藏鸡GEM基因克隆及其时空表达特性分析

摘要

旨在获得藏鸡GEM基因序列,阐明其组织表达和时序表达谱,同时分析基因表达与肌内脂肪(Intramuscular fat,IMF)沉积的关系,为进一步研究藏鸡肉质和脂肪沉积奠定基础.选取1,81,119,154,210日龄健康藏鸡为试验材料,屠宰后分别采集心、肝、脾、肺、肾、胸肌、腿肌和皮下脂肪组织,提取组织总RNA,利用RT-PCR技术克隆藏鸡GEM基因序列,利用实时荧光定量PCR技术检测该基因在藏鸡不同组织以及不同发育时期胸肌和腿肌的表达情况.结果表明,克隆获得藏鸡GEM基因序列940 bp(GenBank登录号:KY747399),其中,CDS为894 bp,5′UTR 24 bp和3′UTR 22 bp,编码297个氨基酸,与原鸡、绿头鸭、人和小鼠GEM氨基酸序列相似性较高(83.50% ~98.32%);组织表达结果显示,GEM在藏鸡肺组织中的表达水平最高,极显著高于其他组织(P<0.01),在脂肪组织和脾组织中也存在较高水平的表达;时序表达结果显示,GEM基因在不同发育阶段藏鸡胸肌和腿肌中的表达模式存在差异,在胸肌中的表达水平随日龄的增加呈下降趋势,而在腿肌中随日龄的增加呈先上升后下降的表达趋势,且在119日龄达到峰值;GEM基因表达水平与不同发育阶段藏鸡胸肌和腿肌中IMF含量呈不同程度的相关,存在性别差异,且在公藏鸡腿肌中达到显著水平(r=0.414,P<0.05).结果表明,GEM基因可能在藏鸡脂质代谢中起重要调控作用.%The aim of this study was to clone GEM gene of Tibetan chicken,determine its expression profiling in different tissues and developmental stages,and analyzed the relationship between the expression level of GEM gene and intramuscular fat (IMF) deposition,which might provide basis for meat quality and fat deposition.1,81, 119,154,210 days Tibetan chicken were selected. After slaughter,the tissue samples of heart,liver,spleen,lung, kidney,breast muscle,thigh muscle and subcutaneous fat were collected for the total RNA extraction. The Tibetan chicken GEM gene was cloned by Reverse Transcription PCR (RT-PCR),the quantitative Real-time quantitative PCR (qPCR) was used to detect the expression level of GEM gene in different tissues and developmental stages of breast muscle and thing muscle. The results showed that the full length of GEM gene was 940 bp(GenBank acces-sion number:KY747399),containing 894 bp CDS,24 bp 5′UTR and 22 bp 3′UTR,encoding 297 amino acids. The Tibetan chicken GEM protein shared higher similarity(83.50%-98.32%) with Gallus gallus,Anas platyrhyn-chos,Homo sapiens and Mus musculu. The tissues expression analysis showed that GEM gene kept the highest ex-pression level in lung of Tibetan chicken,which was remarkably higher than other tissues(P<0.01),and also high-er in spleen and subcutaneous fat. The temporal expression showed that the expression pattern of GEM gene was different in breast muscle and thigh muscle of Tibetan chicken in different stages. The expression level of GEM mR-NA in breast muscle decreased with the increasing daily age,however,in thigh muscle increased first and then de-creased with Tibetan chicken growth,and peaked at the daily age of 119. The expression levels of Tibetan chicken GEM gene in breast muscle and thigh muscle at different developmental stages presented unlike relationship with IMF content,and there were genders differences. And the correlation in thigh muscle of male Tibetan chicken was remarkably stronger(r=0.414,P<0.05). These data indicate that GEM gene may play an important role in lipid metabolism of Tibetan chicken.