| 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):- |
| 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： |
|
| 摘要点击次数: 30 |
| 全文下载次数: 0 |
| Abstract: |
| 【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. |
|
View Fulltext
查看/发表评论 下载PDF阅读器 |
|
|
|
