Cotton is a vital economic crop, and cotton fiber serves as the primary raw material for the textile industry. Lignin in cotton fiber is closely associated with fiber quality. Lignin is synthesized through the phenylpropanoid metabolic pathway, where the cinnamyl alcohol dehydrogenase gene CAD6 plays a significant role. In this study, we obtained successfully transformed overexpression plants by constructing an overexpression vector and performing genetic transformation and tissue culture. To verify the function of the GhCAD6 gene in upland cotton, we analyzed the agronomic traits, fiber quality, cell wall structure, and lignin content of GhCAD6-overexpressing plants. Our results indicate that the GhCAD6 gene is predominantly expressed during the stages of fiber elongation and secondary wall synthesis. Overexpression of the GhCAD6 gene resulted in increased plant lignin content and fiber upper half mean length, boll number per plant, fiber uniformity index, strength, and lint were improved. The fiber surface was smoother, and the fiber cell wall was more compact. These findings demonstrate that the GhCAD6 gene positively regulates lignin synthesis and fiber quality formation, contributing to the enhancement of cotton fiber quality.

Multiple cis-acting elements are present in promoter sequences that play critical regulatory roles in gene transcription and expression. In this study, we isolated the cotton FDH (Fiddlehead) gene promoter (pGhFDH) using a real-time reverse transcription-PCR (qRT-PCR) expression analysis and performed a cis-acting elements prediction analysis. The plant expression vector pGhFDH::GUS was constructed using the Gateway approach and was used for the genetic transformation of Arabidopsis and upland cotton plants to obtain transgenic lines. Histochemical staining and a β-glucuronidase (GUS) activity assay showed that the GUS protein was detected in the roots, stems, leaves, inflorescences, and pods of transgenic Arabidopsis thaliana lines. Notably, high GUS activity was observed in different tissues. In the transgenic lines, high GUS activity was detected in different tissues such as leaves, stalks, buds, petals, androecium, endosperm, and fibers, where the pGhFDH-driven GUS expression levels were 3–10-fold higher compared to those under the CaMV 35S promoter at 10–30 days post-anthesis (DPA) during fiber development. The results indicate that pGhFDH can be used as an endogenous constitutive promoter to drive the expression of target genes in various cotton tissues to facilitate functional genomic studies and accelerate cotton molecular breeding.