Objective This study aimed to develop a compact, efficient, stable and low-sequence-homology bidirectional promoter-based multigene expression system with low sequence homology, providing a high-performance platform for native gene functional validation in conifers.

Method A validated plant short transcriptional enhancer (STE) was tetramerized (4 × STE) and flanked by two non-repetitive minimal core promoters, CaMV 35S and CsVMV, in opposite orientations to minimize source sequence homology. Combined with a P2A self-cleaving peptide-based polycistronic strategy, a single-promoter system for the simultaneous expression of five genes was constructed. The expression efficiency and cross-species applicability of this system were verified in the model plant tobacco (Nicotiana benthamiana) and hypocotyls of the gymnosperm Pinus tabuliformis via Rhizobium-mediated transient transformation.

Result (1) A compact bidirectional promoter was successfully constructed, driving robust and simultaneous expression of both upstream and downstream genes in tobacco and P. tabuliformis, thereby confirming that the system has certain species applicability. (2) The downstream betalain biosynthesis genes (CYP76AD1, DODA, GT) were successfully expressed and catalyzed the conversion of tyrosine into betalains, resulting in distinct red pigmentation in tissues. (3) The expression intensity of the upstream GUS was comparable to that of the 35S promoter and remained stable;furthermore stable luminescent signals were also detected for LUC.

Conclusion The bidirectional promoter-based polygenic expression system established in this study effectively avoids the risk of transgene silencing caused by repetitive sequences in traditional polygenic vectors, resolving the trade-off between vector structural redundancy and expression efficiency. This system provides a novel tool for plant polygenic genetic improvement and exhibits significant application potential in gene function characterization and molecular breeding of recalcitrant species, including conifers.