畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (5): 826-831.doi:

• 临床兽医 • 上一篇    下一篇

山羊溶酶体αAMA基因的克隆、生物信息学及组织表达谱分析

孔祥雅,李义,程敏,荆新堂,李勤凡*   

  1. 西北农林科技大学动物医学院,杨凌 712100
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2012-05-28 发布日期:2012-05-28
  • 通讯作者: 李勤凡

Cloning, Tissue Specific Expression and Bioinformatics Analysis of Goat Lysosomal αAMA Gene

KONG Xiangya, LI Yi, CHENG Min, JING Xintang, LI Qinfan*   

  1. College of Veterinary Medicine, Northwest A & F University, Yangling 712100, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2012-05-28 Published:2012-05-28
  • Contact: LI Qinfan

摘要: 本研究旨在对山羊溶酶体α甘露糖苷酶(αAMA)基因进行组织表达谱和生物信息学分析。参考牛αAMA基因序列设计引物,采用PCR技术克隆山羊αAMA基因序列,并利用荧光定量RTPCR进行组织表达谱分析以及进行生物信息学预测。首次获得了山羊αAMA基因,含有完整CDS编码区3 000 bp,编码999个氨基酸,其中前50个氨基酸为信号肽序列。其编码区的核苷酸序列和预测氨基酸序列与牛的αAMA相似性最高,分别为95.93%和94.79%。组织表达谱分析表明αAMA在山羊各组织均不同程度的表达,其中在肺脏、肝脏、小脑表达量较高。生物信息学预测发现,αAMA蛋白属于糖苷水解酶38家族成员,有2个保守的结构域,存在9个N糖基化位点。SWISSMODEL同源建模山羊αAMA具有良好的可信度。本研究为探讨酶的作用机理及疯草解毒剂的研制提供了理论依据。

Abstract: Goat lysosomal αAMA gene was amplified using RTPCR and the tissue specific expression profile, bioinformatics characteristic of αAMA were studied. Primers were designed based on the sequence of bovine lysosomal αAMA gene and were used in amplifying the goat αAMA, the tissue specific expression profile was analyzed by qRTPCR, and the bioinformatics analysis of αAMA was conducted. The results showed that CDS sequence of goat αAMA was 3 000 bp, encoding a deduced protein containing 999 amino acid residues in which the first 50 residues were signal peptide, and this nucleotide sequence of CDS and the deduced amino acids shared 95.93% and 94.79% homology with the αAMA mRNA of cattle. The qRTPCR revealed that the αAMA gene was expressed in various tissues at different levels. The expression level of this gene was higher in the lung, liver, and cerebellum. It was predicted that αAMA was belonged to Glycosyl hydrolases family 38, and composed of two conserved domains. There were thirtyfive phosphorylation sites, one phosphorylation site of specific protein kinase and nine Nglycosylation sites in αAMA protein. By SWISSMODEL homologous modeling, the predicted structure of goat was similar to the template and predictive. These results provide the theoretic basis for the mechanism research of αAMA and development of toxin antidote toward locoweed.