西北植物学报2024，Vol.44Issue(3)：443-450,8.DOI:10.7606/j.issn.1000-4025.20230387
山黧豆AAE超家族基因鉴定及LsAAE3的酶活检测
Gene identification of the AAE superfamily and AAE3 activity determination in Lathyrus sativus L.
摘要
Abstract
[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.关键词
山黧豆/三七素/AAE超家族/草酰辅酶A合成酶/酶活测定Key words
Lathyrus sativus/dencicine/AAE superfamily/oxalyl-CoA synthase/enzyme activity detection分类
农业科技引用本文复制引用
贾海燕,张颖,陈红,刘嘉怡,焦成瑾,徐全乐..山黧豆AAE超家族基因鉴定及LsAAE3的酶活检测[J].西北植物学报,2024,44(3):443-450,8.基金项目
陕西省自然科学基金项目(2023-JC-YB-152) （2023-JC-YB-152）
细胞活动与逆境适应教育部重点实验室开放基金项目(lzujbky-2021-kb05) （lzujbky-2021-kb05）
甘肃高等学校科学研究项目(2021A-124) （2021A-124）
陇东学院青年科技创新基金项目(XYZK2304) （XYZK2304）
