Guangdong nongye kexue (May 2024)
Screening and Identification of PcPTS Interacting Proteins in Pogostemon cablin
Abstract
【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 8.6×106 colony-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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