Overexpression of Sm4CL1 and Sm4CL2 from Swertia mussotii in Lemna minor Enhances Lignin and Flavonoid Biosynthesis
Description
The phenylalanine metabolic pathway is a crucial route in plants for synthesizing secondary metabolites, such as flavonoids and lignans, which possess diverse pharmacological activities. 4-Coumarate: CoA ligase (4CL) is a pivotal enzyme that channels the phenylalanine metabolic pathway into various secondary metabolic branches. Swertia mussotii Franch., a plant celebrated for its anti-inflammatory and anti-tumor properties, relies on flavonoid compounds as the pharmacological basis for its anti-tumor activity. In this study, we identified the Sm4CL1 and Sm4CL2 genes in S. mussotii through bioinformatics analysis. Using genetic engineering techniques, we constructed the plant expression vectors pCAMBIA1301-Sm4CL1 and pCAMBIA1301-Sm4CL2, which were overexpressed in Lemna minor via Agrobacterium-mediated transformation. This approach elucidated the roles of Sm4CL1 and Sm4CL2 in regulating plant secondary metabolism. Our study provides novel insights into the regulatory mechanisms of secondary metabolism in L. minor driven by the phenylalanine metabolic pathways associated with Sm4CL1 and Sm4CL2. By evaluating the growth, flavonoid, and lignin content in L. minor overexpressing Sm4CL1 and Sm4CL2, we found that these genes significantly enhanced lignin biosynthesis, with Sm4CL2 showing slightly higher accumulation. Sm4CL1 also increased specific flavonoid accumulation, particularly under L-phenylalanine feeding, suggesting dual roles in lignin and flavonoid pathways. Optimal L-phenylalanine concentrations promoted growth and metabolite production, highlighting the importance of precursor supply. Our findings highlight the potential of L. minor as a chassis for producing valuable secondary metabolites through genetic engineering. Future work should focus on elucidating the detailed mechanisms of Sm4CL1 and Sm4CL2 and exploring their applications in synthesizing bioactive compounds.
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Total RNA from transgenic L. minor lines overexpressing Sm4CL1 and Sm4CL2 was extracted using an RNA extraction kit and subsequently reverse-transcribed into cDNA. Specific primers were designed (Table S1), and quantitative real-time PCR (qRT-PCR) was conducted using SuperReal Fluorescence Quantification Premixed Reagent Enhanced (SYBR Green) (Tiangen, Beijing, China). Actin served as the internal reference gene. The reaction mixture totaled 20 µL, consisting of 10 μL of 2×SuperReal PreMix Plus (SYBR Green), 0.6 μL each of forward and reverse primers (10 μmol/L), 1 μL of cDNA template, and 7.8 μL of nuclease-free water. qRT-PCR was carried out on a Bio-Rad instrument, following a two-step protocol. Each sample was analyzed in triplicate, and relative expression levels were calculated using the 2−ΔΔCT method. The 4CL enzyme activity of each lines of L. minor was measured using the Plant 4-Coumarate-CoA Ligase (4CL) ELISA kit (Jingkang, Shanghai, China).