Molecular characterization and transcriptional regulation analysis of the squalene synthase gene involved in sitosterol biosynthesis and drought response.

The kernel of cv. 'Merrillii' (Cephalotaxaceae) is a rare nut with a variety of bioactive compounds and a high economic value. β-sitosterol is not only the most abundant plant sterol but also has various biological effects, such as antimicrobial, anticancer, anti-inflammatory, lipid-lowering, antioxidant, and antidiabetic activities. In this study, a squalene synthase gene from , , was identified and functionally characterized. encodes a deduced protein of 410 amino acids. Prokaryotic expression of the TgSQS protein could catalyze farnesyl diphosphate to produce squalene. Transgenic plants overexpressing showed a significant increase in the content of both squalene and β-sitosterol; moreover, their drought tolerance was also stronger than that of the wild type. Transcriptome data from seedlings showed that the expression levels of sterol biosynthesis pathway-related genes, such as , , , , , , , and , increased significantly after drought treatment. We also demonstrated that TgWRKY3 directly bound to the promoter region and regulated its expression through a yeast one-hybrid experiment and a dual luciferase experiment. Together, these findings demonstrate that has a positive role in β-sitosterol biosynthesis and in protecting against drought stress, emphasizing its importance as a metabolic engineering tool for the simultaneous improvement of β-sitosterol biosynthesis and drought tolerance.