Using The Mechanism Of Plant Photosynthesis, The Assimilation Of Formaldehyde

The formaldehyde (HCHO) is produced in the early steps of C1 metabolisms in all of the prokaryotes and eukaryotes which can utilize C1 compounds (methane, methanol, formaldehyde and methylated amines). The cells of these organisms posses HCHO assimilation pathways which convert HCHO, produced from C1 compounds, into cell constituents and HCHO dissimilation pathways which oxidize HCHO to produce energy for cell growth. There is a highly efficient metabolism for the utilization of the one-carbon compound methanol in methylotrophic yeast species, such as Candida boidinii, HansenuLa polymorpha and Pichia pastoris. In methylotrophic yeasts, formaldehyde is the key intermediate in methanol metabolism since it stands at the branchpoint of pathways for methanol assimilation and dissimilation. Formaldehyde is generated from methanol via alcohol oxidase. A portion of the formaldehyde is directly fixed with xyluLose-5-phosphate (Xu5P) to produce dihydroxyacetone (DHA) and glyceraldehydes 3-phosphate (GAP). DAS is a type of thiamine pyrophosphate-dependent transketolase and catalyzes the first reaction in the HCHO-assimilation pathway. Recently, the DAS gene (DAS1) was cloned from the methylotrophic yeasts, C. boidinii and H. polymorpha. DHA is toxic to yeast cells and dihydroxyacetone kinase (DAK) is involved in the detoxification of DHA. The reaction catalyzed by DAK converts DHA into dihydroxyacetone phosphate (DHAP) which is no toxic and can be ustilized by yeast cells. DAK presents in most organisms. DAK genes in the Saccharmyces cerevisiae and P. pastoris were cloned and characterized by a combined genetic and biochemical approach and the proteins encoded by these DAK genes are homodimer.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. Since the substrates and products of DAS and DAK, Xu5P, DHAP and GAP are the intermediates of the Calvin cycle, the present study attempted to express DAS and DAK from the yeasts into plant chloroplasts and construct a HCHO-fixing pathway with DAS and DAK. Thus theHCHO-fixation pathway from yeasts was incorporated into the Calvin cycle as a bypass in plants. This wouLd create a new strategy and method for phytoremediation of HCHO pollution. In order to apply this new strategy to ornamental for enhancement of their ability to metabolize formaldehyde and generation of functional ornamental in the future as soon as possible, the tissue cuLture for some important ornamentals such as new lily species, Conca D 'or and La Mancha was conducted in this study for accumuLation of experimental materials. This study also tried to isolate yeast strains which can use C1 compounds with local source for cloning of genes related to C1 metabolism and thus established a foundation for the molecuLar study on methylotrophic yeasts in the future. The main resuLts of this study are concluded as follows:(1) DAS and DAK genes were amplified from the genomic DNA of C.boidinii and P. pastoris , respectively. The plant expression vectors, pK2-Prbcs-DAS and pH2-Prbcs-DAK were constructed for DAS and DAK genes by gateway technology. In these two plant expression vectors, the expression of the DAS and DAK genes was under the control of tomato Rubisco-3c promoter (Prbcs). The Prbcs promoter contains a transit peptide sequence which allows the location of the expressed DAS and DAK into chloroplasts. The DAS and DAK genes were introduced into Nicotiana tabacum and Petunia hybrida by Agrobacterium tumefaciens- mediated procedure. The transgenic plants were selected on MS medium containing antibiotics and identified by PCR. 17 lines of transgenic tobacco and 21 lines of transgenic Petunia, respectively, were obtained for DAS gene, while 20 transgenic tobacco lines and 29 transgenic Petunia lines, respectively, were obtained for DAK gene.The transcriptions of DAS and DAK genes were confirmed by RT-PCR analysis. The transgenic DAS tobacco and petunia were transformed again with pH2-Prbcs-DAK for generation of double gene (DAS/DAK) transgenic plants. The transgenic plants were selected on MS medium containing antibiotics and identified by PCR. 19 lines of transgenic tobacco and 24 lines of transgenic Petunia, respectively, were obtained. The further analysis indicated that the double gene transgenic petunia possessed stronger capacity to take up liquid formaldehyde than the control and the single gene, DAS or DAK, transgenic plants. This resuLt suggests that the expressed DAS and DAK might establish aHCHO-fixation pathway which functions in the double gene transgenic plants.(2) The tissue cuLture for Conca D'or, a new species of lily, was conducted with its ovary. The resuLts showed that the best medium for callus formation was MS+BA 1.0 mg.L^-1+NAA 0.5 mg.L^-1 and the inducing rate for callus was 76%. The best medium for bud initiation was MS+BA 1.0 mg.L^-1+NAA 0.1 mg.L^-1 and the inducing rate was 82.5%. The best medium for root formation was MS, and the rooting rate reached 100%. At the same time, the tissue cuLture for Mancha was also conducted with its ovary. The best medium for callus formation was MS +KT4.0 mg.L^-1 + 2,4-D 4.0 mg.L^-1 and the inducing rate for callus was 84%. The best medium for bud initiation was MS + KT4.0 mg.L^-1 + 2, 4-D 4.0 mg.L^-1 ,and the inducing rate was 70%. The best medium for root formation was MS, and the rooting rate reached 96%(3) Two yeast strains resistant to high HCHO were isolated from sewage samples. The morphological and physiological identification showed one of them was methylotrophic yeast and designated as Yflj3...