鸡BRD2亚细胞定位及其组织表达特性分析

Subcellular localization of chicken bromodomain-containing protein 2(BRD2)and its expression characteristics

【目的】明确含溴结构域蛋白2(BRD2)的亚细胞定位及其表达特征,为后续研究BRD2基因调控鸡组织发育的作用机制提供参考依据。【方法】通过构建鸡BRD2基因重组真核表达载体pEGFP-BRD2,转染人胚胎肾细胞(HEK-293T)后检测重组蛋白EGFP-BRD2的表达情况及亚细胞定位;利用实时荧光定量PCR分别检测鸡胚14胚龄(E14 d)、鸡出壳后1日龄(1 d)、7日龄(7 d)和14日龄(14 d)4个时间点各组织中BRD2基因的表达情况。【结果】重组真核表达载体pEGFP-C1-BRD2转染HEK-293T细胞获得的融合蛋白EGFP-BRD2约115.00 kD,且主要定位在细胞核。实时荧光定量PCR检测结果表明,鸡BRD2基因在E14 d和1 d肌胃中的相对表达量最高,在7和14 d肺脏中的相对表达量最高。BRD2基因在各组织的表达规律为:在眼球内E14 d到14 d的表达先下降后趋于稳定;在脑组织和心脏内的表达在E14 d至14 d间呈先下降后上升的变化趋势;在肝脏内E14 d表达稳定,1 d后开始逐渐增加,至14 d时达最高值;在肺脏中E14 d的表达下降,1 d后急剧上升,至14 d时达最高值;在肌胃和胸肌中,均在7 d时最高,1 d时最低,E14 d和14 d时有较高表达,整体上呈倒N表达模式;在腿肌中,从E14 d到1 d表达减少,而后略有增加,7 d后开始下降,至14 d时降到最低值。【结论】鸡BRD2基因重组真核表达载体能在HEK-293T细胞中正确表达,且融合蛋白主要定位在细胞核;BRD2基因在鸡不同发育阶段的各组织中均有表达,但其表达量呈现不同的变化趋势。

【Objective】This study aimed to investigate the subcellular localization of chicken(Gallus gallus)bromodomain-containing protein 2(BRD2)and analyze its expression characteristics in different tissues of chickens,which could provide the reference for subsequent research on the mechanism of BRD2 gene regulating chicken tissue development.【Method】The recombinant eukaryotic expression vector PEGFP-BRD2 of chicken BRD2 gene was constructed and transfected into human embryonic kidney cells(HEK-293T).The expression and subcellular localization of the recombinant protein EGFP-BRD2 were examined by Western blot and inverted fluorescence microscope,respectively.In addition,real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression patterns of BRD2 gene in different tissues at chicken embryo 14 days(E14 d),1-day-old(1 d),7-day-old(7 d),and 14-day-old(14 d).【Result】The results showed that pEGFP-C1-BRD2 transfected HEK-293T cells,and the obtained fusion protein EGFP-BRD2 was about 115.00 kD and localized mainly in the nucleus.The qRT-PCR results showed that BRD2 gene had the highest relative expression in the stomach of chicken at E14 d and 1 d,and the lung at 7 d and 14 d.The expression pattern of BRD2 gene in various tissues revealed that the expression of BRD2 gene in the eye ball was first decreased and then stabilized from E14 d to 14 d,while in the brain and heart between E14 d to 14 d showed a trend from decline to increase.As for the liver and lung,it showed the stable expression in the liver on E14 d,gradually increased after 1 d and reached the peak on 14 d;it decreased expression in the lung at E14 d,began to increase sharply after 1 d and reached the highest level at 14 d.In the stomach and pectoral muscle,the expression pattern of BRD2 gene was inverted N,showing that it was the highest at 7 d and the lowest at 1 d,but had high expression at E14 d and 14 d.However,in leg muscles,the expression of BRD2 decreased from the E14 d to 1 d,then increased slightly,and began to decrease at 7 d and reached the lowest level at 14 d.【Conclusion】The chicken BRD2 gene recombinant eukaryotic expression vector is correctly expressed in HEK-293T cells and the fusion protein is mainly located in the nucleus.The qRT-PCR result shows that the chicken BRD2 gene is expressed in various tissues at different stages of chicken development,but its expression level shows different trends.