Authors: Tian Yuan JiRui Gu WenBo Gu Jiang Wu ShaoRong Ge Heng Xu
Publish Date: 2010/09/21
Volume: 38, Issue: 3, Pages: 2059-2065
Abstract
Adenylosuccinate lyase ADSL is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling In the present study we have constructed a grass carp Ctenopharyngodon idella intestinal cDNA library that has over 23 × 105 primary clones An expressed sequence tag EST of grass carp adenylosuccinate lyase gcADSL gene was screened from this library Both 5′RACE and 3′RACE were carried out in order to obtain the complete cDNA sequence which contains a 1446 bp open reading frame encoding 482 amino acids about 54552 kDa The deduced amino acid sequence shares high homology with its vertebrate counterparts which shares 94 similarity with zebrafish 81 with African clawed frog as well as chicken 77 with human and 76 with mouse This gcADSL genomic sequence consisted of 13 exons and 12 introns is 8557 bp in size Realtime quantitative PCR analysis revealed that the highest expression level of gcADSL was detected in muscle and the lowest in gill In western blotting analysis His6tagged gcADSL protein expressed in Escherichia coli could be recognized not only by an antiHis6tag monoclonal antibody but also by an antihuman ADSL polyclonal antibody indicating immunological crossreactivity occurs between grass carp and human ADSL protein 1082 bp 5′flanking region sequence was cloned and analyzed
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