Identification of candidate key genes involved in natural rubber biosynthesis in Periploca sepium

Natural rubber is a critical industrial material facing supply shortages. Periploca sepium has emerged as a promising alternative for producing the rubber component cis-polyisoprene. To investigate its biosynthesis, we performed genome and transcriptome sequencing. Our results indicate that this plant possesses complete mevalonic acid (MVA), 1-deoxy-D-xylulose-5-phosphate/2-C-methyl-D-erythritol-4-phosphate (DXP/MEP), and polyisoprene pathways, as well as contains corresponding rubber particle membrane proteins. Using weighted gene co-expression network analysis, we identified 11 key genes positively correlated with levels of cis-polyisoprene and precursor allylic diphosphates (IPP/DMAPP, GPP, FPP, GGPP). Overexpressing acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and 1-deoxy-D-xylulose-5-phosphate reductoisomerase genes from the MVA and DXP/MEP pathways in P. sepium increased IPP/DMAPP levels. Molecular docking and enzyme assays indicated that cis-prenyltransferase exhibits the highest affinity for FPP, suggesting it is the dominant initiator of IPP condensation in cis-polyisoprene biosynthesis. Overexpressing the FPP synthase gene enhanced FPP levels, leading to increased cis-poly7isoprene content. Overexpressing cis-prenyltransferase, small rubber particle protein, and rubber elongation factor genes, especially in combination, significantly raised cis-polyisoprene content and high-molecular-weight proportions. These findings demonstrate that targeted gene up-regulation can improve both rubber yield and quality in P. sepium. The candidate key genes involved in rubber biosynthesis in P. sepium were identified via genome and transcriptome sequencing combined with WGCNA. Up-regulation of the key genes by overexpression, especially in combination, can improve both rubber yield and quality in P. sepium.