Screening and functional validation of transcriptional enhancer cis-elements in Medicago sativa L.

Alfalfa (Medicago sativa L.) is a globally important forage crop. Identification of enhancer cis-regulatory elements (CREs) in high-expression gene promoters is essential for precise gene regulation and molecular breeding. This study aimed to systematically screen and functionally validate enhancer CREs in alfalfa. Twenty constitutively highly expressed genes (coefficient of variation < 0.2) were identified from leaf, stem, and root tissues of 15-day-old alfalfa seedlings (nine RNA-seq libraries, n = 3 per tissue). Promoter regions (2,000 bp upstream to 500 bp downstream of the transcription start site) were extracted and analyzed using PlantCARE, FIMO, and Fisher's exact test (adjusted p < 0.05). Ten candidate enhancer CREs (M1–M10) were identified, and an 89-bp fragment containing a CAAT box from the MsEF1-α promoter, named M11, was selected for functional validation. Recombinant promoter-LUC vectors containing M1, M2, M7, and M11 were constructed and transiently expressed in Nicotiana benthamiana. Luciferase (LUC) activity was quantified to assess enhancer function. Deletion of M11 from the MsEF1-α promoter reduced LUC activity by approximately 55%, whereas insertion of M11 into the MsLEA2 promoter increased activity by approximately 183%. M1 and M7 exhibited no significant enhancer activity, while M2 showed unstable enhancing effects. M11 is a robust and promoter-independent enhancer CRE in alfalfa, providing a valuable tool for gene regulation and molecular breeding.