Overexpressions of dihydroxyacetone synthase and dihydroxyacetone kinase in chloroplasts install a novel photosynthetic HCHO-assimilation pathway in transgenic tobacco using modified Gateway entry vectors

Dihydroxyacetone synthase (DAS) and dihydroxyacetone kinase (DAK) are two key enzymes for formaldehyde assimilation in methylotrophic yeasts. In order to using a Gateway LR recombination reaction to construct a plant expression vector that contains the expression cassettes for the das and dak genes and allow the proteins encoded by the two target genes to be localized to the chloroplasts of transgenic plants, the entry vector pEN-L4*-PrbcS-*T-gfp-L3* contained the tomato rbcS 3C promoter (PrbcS) with its transit peptide sequence (*T) and a GFP reporter gene (gfp) was constructed in this study. To verify the applicability of pEN-L4*-PrbcS-*T-gfp-L3*, we generated an entry vector for the dak gene by replacing the gfp gene in this entry vector with the dak gene. We also generated an entry vector for the das gene by replacing the gus gene in another entry vector (pENTR*-PrbcS-*T-gus) with the das gene. Using these entry vectors and pK7m34GW2-8m21GW3, we successfully constructed the pKm-35S-PrbcS-*T-gfp-PROLD-PrbcS-*T-gus and the pKm-35S-PrbcS-*T-dak-PROLD-PrbcS-*T-das expression vectors. Our results showed that high expression of GUS was achieved in leaves, and the expressed GFP, DAS and DAK proteins could be targeted to the chloroplasts after the two expression vectors were used to transform tobacco. The overexpressions of DAS and DAK in the chloroplasts successfully created a novel photosynthetic HCHO-assimilation pathway in transgenic tobacco. By utilizing these expression vectors, we not only successfully expressed two target genes with one transformation but also localized the expressed proteins to chloroplasts via the transit peptide sequence (*T). Therefore, the construction of pEN-L4*-PrbcS-*T-gfp-L3* establishes a technique platform that provides a convenient means for chloroplast genetic engineering.