Gene identification of the AAE superfamily and AAE3 activity determination in Lathyrus sativus L.
[Objective]Dencichine(β-N-oxalyl-L-α,β-diaminopropionic acid,β-ODAP)is a neuroactive substance in species such as grass pea(Lathyrus sativus L.)and Panax notoginseng,and its biosynthesis is closely related to oxalyl-CoA.The aim of the study is to identify the acyl-CoA synthase encoding gene in grass pea based on the whole genome,which lays a foundation for the regulation of β-ODAP biosynthesis.[Methods]Acyl-activating enzyme superfamily genes were identified in the genome of grass pea using the AMP binding domain by hidden Markov model.The acyl-CoA synthase gene was cloned and analyzed via the multiple sequence alignment,prokaryotic expression,and enzyme activity detection.[Results]The re-sults showed that at least 22 AAE genes were located on 7 different branches in grass pea.Phylogenetic analysis indicated that LsAAE3 and Arabidopsis thaliana AtAAE3 clustered at one branch,belonging to acyl-CoA synthetase family.Cloning and sequence analysis showed that the full-length cDNA of LsAAE3 was 1 566 bp,and LsAAE3 and AtAAE3 possessed several conversed domains including AMP binding site,CoA binding site,and AAE consensus motif.The purified protein,which molecular weight was 56 kD,showed a single band as assayed by SDS-PAGE,which was detected in both induced bacterial proteins and purified recombinant proteins by Western blot,suggesting that the fusion protein was LsAAE3.The oxaloyl-CoA synthase activity of LsAAE3 depended on ATP and magnesium ions.[Conclusion]The study identified the acyl-activating enzyme(AAE)superfamily genes in the genome of grass pea and verified the oxaloyl-CoA synthase activity of LsAAE3,paving the way for the analysis of LsAAE3 function in β-ODAP biosynthesis.
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万方数据
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