鸡SPOP和MyD88基因分子特征及其组织表达特性分析

Molecular characteristics and tissue expression analysis of the chicken genes SPOP and MyD88

【目的】掌握SPOP和MyD88基因分子特征及其在鸡不同组织发育过程中的表达特征,为后续研究其调控鸡组织生长发育机理及开展抗病育种提供参考依据。【方法】通过RT-PCR克隆鸡SPOP和MyD88基因编码区(CDS)序列,运用ExPASy、SOPMA、SWISS-MODEL及PSORT II Prediction等在线软件进行生物信息学分析,并以实时荧光定量PCR检测这2个基因在鸡胚14胚龄(E14 d)及出壳后1 d(H1 d)、7 d(H7 d)和14 d(H14 d)各组织中的表达情况。【结果】鸡SPOP、MyD88基因CDS序列长为1125和900 bp,分别编码374和299个氨基酸残基。SPOP蛋白分子式为C_(1866)H_(2926)N_(496)O_(559)S_(28),相对分子量为42 kD,理论等电点(pI)为5.58,为相对不稳定蛋白;MyD88蛋白分子式为C_(1502)H_(2394)N_(410)O_(438)S_(18),相对分子量为33 kD,pI为5.93,为相对不稳定蛋白。鸡SPOP和MyD88蛋白二级结构以α-螺旋和无规则卷曲为主,主要定位于细胞质(占60.9%)。与人类和哺乳动物相比,鸡SPOP蛋白的3个功能结构域(MATH、BTB-POZ和BACK)较保守,而MyD88蛋白的2个功能结构域(Death和TIR)存在多处氨基酸位点变异。SPOP和MyD88基因在鸡不同发育阶段各组织中均有表达,但以肺脏中的相对表达量最高,且二者间的表达差异极显著(P<0.01,下同)。从E14 d发育至H14 d,SPOP基因在眼球和肺脏中的表达整体上呈上升趋势,且至H14 d时肺脏中的表达趋于稳定,在脑组织、心脏和肌胃中的表达呈先上升后下降的变化趋势,在肝脏中的表达呈先下降后上升再下降的变化趋势;MyD88基因在眼球、肝脏和肺脏中的表达均呈先上升后下降再上升的变化趋势,在肌胃和胸肌中的表达呈下降趋势,至H14 d时降至最低值。【结论】SPOP和MyD88基因在鸡胚不同发育阶段肺脏、眼球和肌胃中的表达相对较高,SPOP基因的表达水平均极显著高于MyD88基因,且二者的相对表达量呈负相关,即SPOP基因负调控MyD88基因的表达。

【Objective】To describe the molecular characteristics of SPOP and MyD88 genes and their expression in different tissue during chicken development,and lay the basis for the subsequent study of their regulation of tissue growth and development in chickens and the development of disease-resistance breeding.【Method】The coding regions(CDS)of chicken SPOP and MyD88 genes were cloned by RT-PCR.ExPASy,SOPMA,SWISS-MODEL and PSORT II Prediction were used for bioinformatics analysis.Real-time quantitative PCR(qRT-PCR)technology was used to detect the expres-sion of SPOP and MyD88 genes in various tissues at 14 embryonic days(E14 d)in chick embryos and at 1-day-old(H1 d),7-day-old(H7 d),and 14-day-old(H14 d)after shelling.【Result】The CDS sequences of chicken SPOP and MyD88 genes were 1125 and 900 bp,encoding 374 and 299 amino acid residues,respectively.The molecular formula of the SPOP protein was C_(1866)H_(2926)N_(496)O_(559)S_(28),,and was predicted to have a molecular weight of 42 kD and a isoelectric point(pI)of 5.58.The molecular formula of the MyD88 protein was C_(1502)H_(2394)N_(410)O_(438)S_(18) and was predicted to have a molecular weight of 33 kD and a pI of 5.93.Both SPOP and MyD88 were relatively unstable proteins.The chicken SPOP and MyD88 proteins,were mainly located in the cytoplasm(60.9%)and their secondary structures mainly consisted ofα-he-lixes and random loops.Compared with humans and mammals,the three functional domains of chicken SPOP(MATH,BTB-POz and BACK)were conserved,while the two functional domains of MyD88(Death and TIR)had multiple ami-no acid site variations.SPOP and MyD88 genes were expressed in all tissues of chicken at different developmental stages,but their expression in the lung was relatively the highest.The differences in expression of SPOP and MyD88 was extreme-ly significant(P<0.01,the same below).From E14 d to H14 d,the expression of SPOP gene in the eyeball and lung showed an overall upward trend,but the lung expression of SPOP stabilized before H14 d.The expression of SPOP gene in the brain,heart,stomach muscle and liver showed an upward trend followed by a downward trend.The expression of MyD88 gene in the eyeball,liver and lung was first increased,followed by a decrease and a further increase.The expres-sion of MyD88 gene in stomach and chest muscles decreased and reached the lowest value at H14 d.【Conclusion】The expression levels of SPOP and MyD88 genes are relatively high in lung,eyeball and stomach muscle of chicken embryo at different developmental stages.The expression levels of SPOP are extremely significantly higher than MyD88 and the relative expression levels of SPOP gene and MyD88 gene are negatively correlated,indicating that SPOP gene regulates the expression of MyD88 gene.