广灵驴ADD1基因的克隆和序列分析与组织表达

Cloning,sequence analysis and tissue expression of ADD1 gene of Guangling donkey

运用RT–PCR方法,克隆广灵驴的ADD1基因的CDS区,对其进行序列分析,并通过qRT–PCR技术鉴定ADD1基因在广灵驴心脏、肝脏、脾脏、肺脏、肾脏和背最长肌中的相对表达水平。结果表明:广灵驴ADD1基因完整的CDS序列全长为2223 bp,可编码740个氨基酸,序列已提交到NCBI,登录号为MN_166472,其核苷酸序列与马的核苷酸序列的同源性最高,达99.6%;生物信息学预测ADD1蛋白为结构稳定的亲水性蛋白,理论等电点为5.58,二级结构主要以α–螺旋和无规则卷曲为主,存在1个Ⅱ类醛缩酶和内收蛋白N端超家族结构域,该蛋白没有信号肽及跨膜区域,主要定位在细胞核中,序列中共存在88个磷酸化位点,69个O–糖基化位点和3个N–糖基化位点;实时荧光定量检测结果表明,ADD1基因在广灵驴的6种组织中均有表达,但存在差异,在背最长肌中表达量最丰富,在肝脏中表达量最低。

The CDS region of ADD1 gene of Guangling donkey was cloned by RT-PCR method,and followed with sequence analysis.qRT-PCR was used to identify the relative expression level of ADD1 gene in heart,liver,spleen,lung,kidney and longissimus dorsi muscle.The results showed that the complete CDS sequence of Guangling donkey ADD1 gene was 2223 bp in length and could encode 740 amino acids.The sequence was submitted to NCBI with the login number MN_166472,and its nucleotide sequence had the highest homology with the horse nucleotide sequence,up to 99.6%.Bioinformatics prediction ADD1 protein had stable hydrophilic protein structure,theory of isoelectric point was 5.58,the secondary structure was mainlyα-helix and random coils,and there was a Aldolase_Ⅱsuperfamily domain in the protein sequence.The protein had no signal peptide and transmembrane region,and was mainly located in the cell nucleus.There were 88 phosphorylation sites,69 O-glycosylation sites and 3 N-glycosylation sites in the sequence.The results of real-time fluorescence quantitative detection showed that ADD1 gene was expressed in all studied 6 tissues,but there were differences in expression.The expression of ADD1 gene was the most abundant in longissimus dorsi muscle and the lowest in liver.