鸡类胰岛素生长因子-Ⅰ基因单碱基突变对该基因转录、表达以及对生长、屠体性状的影响
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摘要
鸡类胰岛素生长因子-Ⅰ(IGF-Ⅰ)对生物体的生长发育具有重要的调控作用,直接发挥生长激素的促生长发育功能。前人的研究结果表明:在鸡的1号染色体长臂近着丝粒附近存在一个影响青年鸡体重的数量性状座位(QTL),IGF-Ⅰ基因处于该区域内。我们认为IGF-Ⅰ基因是产生这种QTL效应的主要因素。
通过对丝羽乌骨鸡和肉鸡IGF-Ⅰ基因的部分碱基序列测定,发现在其5’端调控区存在一处单碱基突变:C→A。肉鸡中大部分都是突变型(A),而丝羽乌骨鸡则全部是野生型(C)。在丝羽乌骨鸡和肉鸡杂交产生的F2代群体中进行基因型鉴定,扩大样本量。最后的统计分析结果显示:该突变位点与一些生长、屠体性状相关显著,对生长速度有显著影响。
该突变位于IGF-Ⅰ基因的启动子区域,距转录启始位点和“TATA”盒较近,而且该区段在几个物种中高度保守。转录因子结合位点预测分析结果表明:突变导致基因5’端启动子区域增加一个转录因子CdxA与之结合。CdxA是鸡体内特异表达的同源异形盒基因,其对消化器官,尤其是肠发育有很重要的调节作用。纯化的外源表达的融合蛋白GST—CdxA与突变和非突变寡核苷酸序列相互作用的电泳迁移率变动实验(EMSA)体外证明了突变确实增加了转录因子CdxA与其结合。相关文献报道:转录因子cDxA能够使报告基因的转录水平显著增加。因此,突变型个体IGF-Ⅰ基因的转录、表达水平也要比非突变个体的水平显著增加。由于转录因子CdxA在鸡出生后仅在肠上皮细胞中表达,虽然IGF-Ⅰ基因在肝脏及其全身多种组织、器官表达,但除了肠上皮细胞外,其他组织、器官没有转录因子与IGF-Ⅰ基因相结合,所以突变型个体仅在肠组织中IGF-Ⅰ基因的转录、表达水平上升,其它组织中则无变化。通过荧光实时定量PCR和放射性免疫实验技术,我们检测了突变和非突变型个体IGF-Ⅰ基因在肝脏、小肠中的转录、表达情况。在肝脏中,IGF-Ⅰ基因的转录、表达水平无显著的基因型差异。在小肠中,突变型个体IGF-Ⅰ基因的转录水平在三周龄时比非突变型个体高出约1倍,表达水平高出约30%。6周龄和9周龄时转录、表达水平没有显著的差异。
IGF-Ⅰ基因能促进人的小肠平滑肌细胞生长,我们的实验结果证明IGF-Ⅰ基因同样能促进鸡的小肠平滑肌细胞生长。由于突变型个体肠组织中IGF-Ⅰ的含量比非突变型个体高出约30%,因此其平滑肌细胞、整个肠组织生长、发育更快。最后导致小肠长度比非突变型个体长约3%。小肠是重要的消化、吸收器官,由于突变型个体的消化、吸收面积增大,使其在同样的条件下获得更多的营养成份,因而其生长速度加快,获得了较高的体重和其它一些屠体性状均值。
在畜禽中,长期对生长速度进行人工选择,可以导致该品种(系)消化器官相对比例的上升,使其消化、吸收能力增强,我们的实验结果验证了这种观点,同时也可以作为解释肉鸡为什么具有很高生长速度的多个因素之一。可以应用于将来的分子育种工作中。数量性状座位(QTL)是基因组上的一段区域,含有一个或几个影响数量性状的基因。某一数量性状一般受多个基因以及环境因素的影响,每个基因对该性状的影响程度相对较小,加上环境因素的作用,寻找、证明QTL基因比较困难。但近年随着一些新的技术手段、统计分析工具的发展以及对基因组的更深入的研
A QTL affecting body weight of young chickens was mapped to the short arm of chromosome 1 near the centromere using an F2 population crossing of a broiler sire-line and egg-lying line previously. Searching for genes or putative genes in this region, we found that the insulin-like growth factor-I gene is the strongest candidate gene for this effect base on its crucial role in growth and development.We identified a C-A mutation nearing the putative 'TATA' box and the transcriptional start site. Association study demonstrated that the regulatory polymorphism is strongly associated with growth and carcass traits. Birds with A A genotype have more 6 week, 9 week and 12 week body weight than birds with CC and CA genotype, in others traits, the AA birds have also higher traits means than AC and CC birds, such as live weight, carcass weight, eviscerated yield with giblet, eviscerated yield, breast muscle weight, leg muscle weight, heart weight, abdominal fat weight, small intestine length, etc.The mutation occurs in the regulatory region which is highly conserved in several species. The mutation increases a transcription factor of CdxA in vitro and in vivo, which has activity of enhancing transcription level of reporter plasmids in CDXA expressing cells, and expressed exclusively in chicken gut after hatch. We analyzed the transcriptional and expressional level of IGF-I gene in liver and in small intestine between the mutant and the wild-type birds at 3, 6, 9 week by real-time quantitative PCR and radioimmunoassay. In liver, there were no transcriptional and expressional differences between the mutant and wild-type birds at 3, 6, 9 week age; in small intestine, the mutant birds had about one-fold mRNA and 30% protein of IGF-I higher than wild type birds at 3 week age, and no significant genotype difference at 6 and 9 week age. Thus the mutation results in the increase of expressional efficiency in vivo. The mutant birds have higher IGF-I in small intestine, and high IGF-I can promote intestine cell growth faster, then these birds favor the longer small intestine length, with more nutrition digested in and absorbed from intestine the mutant birds finally result in higher growth rate and others carcass traits means.Our results supported the view that selection for high growth rate in birds is linked to an increase in the relative size of the digestive organs and rapid early development of the digestive organs. Our results may be also an example of a complex genetic traits created by multiple susceptibility genes. Identification of QTL gene can provide suggestive information about normal development and disease processes, and now with the development of new genetic technique, statistical tools and better characterized genome, find QTL gene is becoming easier than before. The results are also useful in practice of chicken molecular breeding programme in the near future.
通过对丝羽乌骨鸡和肉鸡IGF-Ⅰ基因的部分碱基序列测定,发现在其5’端调控区存在一处单碱基突变:C→A。肉鸡中大部分都是突变型(A),而丝羽乌骨鸡则全部是野生型(C)。在丝羽乌骨鸡和肉鸡杂交产生的F2代群体中进行基因型鉴定,扩大样本量。最后的统计分析结果显示:该突变位点与一些生长、屠体性状相关显著,对生长速度有显著影响。
该突变位于IGF-Ⅰ基因的启动子区域,距转录启始位点和“TATA”盒较近,而且该区段在几个物种中高度保守。转录因子结合位点预测分析结果表明:突变导致基因5’端启动子区域增加一个转录因子CdxA与之结合。CdxA是鸡体内特异表达的同源异形盒基因,其对消化器官,尤其是肠发育有很重要的调节作用。纯化的外源表达的融合蛋白GST—CdxA与突变和非突变寡核苷酸序列相互作用的电泳迁移率变动实验(EMSA)体外证明了突变确实增加了转录因子CdxA与其结合。相关文献报道:转录因子cDxA能够使报告基因的转录水平显著增加。因此,突变型个体IGF-Ⅰ基因的转录、表达水平也要比非突变个体的水平显著增加。由于转录因子CdxA在鸡出生后仅在肠上皮细胞中表达,虽然IGF-Ⅰ基因在肝脏及其全身多种组织、器官表达,但除了肠上皮细胞外,其他组织、器官没有转录因子与IGF-Ⅰ基因相结合,所以突变型个体仅在肠组织中IGF-Ⅰ基因的转录、表达水平上升,其它组织中则无变化。通过荧光实时定量PCR和放射性免疫实验技术,我们检测了突变和非突变型个体IGF-Ⅰ基因在肝脏、小肠中的转录、表达情况。在肝脏中,IGF-Ⅰ基因的转录、表达水平无显著的基因型差异。在小肠中,突变型个体IGF-Ⅰ基因的转录水平在三周龄时比非突变型个体高出约1倍,表达水平高出约30%。6周龄和9周龄时转录、表达水平没有显著的差异。
IGF-Ⅰ基因能促进人的小肠平滑肌细胞生长,我们的实验结果证明IGF-Ⅰ基因同样能促进鸡的小肠平滑肌细胞生长。由于突变型个体肠组织中IGF-Ⅰ的含量比非突变型个体高出约30%,因此其平滑肌细胞、整个肠组织生长、发育更快。最后导致小肠长度比非突变型个体长约3%。小肠是重要的消化、吸收器官,由于突变型个体的消化、吸收面积增大,使其在同样的条件下获得更多的营养成份,因而其生长速度加快,获得了较高的体重和其它一些屠体性状均值。
在畜禽中,长期对生长速度进行人工选择,可以导致该品种(系)消化器官相对比例的上升,使其消化、吸收能力增强,我们的实验结果验证了这种观点,同时也可以作为解释肉鸡为什么具有很高生长速度的多个因素之一。可以应用于将来的分子育种工作中。数量性状座位(QTL)是基因组上的一段区域,含有一个或几个影响数量性状的基因。某一数量性状一般受多个基因以及环境因素的影响,每个基因对该性状的影响程度相对较小,加上环境因素的作用,寻找、证明QTL基因比较困难。但近年随着一些新的技术手段、统计分析工具的发展以及对基因组的更深入的研
A QTL affecting body weight of young chickens was mapped to the short arm of chromosome 1 near the centromere using an F2 population crossing of a broiler sire-line and egg-lying line previously. Searching for genes or putative genes in this region, we found that the insulin-like growth factor-I gene is the strongest candidate gene for this effect base on its crucial role in growth and development.We identified a C-A mutation nearing the putative 'TATA' box and the transcriptional start site. Association study demonstrated that the regulatory polymorphism is strongly associated with growth and carcass traits. Birds with A A genotype have more 6 week, 9 week and 12 week body weight than birds with CC and CA genotype, in others traits, the AA birds have also higher traits means than AC and CC birds, such as live weight, carcass weight, eviscerated yield with giblet, eviscerated yield, breast muscle weight, leg muscle weight, heart weight, abdominal fat weight, small intestine length, etc.The mutation occurs in the regulatory region which is highly conserved in several species. The mutation increases a transcription factor of CdxA in vitro and in vivo, which has activity of enhancing transcription level of reporter plasmids in CDXA expressing cells, and expressed exclusively in chicken gut after hatch. We analyzed the transcriptional and expressional level of IGF-I gene in liver and in small intestine between the mutant and the wild-type birds at 3, 6, 9 week by real-time quantitative PCR and radioimmunoassay. In liver, there were no transcriptional and expressional differences between the mutant and wild-type birds at 3, 6, 9 week age; in small intestine, the mutant birds had about one-fold mRNA and 30% protein of IGF-I higher than wild type birds at 3 week age, and no significant genotype difference at 6 and 9 week age. Thus the mutation results in the increase of expressional efficiency in vivo. The mutant birds have higher IGF-I in small intestine, and high IGF-I can promote intestine cell growth faster, then these birds favor the longer small intestine length, with more nutrition digested in and absorbed from intestine the mutant birds finally result in higher growth rate and others carcass traits means.Our results supported the view that selection for high growth rate in birds is linked to an increase in the relative size of the digestive organs and rapid early development of the digestive organs. Our results may be also an example of a complex genetic traits created by multiple susceptibility genes. Identification of QTL gene can provide suggestive information about normal development and disease processes, and now with the development of new genetic technique, statistical tools and better characterized genome, find QTL gene is becoming easier than before. The results are also useful in practice of chicken molecular breeding programme in the near future.
引文
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