College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China.
School of Pharmacy, Anhui Medical University, Hefei 230032, China.
Protein Pept Lett. 2022;29(2):156-165. doi: 10.2174/0929866528666211126162838.
Cangzhu (Atractylodes lancea), a valuable and common traditional Chinese medicinal herb, is primarily used as an effective medicine with various health-promoting effects. The main pharmacological bioactive ingredients in the rhizome of A. lancea are terpenoids. Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the terpenoid synthesis pathway and catalyzes two units of acetyl-CoA into acetoacetyl-CoA.
The objective of the present work was to clone and identify function of AlAACT from Atractylodes lancea.
A full-length cDNA clone of AlAACT was isolated using PCR and expressed in Escherichia coli. The expressed protein was purified using Ni-NTA agarose column using standard protocols. AlAACT was transiently expressed in N. benthamiana leaves to determine their subcellular location. The difference in growth between recombinant bacteria and control bacteria under different stresses was observed using the droplet plate experiment.
In this study, a full-length cDNA of AACT (AlAACT) was cloned from A. lancea, which contains a 1,227 bp open reading frame and encodes a protein with 409 amino acids. Bioinformatic and phylogenetic analysis clearly suggested that AlAACT shared high similarity with AACTs from other plants. The recombinant protein pET32a(+)/AlAACT was successfully expressed in Escherichia coli BL21 (DE3) cells induced with 0.4 mM IPTG at 30°C as the optimized condition. The recombinant enzyme pET-32a-AlAACT was purified using the Ni-NTA column based on the His-tag, and the molecular weight was determined to be 62 kDa through SDS-PAGE and Western Blot analysis. The recombinant protein was eluted with 100, 300, and 500 mM imidazole; most of the protein was eluted with 300 mM imidazole. Under mannitol stress, the recombinant pET-32a- AlAACT protein showed a substantial advantage in terms of growth rates compared to the control. However, this phenomenon was directly opposite under NaCl abiotic stress. Subcellular localization showed that AlAACT localizes to the nucleus and cytoplasm.
The expression and purification of recombinant enzyme pET-32a-AlAACT were successful, and the recombinant strain pET-32a-AlAACT in showed better growth in a drought stress. The expression of AlAACT-EGFP fusion protein revealed its localization in both nuclear and cytoplasm compartments. This study provides an important foundation for further research into the effects of terpenoid biosynthesis in A. lancea.
苍术（Atractylodes lancea）是一种有价值且常见的传统中药草，主要用作具有多种保健功效的有效药物。苍术根茎中的主要药理生物活性成分是萜类化合物。乙酰辅酶 A C-乙酰基转移酶（AACT）是萜类化合物合成途径中的第一个酶，催化两个单位的乙酰辅酶 A 转化为乙酰乙酰辅酶 A。
本工作旨在从苍术中克隆和鉴定 AlAACT 的功能。
采用 PCR 法从苍术中分离得到全长 cDNA 克隆的 AlAACT，并在大肠杆菌中表达。采用 Ni-NTA 琼脂糖柱按照标准方案对表达蛋白进行纯化。采用瞬时表达法在 N. benthamiana 叶片中确定其亚细胞定位。采用液滴板实验观察重组菌和对照菌在不同应激条件下的生长差异。
本研究从苍术中克隆得到全长 cDNA 的 AACT（AlAACT），包含 1227bp 的开放阅读框，编码 409 个氨基酸的蛋白质。生物信息学和系统发育分析清楚地表明，AlAACT 与其他植物的 AACTs 具有高度相似性。在优化条件下，即 30°C 时用 0.4mM IPTG 诱导大肠杆菌 BL21（DE3）细胞，成功表达了重组蛋白 pET32a（+）/AlAACT。采用 Ni-NTA 柱基于 His 标签纯化重组酶 pET-32a-AlAACT，通过 SDS-PAGE 和 Western blot 分析确定分子量为 62kDa。用 100、300 和 500mM 咪唑洗脱重组蛋白，其中大部分蛋白用 300mM 咪唑洗脱。在甘露醇胁迫下，与对照相比，重组 pET-32a-AlAACT 蛋白的生长速度具有明显优势。然而，在非生物胁迫 NaCl 下，这种现象则相反。亚细胞定位显示 AlAACT 定位于细胞核和细胞质。
成功表达和纯化了重组酶 pET-32a-AlAACT，重组菌 pET-32a-AlAACT 在干旱胁迫下表现出更好的生长。AlAACT-EGFP 融合蛋白的表达表明其定位于核和细胞质区室。本研究为进一步研究苍术中萜类化合物生物合成的影响提供了重要基础。
