摘要： 在单、双羔蒙古羊卵巢组织抑制性消减杂交的基础上,以其差异表达片段ADAMTS1基因序列为种子序列,采用GenBank的BLAST检索系统首先对蒙古羊的ADAMTS1基因序列片段进行了电子延伸,其次在获得蒙古羊AD-AMTS1基因的cDNA全序列后设计引物,采用RT-PCR方法进一步克隆了蒙古羊ADAMTS1基因。结果显示,试验所得序列与电子延伸序列的同源性为99.4%,序列长为2 420 bp包括836 bp 3’UTR区域和1 578 bp的完整开放读码框,共编码525个氨基酸。Blastn比对发现蒙古羊ADAMTS1基因序列与牛、猪和人的同源性分别为94%,87%,82%;对氨基酸序列进行blastp比对发现与牛、猪和人的同源性分别为91%,90%,87%。采用SMART程序预测其结构功能域发现蒙古羊ADAMTS1蛋白质有4个结构功能域:1个富半胱氨酸结构域和3个血小板反应蛋白。
关键词:
蒙古羊,
ADAMTS1基因,
电子克隆,
RT-PCR
Abstract: The study firstly obtained Mongolian SheePADAMTS1 gene cDNA sequences by BLAST in the Gen-Bank used differentially expressed ADAMTS1 gene fragment based on the suppression subtractive hybridization of Mongolian Sheep Ovary tissue.Based on the cDNA sequence primers were designed and cloned Mongolian SheePADAMTS1 gene by RT-PCR.The results showed that the cDNA homology between RT-PCR and in Silico Cloning is 99.4%, the sequence is 2 420 bp and included 836 bp 3' UTR and the opening reading frame( ORF) is 1 578 bp, encoding 525 amino acids; The sequence homology intercomparison with cattle,pig,human is 94%, 87%, 82%, respectively by Blastn and the amino acids homology intercomparison with cattle, pig, human is 91%, 90%, 87%, respectively by Blastp.The Mongolian SheePADAMTS1 protein have one ADAM Cysteine-Rich domain and three Thrombospondin type 1 domains by SMART program．
Key words:
Mongolian sheep,
ADAMTS1 Gene,
In Silico Cloning,
RT-PCR
中图分类号:
何小龙, 刘永斌, 王峰, 田春英, 达赖, 荣威恒. 蒙古羊卵巢组织差异表达基因ADAMTS1的电子克隆及RT-PCR验证[J]. 华北农学报, 2010, 25(3): 47-51. doi: 10.7668/hbnxb.2010.03.011.
HE Xiao-long, LIU Yong-bin, WANG Feng, TIAN Chun-ying, DA Lai, RONG Wei-heng,. In Silico Cloning and Verification by RT-PCR of Differentially Expressed ADAMTS1 Gene between Mongolian Sheep Ovary[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2010, 25(3): 47-51. doi: 10.7668/hbnxb.2010.03.011.