| 陈立凯，吴带娣.广藿香醇合酶 PcPTS 互作蛋白的筛选与鉴定分析[J].广东农业科学,2024,51(5):1-15 |
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广藿香醇合酶 PcPTS 互作蛋白的筛选与鉴定分析 |
| Screening and Identification of PcPTS Interacting Proteins in Pogostemon cablin |
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| DOI：10.16768/j.issn.1004-874X.2024.05.001 |
| 中文关键词: 药用成分 生物合成调控 植物代谢 酵母双杂交 广藿香醇合酶 互作蛋白 |
| 英文关键词: medicinal ingredient biosynthetic regulation plant metabolism yeast two-hybrid patchouli alcohol synthase interacting protein |
| 基金项目:国家自然科学基金（82373976）；广东省乡村振兴战略专项资金种业振兴项目（GDNK20230331）；云浮市科技创新战略专项（202301） |
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| 中文摘要: |
| 【目的】广藿香〔Pogostemon cablin（Blanco）Benth.〕是临床常用的芳香化湿药，其质量指标成分广藿香醇具有良好的抗病毒、抗炎症等活性。广藿香醇合酶（Patchouli alcohol synthase，PcPTS）在广藿香醇生物合成途径中起关键作用，但其在蛋白互作层面调控广藿香醇合成的机制尚不清楚。旨在筛选 PcPTS 的互作蛋白，以揭示 PcPTS 在广藿香醇生物合成途径中的调控机制和功能。【方法】利用酵母双杂交技术和 GST pull-down 联合液相色谱 - 串联质谱（LC-MS/MS）技术筛选可能与 PcPTS 互作的蛋白，利用 MASCOT 软件和 BLAST 分析注释互作蛋白，选取有文献报道参与其他蛋白互作、影响蛋白转运、修饰或降解、参与次生代谢调控的蛋白，利用酵母双杂交技术初步验证它们与 PcPTS 蛋白的互作关系并对互作蛋白进行生物信息学分析。【结果】通过 PCR 技术成功克隆了 PcPTS 的 cDNA 序列，开放阅读框（ORF）为 1 659 bp，编码 522 个氨基酸。构建了广藿香 cDNA 初级文库和酵母杂交文库，初级和次级文库容量分别为 7.6×106 CFU 和 8.6×106 CFU，初级和次级文库的重组率均为 100%，且插入片段平均长度均大于 800 bp。构建的诱饵载体 pGBKT7-PcPTS 对酵母菌株不产生毒性，且无自激活活性。利用酵母双杂交筛选得到阳性克隆并进行测序和 BLAST 分析，获得 17 个候选蛋白。成功构建了 GST-PcPTS 表达载体，诱导表达纯化获得 GST-PcPTS 诱饵蛋白，利用诱饵蛋白从广藿香总蛋白中捕获互作蛋白，共鉴定出 98 个候选互作蛋白。从候选蛋白中筛选 14 个可能与 PcPTS 互作的蛋白，利用酵母双杂交进行点对点验证，结果发现PcC3H6、PcENO3、PcACR11 和 PcHAD 与 PcPTS 存在相互作用。生物信息分析表明，四者分别属于 CCCH 锌指蛋白家族、烯醇化酶蛋白、ACR 亚家族和 HAD-like 超家族。【结论】初步筛选验证获得与 PcPTS 存在相互作用的 4个蛋白 PcC3H6、PcENO3、PcACR11 和 PcHAD，有助于揭示 PcPTS 在广藿香醇生物合成途径中的作用机制，也为进一步研究广藿香醇的合成调控机制奠定了坚实基础。 |
| 英文摘要: |
| 【Objective】Pogostemon cablin (Blanco) Benth. is a frequently-used aromatic herb eliminating dampness in clinical settings, with its primary bioactive compound, patchouli alcohol, demonstrating potent antiviral and anti-inflammatory and other beneficial properties. Patchoulialcohol synthase (PcPTS) plays an essential role in the biosynthesis pathway of patchouli alcohol. However, the precise mechanisms by which PcPTS modulates patchouli alcohol production at the level of protein-protein interactions is still unclear. This study aims to identify interacting proteins of PcPTS in order to elucidate its regulatory mechanisms and functions in the biosynthesis pathway of patchouli alcohol.【Method】The yeast two-hybrid technique and GST pull-down combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed to identify potential interacting proteins of PcPTS. The annotation of these interacting proteins was conducted by the MASCOT software and BLAST analysis. Proteins known to be involved in protein translocation, modification, degradation and regulation of secondary metabolism were selected for further analysis. Their interactions with PcPTS proteins were initially confirmed through yeast two-hybrid technology, and subsequently analyzed by using bioinformatics tools. 【Result】The cDNA sequence of PcPTS was successfully cloned by PCR, resulting in an open reading frame (ORF) of 1 659 bp, encoding 552 amino acids. The primary cDNA library and the yeast hybrid cDNA library were constructed and obtained, with capacities of 7.6×106 and 8.6×106colony-forming units (CFU), respectively. Both libraries achieved a 100% recombination rate, with an average length of the inserted fragment greater than 800 bp. The constructed bait vector pGBKT7-PcPTS exhibited no toxicity towards yeast strains and did not exhibit any self-activating activity in yeast cells. The positive clones identified through yeast two-hybrid screening were subjected to sequencing and BLAST analysis, resulting in the identification of 17 candidate proteins. Subsequently, the GST-PcPTS expression vector was effectively constructed, and the GST-PcPTS bait protein was obtained through induced expression purification. This bait protein was utilized to capture interacting proteins from the total protein of Pogostemon cablin, and 98 candidate interacting proteins were identified. Among these candidates, 14 proteins potentially interacting with PcPTS were selected and confirmed through point-to-point verification with yeast two-hybridization. The results indicated that PcC3H6, PcENO3, PcACR11 and PcHAD interacted with PcPTS. Bioinformatics analysis indicated that they belonged to the CCCH zinc finger protein family, enolase proteins, ACR subfamily and HAD-like superfamily, respectively. 【Conclusion】Four proteins (PcC3H6, PcENO3, PcACR11, and PcHAD) were identified as interacting with PcPTS through initial screening and validation. This finding aids in elucidating the role of PcPTS in the biosynthesis pathway of patchouli alcohol and provides a robust basis for future investigations into the regulatory mechanisms of patchouli alcohol synthesis. |
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