琅琊鸡胚MSX2基因克隆及发育性表达变化

Cloning and Developmental Expression of MSX2 Gene in Langya Chicken Embryo

【目的】克隆鸡Msh同源框2(Msh-homeobox 2,MSX2)基因,并对其进行生物信息学和胚胎期表达模式分析,为进一步研究MSX2基因的结构和功能提供支持。【方法】对80枚琅琊鸡种蛋进行孵化,分别于孵化期第1、2、3、4、5、6、9、12、15、18天采集样品(1~6胚龄采集整胚,9、12、15、18胚龄分别采集心脏和肝脏),提取总RNA,利用RT-PCR方法扩增并克隆MSX2基因,对其进行生物信息学分析;利用实时荧光定量PCR技术分析MSX2基因在琅琊鸡鸡胚和组织中的表达水平。【结果】成功克隆了琅琊鸡胚MSX2基因,序列长度为818 bp,开放阅读框为780 bp,编码259个氨基酸。琅琊鸡MSX2氨基酸序列与日本鹌鹑、秃鹰和仓鸮的相似性最高,均为99.23%,与山羊的相似性最低,为74.54%;系统进化树分析结果显示,琅琊鸡与日本鹌鹑聚为一类。生物信息学分析表明,MSX2蛋白分子质量为28235.18 u,等电点(pI)为9.71,没有信号肽和跨膜域,为不稳定的亲水性蛋白,主要分布在细胞核(60.9%);存在37个磷酸化位点、8个O-糖基化位点和1个N-糖基化位点;二级结构主要由无规则卷曲(63.32%)和α-螺旋(28.19%)组成。实时荧光定量PCR分析表明,MSX2基因在整胚(1~6胚龄)中的表达水平呈现先上升后下降趋势,且在4胚龄时表达水平最高,极显著高于1、2、3胚龄(P<0.01);MSX2基因在12胚龄鸡心脏中的表达水平极显著低于9胚龄(P<0.01);在9~18胚龄鸡肝脏中的表达量呈逐渐下降趋势,且在9胚龄鸡肝脏中表达水平最高(P<0.05)。【结论】试验成功克隆了琅琊鸡MSX2基因序列,其表达模式在不同胚龄和同一胚龄不同组织间存在差异,为进一步探索琅琊鸡MSX2基因的结构和功能奠定了基础。

【Objective】The aim of this study was to clone Msh-homeobox 2(MSX2)gene in chickens,and analyze its bioinformatics and embryonic expression patterns,so as to provide support for further study on the structure and function of MSX2 gene.【Method】The 80 Langya chicken eggs were incubated,the samples were collected on 1st,2nd,3rd,4th,5th,6th,9th,12th,15th and 18th days of incubation,the whole embryos were collected from 1 to 6 embryonic age,the heart and liver were collected from 9,12,15 and 18 embryonic age,respectively.Total RNA was extracted,MSX2 gene was amplified by RT-PCR and cloned,bioinformatics analysis was carried out.Real-time quantitative PCR was used to analyze its expression level in embryo and tissues of Langya chickens.【Result】MSX2 gene was successfully cloned from Langya chicken embryo with a sequence length of 818 bp,and an open reading frame of 780 bp,encoding 259 amino acids.The MSX2 amino acid sequence of Langya chickens was 99.23%with that of Coturnix japonica,Haliaeetus leucocephalus and Tyto alba alba,and the lowest with that of Capra hircus,which was 74.54%.The phylogenetic tree analysis results showed that Langya chickens and Coturnix japonica was clustered together.Bioinformatics analysis showed that the molecular weight of MSX2 gene was 28235.18 u,and the isoelectric point(pI)was 9.71.MSX2 protein had no signal peptide and transmembrane domain,and were unstable hydrophilic proteins,and the majority of MSX2 protein was distributed in nucleus(60.9%).The MSX2 protein had 37 phosphorylation sites,8 O-glycosylation sites and 1 N-glycosylation site.The secondary structure of MSX2 protein was mainly composed of random coil(63.32%)and alpha helix(28.19%).Real-time quantitative PCR analysis showed that the expression of MSX2 gene in whole embryo(1-6 embryonic ages)were firstly increased and then decreased,and the expression were the highest at 4 embryonic age,and was extremely significantly higher than 1,2 and 3 embryonic ages(P<0.01).The expression of MSX2 gene in heart of 12 embryonic age were extremely significantly lower than that of 9 embryonic age(P<0.01).However,it showed a gradually decreasing trend in liver of 9-18 embryonic ages,and the expression were the highest in liver of 9 embryonic age(P<0.05).【Conclusion】The sequence of MSX2 gene in Langya chickens were successfully cloned.The expression patterns of MSX2 gene were different in different embryonic ages and different tissues of the same embryonic age,which provided a foundation for further exploring the structure and function of MSX2 gene in Langya chickens.