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.

【目的】三七素(β-N-草酰-L-α,β-二氨基丙酸,β-ODAP)是山黧豆、三七等物种中的神经活性物质,其生物合成与草酰辅酶A密切相关。研究基于全基因组鉴定山黧豆草酰辅酶A合成酶的编码基因,为β-ODAP生物合成的调控研究奠定基础。【方法】用AMP结合结构域隐马尔科夫模型在山黧豆基因组中鉴定酰基激活酶超家族(acyl-activating enzyme superfamily,AAE)基因,在此基础上,克隆山黧豆草酰辅酶A合成酶基因LsAAE3,用多序列比对、原核表达及酶活检测等方法分析LsAAE3基因功能。【结果】山黧豆中至少存在22条AAE家族基因;系统发育分析表明:LsAAE3与拟南芥AtAAE3等聚为一支,隶属于酰基辅酶A合成酶家族。基因克隆及序列分析表明:LsAAE3基因cDNA全长为1566 bp;LsAAE3和AtAAE3的保守结构域类似,均含有AMP结合位点、CoA结合位点及AAE保守结构域。SDS-PAGE检测表明所获融合蛋白条带单一,大小在56 kD左右;用His标签抗体进行Western blot分析发现,诱导后和纯化的融合蛋白均能检测到特征条带,说明所获融合蛋白为LsAAE3。体外酶活检测表明:LsAAE3具有草酰辅酶A合成酶活性,其活性发挥依赖于ATP、Mg^(2+)。【结论】研究在全基因组水平鉴定山黧豆AAE超家族基因,并验证了LsAAE3具有草酰辅酶A合成酶活性,为进一步分析山黧豆LsAAE3在β-ODAP生物合成过程中的功能奠定基础。

[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 ofβ-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 1566 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 inβ-ODAP biosynthesis.