雪山草鸡血管紧张素转换酶2(ACE2)基因克隆、结构特性分析及其表达特征研究

Molecular Cloning,Sequence Analysis and Expression Characteristics of Angiotensin Converting Enzyme 2(ACE2)Gene in Xueshan Chicken

旨在克隆血管紧张素转换酶2(angiotensin converting enzyme 2,ACE2)基因,分析其结构特性和表达特征。本试验以45和90日龄的健康雪山草鸡为研究对象,利用PCR技术扩增ACE2基因的编码区序列;运用生物信息学方法对其结构特征进行分析;通过TA克隆构建黄羽肉鸡ACE2编码区序列质粒标准品,使用绝对定量方法测定ACE2基因在45和90日龄雪山草鸡公母鸡各组织中的表达特性。结果,克隆得到2427 bp的编码区序列,共编码808个氨基酸,与白羽肉鸡一致,但是在黄羽肉鸡序列中存在大量突变位点造成编码氨基酸发生变化。同源性比较发现,其与红色原鸡的同源性为99%,其次为绿头鸭88%,与其它动物的同源性为73%~76%。生物信息学分析发现,ACE2属于I型跨膜蛋白,即分泌型蛋白,信号肽位于1~17 aa,含有46个蛋白质磷酸化位点。定量结果发现,小肠中ACE2表达量显著高于其它组织。法氏囊、气管、胆囊和脾中ACE2表达量较低,脾中表达量最低。母鸡空肠、盲肠、脾和肺中ACE2基因表达量显著高于公鸡。公鸡肾和心中ACE2基因表达量显著高于母鸡。90日龄雪山草鸡的十二指肠、空肠、肺、脾和回肠组织ACE2基因表达量均显著低于45日龄雪山草鸡,在法氏囊和肾组织中结果呈相反趋势。通过克隆获得了雪山草鸡ACE2基因编码区序列,生物信息学分析发现,雪山草鸡ACE2基因在家禽中较为保守;ACE2基因表达规律呈现出在雪山草鸡肠道组织中高于其他组织,母鸡中高于公鸡,随着时间表达水平呈下降趋势。研究结果为开展ACE2基因在黄羽肉鸡肠道中的功能研究奠定了理论基础。

The study aimed to clone the angiotensin converting enzyme 2(ACE2)gene,analyze its structural and expressing characteristics.In this experiment,the Xueshan chicken was selected as the research objects,the coding region of ACE2 gene was amplified by PCR,the structural characteristics were analyzed by the method of bioinformatics,the standard plasmid of ACE2 gene was constructed by TA cloning,and the expression characteristics of ACE2 gene was determined in the tissues of Xueshan chicken by absolute quantitative method.The results showed that CDS(2427 bp)of ACE2 was cloned and encoded 808 amino acids,it was consistent with the ACE2 gene in white-feathered broilers,but a large number of mutations were found in yellow-feathered broilers which led to changes in amino acids.The homology analysis found that sequence of ACE2 gene of Xueshan chicken had 99%homology with red jungle fowl,followed by mallard duck 88%,and 73%-76%with other animals.The bioinformatics analysis showed that ACE2 belonged to type I transmembrane protein(secretory protein),and signal peptide was located in 1-17aa,contained 46 protein phosphorylation sites.The quantitative results found that the expression of ACE2 in the small intestine was significantly higher than that in other tissues.The expression of ACE2 in bursa,trachea,gallbladder and spleen was lower,and the expression in the spleen was the lowest.The expression of ACE2 in jejunum,cecum,spleen and lung of hens were significantly higher than those of roosters.The expression of ACE2 in kidney and heart of roosters were significantly higher than those of hens.The expression of ACE2 in the duodenum,jejunum,lung,spleen and ileum tissues of 90-day-old Xueshan chicken were significantly lower than those of 45-day-old Xueshan chicken,and the results showed an opposite trend in the bursa and kidney.The coding region of ACE2 gene was obtained in Xueshan chicken.Bioinformatics analysis showed that ACE2 gene was conservative in poultry.The expression level of ACE2 gene was higher in the intestinal tissues than that in other tissues,higher in hen than that in rooster,and the expression level decreased with time.The results laid a foundation for the study of function of ACE2 gene in the gut of yellow feather broilers.