菘蓝中松脂醇-落叶松脂素还原酶编码基因IiPLR2的克隆与功能分析

Cloning and functional characterization of the pinoresinol-lariciresinol reductase gene IiPLR2 in Isatis indigotica

摘要:

松脂醇-落叶松脂素还原酶(pinoresinol-lariciresinol reductase, PLR)是植物中木脂素生物合成的关键酶，能连续催化两步反应分别生成落叶松脂素和开环异落叶松脂素。落叶松脂素等木脂素类成分是中药板蓝根发挥抗病毒活性的有效成分。为了研究板蓝根基原植物菘蓝中落叶松脂素生物合成关键酶PLR的功能，从菘蓝(Isatis indigotica)中克隆得到IiPLR2基因序列，全长954 bp，编码317个氨基酸；多序列比对分析显示IiPLR2具有保守的还原型烟酰胺腺嘌呤二核苷酸磷酸(reduced nicotinamide adenine dinucleotide phosphate, NADPH)结合基序；进化树分析显示IiPLR2与来自拟南芥的AtPrR1聚在同一分支；构建原核表达载体pET32a-IiPLR2，转化至大肠杆菌BL21(DE3)中进行蛋白诱导表达纯化；体外酶活实验检测发现IiPLR2能够催化松脂醇生成落叶松脂素，并能进一步催化落叶松脂素生成开环异落叶松脂素。IiPLR2编码基因的克隆、序列分析及催化功能研究丰富了菘蓝中该类功能蛋白的认识，完善了菘蓝木脂素生物合成途径，为开展木脂素类成分的代谢调控和合成生物学研究提供了功能元件，也为深入研究该类蛋白的空间结构与催化功能的关系等奠定了基础。

Abstract:

The pinoresinol-lariciresinol reductase (PLR), a crucial enzyme in the biosynthesis of lignans in plants, catalyzes a two-step reaction to produce lariciresinol and secoisolariciresinol. Lignans such as lariciresinol are the effective components of traditional Chinese medicine Radix Isatidis in exerting antiviral activity. In order to study the function of the key enzyme PLR in the biosynthesis of lariciresinol in Isatis indigotica, the original plant of Radix Isatidis, IiPLR2 was cloned from I. indigotica, with a full length of 954 bp, encoding 317 amino acids. Multiple sequence alignment showed that IiPLR2 contained a conserved nicotinamide adenine dinucleotide phosphate (NADPH)-binding motif. The phylogenetic tree showcased that IiPLR2 shared the same clade with AtPrR1 from Arabidopsis thaliana. The prokaryotic expression vector pET32a-IiPLR2 was constructed and then transformed into Escherichia coli BL21(DE3) competent cells for protein expression. The purified enzyme IiPLR2 could catalyze the conversion of pinoresinol to lariciresinol and the conversion of lariciresinol to secoisolariciresinol. The cloning, sequencing, and catalytic functional analysis of IiPLR2 in this study enrich the understanding of this kind of functional proteins in I. indigotica and supplement the biosynthesis pathways of lignans. Moreover, this study provides a functional module for further research on metabolic regulation and synthetic biology and lays a foundation for comprehensively revealing the relationship between the spatial structures and catalytic functions of such proteins.