Construction of yeast two-hybrid cDNA library and identification of interacting protein with McHDRa/b in Matricaria chamomilla L.

German chamomile (Matricaria chamomilla L.) is a traditional aromatic medicinal plant, its flower contains volatile aromatic oil (essential oil). The main sesquiterpene components of the essential oil are (E)-β-farnesene, chamazulene, and α-bisabolol, these components have significant medicinal value and are used in food, cosmetics, and pharmaceuticals. However, the German chamomile genome has not yet been cataloged in any database; consequently, research on the intricate regulatory network and interaction mechanisms among proteins in German chamomile remains limited. Furthermore, no study has thus far developed a yeast cDNA library for German chamomile. Therefore, we constructed a homogenized yeast cDNA library using different tissues of German chamomile, this yeast cDNA library had a titer of 1.444 × 10⁸ colony-forming units/mL, an average insert size of > 1,000 bp, and a positive rate of 100%. In addition, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS) that interacted with Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) involved in the final step of the methylerythritol 4-phosphate (MEP) pathway was verified through the yeast two-hybrid (Y2H) assay and bimolecular fluorescence complementation (BiFC). At the same time, the expression pattern and function of McHDS were further analyzed. In conclusion, we successfully constructed a yeast cDNA library of German chamomile for the first time, and McHDS interacting with McHDRa/b was successfully screened, providing a reliable theoretical foundation for investigating the molecular mechanism of its coordination with McHDRa/b to regulate the biosynthesis of (E)-β-farnesene in German chamomile. Which lays the groundwork for our comprehensive understanding of the protein interaction network involved in sesquiterpene synthesis of German chamomile.