Improve The Genetic Manipulation Of Plant Metabolic Capacity Of Formaldehyde

Formaldehyde (HCHO) is one of the main indoor air pollutants, present in tobacco smoke, furniture, industrial adhesives, and varnishes. It is also a highly reactive compound that has a toxic effect on all organisms through its nonspecific reactivity with protein, nucleic acid and lipid. Plants are thought to be the best choice to remove indoor HCHO for its convinience and environmental protection, but the absorption ability of plants is greatly limited. Thus, we should enhance the absorption competence of plants by genetic engineering.RuMP (Ribulose monophosphate pathway) is one of bacterial HCHO assimilation pathways. HPS O-hexulose-6-P synthase) and PHI (6-phospho-3-hexuloisomerase) are two key enzymes in RuMP pathway. Recent studies reveal that over-expressing of HPS and PHI from Mycobacterium gastri MB19 in plant chloroplasts enhances the tolerance of transgenic plants to HCHO and their ability to uptake evaporated and liquid HCHO. Furthermore, the fused gene rmpAB which consists of the two genes encoding HPS and PHI, respectively, was expressed in Escherichia coli, and the recombinant fused protein Hps-Phi exhibits functions corresponding to the individual enzyme activities. On the other hand, Arabidopsis thaliana over-expressing glutathione-dependent formaldehyde dehydrogenase (FALDH) which involved in HCHO dissimilation in plants, showed a 25% increase in its efficiency to take up exogenous HCHO. This study attempted to express Hps-Phi and FALDH, respectively, in transgenic tobacco and compared their effects on HCHO tolerance. The better strategy will be useful for molecular breeding to elevate HCHO metabolism capacity of plants.The plant expression vectors, pK2-r-T-rmpAB and pH2-r-adh for rmpAB and FALDH gene (adh) respectively, were constructed by the Gateway technology. The expression of rmpAB and adh gene were expressed under the control of tomato rbcS-3C promoter. The pK2-r-T-rmpAB harbored a transit peptide sequence which would locate the fused Hps-Phi into chloroplasts. The expressed FALDH wouldbe located in cytoplasm for the lack of the locating sequence in pH2-r-adh. Tobacco was transformed with pK2-r-T-rmpAB and pH2-r-adh. The transgenic plants were selected on MS medium containing antibiotics and verifyed by PCR. 13 rmpAB transgenic lines and 17 adh transgenic lines were obtained. RT-PCR analysis demonstrated these two genes were transripted successfully in transgenic tobacco. Western blot analysis showed that the fused Hps-Phi was expressed. The transgenic rmpkB lines exhibited 1- to 5-fold the wild-type Hps-Phi activity. 3- to 6-fold the wild-type FALDH activity for the adh transgenic lines were observed. The transgenic tobacco over-expressing Hps-Phi (Hps-Phi tobacco) showed higher tolerance to HCHO than the transgenic tobacco over-expressing FALDH (FALDH tobacco) when they were grown on MS medium with 10 mmol/L HCHO or exposed to gaseous HCHO.Hps-Phi tobacco detoxified HCHO more efficiently when compared with FALDH tobacco. To test if this strategy would function well or not in ornamentals, petunia was transformed with pK2-r-T-rmpAB. 15 transgenic petunia lines were identified by PCR. RT-PCR analysis indicated that all the transgenic lines showed a similar transcriptional level. Western blot analysis indicated that the Hps-Phi was expressed in transgenic petunia, which also exhibited higher Hps-Phi activity than wild-type. The transgenic petunia over-expressing Hps-Phi (Hps-Phi petunia) showed higher tolerance to HCHO than wild-type either on MS medium with HCHO or treatment with gaseous HCHO and possessed stronger capacity to fix HCHO than the control.Expressing of Hps-Phi improves the HCHO tolerance of transgenic tobacco, which exhibited the same phenotype as those expressing both HPS and PHI singly. The Hps-Phi tobacco could detoxify HCHO more efficiently than FALDH tobacco. Furthermore, over-expressing of Hps-Phi enhanced the HCHO detoxification capacity of petunia. This suggests that the application of rmpAB can replace of HPS and PHI gene as a rapid and convenient procedure for generation of functional ornamental plants in one step.