| 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,(5-6):- |
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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 Interaction Proteins in Pogostemon cablin |
| 投稿时间：2024-03-20 修订日期：2024-04-04 |
| DOI： |
| 中文关键词: 广藿香 广藿香醇 PTS 互作蛋白 |
| 英文关键词: Pogostemon cablin Patchouli alcohol PTS Interaction protein |
| 基金项目:国家自然, |
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| 摘要点击次数: 13 |
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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PcPTS在广藿香醇生物合成途径中起关键作用，但PcPTS在转录水平外如蛋白互作层面调控广藿香醇合成的机制尚不清楚。本研究旨在筛选广藿香醇合酶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分析进行互作蛋白的注释，选取有文献报道参与其他蛋白互作、影响蛋白转运、修饰或蛋白降解、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技术成功克隆了广藿香醇合酶PcPTS的cDNA序列，开放阅读框（ORF）为1659 bp，编码了522个氨基酸。构建获得广藿香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，结果表明PcC3H6、PcENO3、PcACR11和PcHAD与PcPTS存在相互作用。生物信息分析表明PcC3H6、PcENO3、PcACR11和PcHAD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，有助于揭示广藿香醇合酶PcPTS在广藿香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 utilized aromatic herbs eliminating dampness in clinical settings, with its primary bioactive compound, patchouli alcohol, demonstrating potent antiviral and anti-inflammatory, and other beneficial properties. Patchoulol synthase(PcPTS), integral to the biosynthesis pathway of patchouli alcohol, is essential for its production. However, the precise mechanisms by which PcPTS modulates patchouli alcohol production, particularly at the post-transcriptional level involving protein-protein interactions, remain unclear. This study aims to identify interacting proteins of PcPTS in order to elucidate its regulatory mechanisms. 【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 using 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 using bioinformatics tools. 【Result】The cDNA sequence of patchouli alcohol synthase (PcPTS) was successfully cloned using PCR, resulting in an open reading frame (ORF) of 1659 bp that encodes 388 amino acids. The primary cDNA library and the yeast hybrid cDNA library were constructed and obtained, with capacities of 7.6×106 and 8.6×106 colony-forming units (CFU) respectively. Both libraries achieved a 100% recombination rate, with average length of the insert greater than 800 bp. The bait vector pGBKT7-PcPTS, used in yeast hybridization, exhibited no toxicity towards yeast strains and did not exhibit any self-activating activity in yeast cells. A total of 17 candidate proteins were identified from a patchouli yeast hybridization library through screening with a yeast two-hybrid system. The positive clones identified through yeast two-hybrid screening were subjected to sequencing and analysis using BLAST, 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 Patchouli, leading to the identification of 98 candidate interworking proteins. Among these candidates, 10 proteins potentially interacting with PcPTS were selected and confirmed through point-to-point verification using yeast two-hybridization. The results indicated that PcC3H6, PcENO3, PcACR11 and PcHAD interacted with PcPTS. Bioinformatic analysis indicated that PcC3H6, PcENO3, PcACR11 and PcHAD belong to the CCCH zinc finger protein family, enolase proteins, ACR subfamily and HAD-like superfamily, respectively. 【Conclusion】Initial screening and validation identified four proteins (PcC3H6, PcENO3, PcACR11, and PcHAD) that interact with PcPTS. This finding serves as a foundational step for further investigations into the regulatory mechanisms of patchouli alcohol synthesis and the role of PcPTS in the biosynthesis pathway of patchouli. |
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