Lu Mi, Yang Yang, Zhou Tao, Zheng Wei, Xu Jiao, He Hua, Shu Guo-Ping, Yuan Qing-Song, Jiang Wei-Ke
Resource Institute for Chinese & Ethnic Materia Medica, Guizhou University of Traditional Chinese Medicine Guiyang 550025, China.
Zhongguo Zhong Yao Za Zhi. 2023 Apr;48(7):1851-1857. doi: 10.19540/j.cnki.cjcmm.20230114.101.
This paper aimed to study the role of asparagine endopeptidase(AEP) gene in the biosynthesis mechanism of cyclic peptide compounds in Pseudostellaria heterophylla. The transcriptome database of P. heterophylla was systematically mined and screened, and an AEP gene, tentatively named PhAEP, was successfully cloned. The heterologous function verification by Nicotiana benthamiana showed that the expression of the gene played a role in the biosynthesis of heterophyllin A in P. heterophylla. Bioinformatics analysis showed that the cDNA of PhAEP was 1 488 bp in length, encoding 495 amino acids with a molecular weight of 54.72 kDa. The phylogenetic tree showed that the amino acid sequence encoded by PhAEP was highly similar to that of Butelase-1 in Clitoria ternatea, reaching 80%. The sequence homology and cyclase active site analysis revealed that the PhAEP enzyme may specifically hydrolyse the C-terminal Asn/Asp(Asx) site of the core peptide in the HA linear precursor peptide of P. heterophylla, thereby participating in the ring formation of the linear precursor peptide. The results of real-time quantitative polymerase chain reaction(RT-qPCR) showed that the expression level of PhAEP was the highest in fruits, followed by in roots, and the lowest in leaves. The heterophyllin A of P. heterophylla was detected in N. benthamiana that co-expressed PrePhHA and PhAEP genes instantaneously. In this study, the PhAEP gene, a key enzyme in the biosynthesis of heterophyllin A in P. heterophylla, has been successfully cloned, which lays a foundation for further analysis of the molecular mechanism of PhAEP enzyme in the biosynthesis of heterophyllin A in P. heterophylla and has important significance for the study of synthetic biology of cyclic peptide compounds in P. heterophylla.
本文旨在研究天冬酰胺内肽酶（AEP）基因在太子参环肽类化合物生物合成机制中的作用。对太子参转录组数据库进行系统挖掘与筛选，成功克隆出一个AEP基因，暂命名为PhAEP。通过本氏烟草进行的异源功能验证表明，该基因的表达在太子参异叶环肽A的生物合成中发挥作用。生物信息学分析表明，PhAEP的cDNA长度为1488 bp，编码495个氨基酸，分子量为54.72 kDa。系统发育树显示，PhAEP编码的氨基酸序列与蝶豆中的Butelase-1高度相似，相似度达80%。序列同源性和环化酶活性位点分析表明，PhAEP酶可能特异性水解太子参HA线性前体肽核心肽的C端Asn/Asp（Asx）位点，从而参与线性前体肽的环化形成。实时定量聚合酶链反应（RT-qPCR）结果表明，PhAEP的表达水平在果实中最高，其次是根，在叶中最低。在瞬时共表达PrePhHA和PhAEP基因的本氏烟草中检测到了太子参的异叶环肽A。本研究成功克隆了太子参异叶环肽A生物合成中的关键酶PhAEP基因，为进一步解析PhAEP酶在太子参异叶环肽A生物合成中的分子机制奠定了基础，对太子参环肽类化合物合成生物学研究具有重要意义。
