Viewing The Genetic Engineering Of Plant Metabolic Pathways Of Formaldehyde

Formaldehyde (HCHO) is a kind of highly reactive compound that has a toxic effect on all organisms. Since HCHO is also a key compound in C1 metabolisms, most organisms have their own system for HCHO detoxification. The HCHO-detoxification system was highly developed in methylotrophic organisms which can assimilate C1 compounds in the course of evolution. RuMP (Ribulose monophosphate pathway) is one of HCHO-assimilation pathways and has great contribution to formaldehyde detoxification in methylotrophs. HPS (3-hexulose-6-P synthase) and PHI (6-phospho-3-hexuloisomerase) are the key enzymes in RuMP pathway. Recent research reveals that HCHO assimilation pathways are wildly exist in bacteria and the HPS and PHI orthologs are also found in many non- methylotrophic organisms. The HPS and PHI genes have been cloned from many methylotrophic bacteria and non-methylotrophic bacteria recently.Formaldehyde is becoming a major indoor air pollutant and also toxic to all plants. The vast majority of plants around us photosynthetically assimilate carbon dioxide via the Calvin cycle pathway and they have very limited capacity to up-take HCHO gas (3). Recent studies demonstrate that HPS and PHI from a methylotrophic bacterium were introduced into plants by genetic engineering and the expression of HPS and PHI in plant chloroplasts enhanced the tolerance of transgenic plants to formaldehyde and the ability of the plants to absorb exogenous formaldehyde. On the other hand, transgenic Arabidopsis overexpressing glutathione-dependent formaldehyde dehydrogenase (FALDH), which involved in HCHO-dissimilation in plants, shows a 25% increase in its efficiency to take up exogenous formaldehyde. These strategies may be used to molecular breeding of ornamentals. The present study tried to isolate bacteria which resist to HCHO and can use one carbon compounds from local resource. The genomic DNA libraries of the isolated strains were constructed for the cloning of novel genes related to C1-metabolism, which will be use for molecular breeding of ornamentals. Furthermore, the present study also conducted a study to augment the ability of ornamentals to metabolize HCHO with the identified genes which involve in HCHO-assimilation and -dissimilation pathways. The results of the present study are concluded as following:The strain A and B with CH₃OH-utilization ability were isolated from sewage samples. By sequence analyses for 16S rDNA, the two isolated strains were identified as Bacillus sp. XSZQ05-02A and Klebsiella pneumoniae KGSH05-01B, respectively. The test with formaldehyde showed they were well tolerant to formaldehyde and could grow on LB medium with 8～15 mmol/L formaldehyde. Southern hybridization analyses revealed that the homologs of HPS and PHI genes from a facultative methylotrophic bacterium, Mycobacterium gastri MB19, were presented in the genomes of the isolated strains. Using pUC118 as the vector, genomic DNA libraries were constructed for the isolated strains. The further analyses showed that the quality of the libraries was satisfied the requirements for gene screening.The fused gene rmpAB, made from HPS and PHI gene, was shown to be expressed in Ecoli, and the recombinant protein Hps-Phi exhibited both HPS and PHI enzyme activities. The present study transformed ornamental plants, Pelargonium sp.frensham and Petunia with the plant expressional vectors, pK2- r- T-rmpAB and pH2-r-adh (FALDH). In the two plant expression vectors, the expression of both rmpAB and adh were under the control of rbcS-3c promoter from tomato. The pK2- r-T-rmpAB harbors a transit peptide sequence which allowed the location of the expressed protein into chloroplasts, while in the pH2-r-adh, the expressed FALDH were located in cytoplasm. The transgenic plants were selected on MS medium containing antibiotics and identified by PCR. 20 lines of transgenic Pelargonium and 12 lines of transgenic Petunia, respectively, were obtained for rmpAB, while 13 adh transgenic Pelargonium lines and 7 transgenic Petunia lines, respectively, were obtained for adh.The rmpAB transgenic Pelargonium showed good growth than the control when grown on MS medium with 7 mmol/L formaldehyde or exposed to gaseous formaldehyde, while the transgenic adh Pelargonium exhibited similar growth as the control. These results indicate that the rmpAB transgenic Pelargonium is much more tolerant to formaldehyde than adh transgenic Pelargonium. The further analysis indicated that the rmpAB transgenic Pelargonium possessed stronger capacity to take up liquid formaldehyde and fix HCHO than the control.RT-PCR analysis showed that both rmpAB and adh were successfully transcripted intransgenic rmpAB and adh Petunia, respectively. Western blot analysis indicates that the Hps-Phi is expressed in the rmpAB transgenic Petunia hybrida, which exhibited about 4 fold the wild-type Hps-Phi activity. The adh transgenic Petunia exhibited about 4 fold the wild-type FALDH activity. The rmpAB transgenic Petunia also showed an increase in its efficiency to take up liquid formaldehyde than the adh transgenic Petunia and wild type. Tracer experiment showed the HCHO-fixation ability of the rmpAB transgenic Petunia was higher than wild-type.In this research, two facultative methylotrops were isolated from the local resource and their genomic DNA libraries were constructed. This established a basis for the cloning the key enzyme genes involved in HCHO metabolism in the future. The study generated two functional ornamentals, transgenic Pelargonium and Petunia over-expressing rmpAB and demonstrated that the fused rmpAB gene can be applied to the molecular breeding for functional- ornamentals.