle to provide the molecular basis for scientific breeding.
The phenotype data of carcass and meat quality traits of 60 cattle (30 bulls and 30 steers) were collected after breeding and they were slaughtered at same conditions. Meat quality and carcass traits were compared between bulls and steers. Bovine longissimus dorsi muscle (LD) samples from 3 bulls and 3 steers were collected to research the different gene expression profile of LD to find differential expression genes in our experiment by gene chip. The results were verified by fluorescence quantitative PCR (FQ-PCR).
The results were as follows:
In carcass traits, body weight before slaughter, net yield of meat,daily weight gains,carcass weight, meat percentage of carcass, dressing percentage, meat percentage in bulls were significantly higher than that of steers (p<0.05). In the top grade beef, sirloin, the fillet, big topside, and little silverside in bulls were significantly higher than that of steers (p<0.05). In meat quality traits, the dehydration rate in bulls were significantly higher than that of steers (P<0.01). The shear force and muscle fibre diameter in bulls were significantly higher than that of steers (p<0.05).In analysis of nutritional component of meat, dry matter and crude protein in bulls were signifieantly lower than that of steers and the original moisture was significantly higher than that of steers (P<0.05) . In the composing of amino the acid, glutamic acid, serine, histidine, valine, methionine, Phenylalanine, lysine, palmitoleic acid, oleic acid , linoleic acid , linolenic acid and total of amino acid are lower than that of steers (P<0.05). Content of the tetradecylic acid in steers was higher and content of hexadecanoic acid and stearic acid were lower than that of bulls (P<0.05) .
Red Steppes bulls(n=3) and steers (n=3) were selected randomly to analyze the LD differentially expressed genes on growth and development and fat metabolism by Affymetrix gene chip, numbers of 22810 probes were detected, numbers of 16321 have hybridization signal, it holds 71.6%. 403 genes that have relative high amino acid similarity with known functional gene, it holds 76.18%, while 126 genes whose functions are still unknown after comparison analysis, which were 23.82%.
The results of 529 differential expression gene by GO analysised were showed 294 gene can find notations on biological process, 121 gene can find notations on cellular component, 243 gene can find notations on molecullar function, 126 gene was not found notations. Each functional group can be independently divided into different sub-groups which are also divided downward, all these groups comprise branch architecture of Ontologics and each has different biological function. Total 25 genes which relating with growth and fat metabolism were founded from the results of GO classify by gene comment.
ADH5, OXSM, FABP3, ARPC3, BDKRB2 had founded in the pathway of Fatty Acid Metabolism, Bile Acid Biosynthesis, Fatty Acid Biosynthesis, PPAR Signaling Pathway, Regulation of actin cytoskeleton. They are correlated with fatty acid metabolism, muscle development and growth regulation.
Five representative genes were selected to verify the result of gene chip by FQ-PCR. The results of FQ-PCR are consistent with gene chip. Gene expression changes were consistent with the physiological characteristics, the difference genes were accurated.
IGF1, IGF2, IGF1R, IGFBP3, IGFBP5 mRNA expression variance of 8 tissues (heart, liver, spleen, lung, kidney, rumen, duodenum, longissimus dorsi) were analyzed, the results were different, which may be related with male hormone secretion reduced after castrated caused liver IGFs expression changed.
In this research, gene chip was employed to construct the bovine longissimus dorsi muscle tissue gene expression profile and analyze the different gene expression between bulls and steers. We use the FQ-PCR to verify parts of the genes' expression. The results will build solid base for the study of major genes with meat quality and carcass traits.
研究以草原红牛为研究对象,在生产性能评定的基础上,采用AffymetrixBovine GeneChip进行表达谱分析,通过表达谱的构建选出与肉牛生长、代谢相关的功能基因。同时采用实时荧光定量PCR的方法比较了公牛与阉牛不同组织中IGF1、IGF2、IGF1R、IGFBP3、IGFBP5的表达差异,研究结果为探索牛肉质性状的主效基因奠定了分子生物学基础,也为肉牛的育种改良提供了依据。
参照国家肉牛屠宰标准及肉质分析方法,分析了草原红牛公牛与阉牛的屠宰性能、肉常规营养组成、肉质性状、氨基酸含量、脂肪酸含量进行了分析及比较发现:屠宰性状中,宰前活重、净肉重、日增重、胴体重、胴体产肉率、屠宰率、净肉率显著差异;高档肉块分割上,牛柳、西冷、大米龙、小黄瓜条肉差异显著;肉质性状上失水率差异极显著;剪切力值和肌纤维直径差异显著:肉常规营养成分分析中,干物质含量及粗蛋白质含量、初水分含量差异显著;氨基酸组成上公牛的谷氨酸、丝氨酸、组氨酸、缬氨酸、蛋氨酸、苯丙氨酸、赖氨酸、肉豆蔻酸、棕榈油酸、油酸、亚油酸、亚麻酸、棕榈酸、硬脂酸以及氨基酸总和差异显著。
利用Affymetrix牛基因组芯片对成年公牛和阉牛背最长肌组织进行生长发育、脂肪代谢等性状的差异表达基因进行筛选,共有529个探针在公牛和阉牛中差异表达倍数在1.5倍以上:上调基因435个,下调基因94个。探针中403个基因与已知功能基因具有较高的氨基酸相似性。对差异表达基因GO分析发现大部分基因均能找到注释:生物过程294个,细胞组成121个,分子功能243个,未分类126个。通过基因注解结合关键词筛选出与生长、发育、脂肪代谢相关的基因25个。通过KEGG pathway通路分析,在脂肪酸生物合成、胆汁酸生物合成、脂肪酸代谢、过氧化物酶体增生物激活受体信号通路、肌动蛋白细胞骨架调控5条pathway中筛选到ADH5、OXSM、FABP3、ARPC3、BDKRB2基因分别与脂肪酸代谢、肌肉发育、生长调节等相关。
对草原红牛公牛、阉牛不同组织(心、肝、脾、肺、肾、瘤胃、十二指肠、背最长肌)中IGF1、IGF2、IGF1R、IGFBP3、IGFBP5 mRNA表达量进行了比较,结果发现部分组织中的表达量存在一定的差异。
Meat quality and carcass traits are comprehensive, including a series of evaluation index. There are so many factors affecting the meat quality and carcass traits and we know little on meat quality, flavor, and taste. For thoroughly understanding the factors on genetic mechanisms of meat quality, it requires rapid and efficient methods to find major specific expression genes in bovine muscle to provide the molecular basis for scientific breeding.
The phenotype data of carcass and meat quality traits of 60 cattle (30 bulls and 30 steers) were collected after breeding and they were slaughtered at same conditions. Meat quality and carcass traits were compared between bulls and steers. Bovine longissimus dorsi muscle (LD) samples from 3 bulls and 3 steers were collected to research the different gene expression profile of LD to find differential expression genes in our experiment by gene chip. The results were verified by fluorescence quantitative PCR (FQ-PCR).
The results were as follows:
In carcass traits, body weight before slaughter, net yield of meat,daily weight gains,carcass weight, meat percentage of carcass, dressing percentage, meat percentage in bulls were significantly higher than that of steers (p<0.05). In the top grade beef, sirloin, the fillet, big topside, and little silverside in bulls were significantly higher than that of steers (p<0.05). In meat quality traits, the dehydration rate in bulls were significantly higher than that of steers (P<0.01). The shear force and muscle fibre diameter in bulls were significantly higher than that of steers (p<0.05).In analysis of nutritional component of meat, dry matter and crude protein in bulls were signifieantly lower than that of steers and the original moisture was significantly higher than that of steers (P<0.05) . In the composing of amino the acid, glutamic acid, serine, histidine, valine, methionine, Phenylalanine, lysine, palmitoleic acid, oleic acid , linoleic acid , linolenic acid and total of amino acid are lower than that of steers (P<0.05). Content of the tetradecylic acid in steers was higher and content of hexadecanoic acid and stearic acid were lower than that of bulls (P<0.05) .
Red Steppes bulls(n=3) and steers (n=3) were selected randomly to analyze the LD differentially expressed genes on growth and development and fat metabolism by Affymetrix gene chip, numbers of 22810 probes were detected, numbers of 16321 have hybridization signal, it holds 71.6%. 403 genes that have relative high amino acid similarity with known functional gene, it holds 76.18%, while 126 genes whose functions are still unknown after comparison analysis, which were 23.82%.
The results of 529 differential expression gene by GO analysised were showed 294 gene can find notations on biological process, 121 gene can find notations on cellular component, 243 gene can find notations on molecullar function, 126 gene was not found notations. Each functional group can be independently divided into different sub-groups which are also divided downward, all these groups comprise branch architecture of Ontologics and each has different biological function. Total 25 genes which relating with growth and fat metabolism were founded from the results of GO classify by gene comment.
ADH5, OXSM, FABP3, ARPC3, BDKRB2 had founded in the pathway of Fatty Acid Metabolism, Bile Acid Biosynthesis, Fatty Acid Biosynthesis, PPAR Signaling Pathway, Regulation of actin cytoskeleton. They are correlated with fatty acid metabolism, muscle development and growth regulation.
Five representative genes were selected to verify the result of gene chip by FQ-PCR. The results of FQ-PCR are consistent with gene chip. Gene expression changes were consistent with the physiological characteristics, the difference genes were accurated.
IGF1, IGF2, IGF1R, IGFBP3, IGFBP5 mRNA expression variance of 8 tissues (heart, liver, spleen, lung, kidney, rumen, duodenum, longissimus dorsi) were analyzed, the results were different, which may be related with male hormone secretion reduced after castrated caused liver IGFs expression changed.
In this research, gene chip was employed to construct the bovine longissimus dorsi muscle tissue gene expression profile and analyze the different gene expression between bulls and steers. We use the FQ-PCR to verify parts of the genes' expression. The results will build solid base for the study of major genes with meat quality and carcass traits.
引文
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