Team:UESTC-China/Design

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<h1 style="color:#1b1b1b; position:relative; left:0px; padding:15 5px; font-size:40px; font-family: calibri, arial, helvetica, sans-serif; font-weight: bold;font-style: Italic; text-align:center; width:1140px;">Design</h1>
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<p style="color:#1b1b1b;">In order to further increase the plant ability of formaldehyde uptake and metabolism by synthesis biology technology, we choosed four enzyme-coding genes related to formaldehyde metabolic pathways from microorganism and plant: they are 3-hexulose-6-phosphate (HPS), 6-phospho-3-hexuloisomerase (PHI), formaldehyde dehydrogenase (FALDH) and formate-dehydrogenase (FDH). These genes are transformed into plants and will promote formaldehyde metabolism. For security reasons, we also induce <i>AdCP</i> gene into our plans because of its capability to lead to pollen abortion. At the same time, chloroplast transformation is taken into consideration to avoid gene flow and improve gene expression.
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<p style="color:#1b1b1b;">In order to further increase the plant ability of formaldehyde uptake and metabolism by synthetic biology technology, we choosed four enzyme-coding genes related to formaldehyde metabolic pathways from microorganism and plant: 3-hexulose-6-phosphate (HPS), 6-phospho-3-hexuloisomerase (PHI), formaldehyde dehydrogenase (FALDH) and formate-dehydrogenase (FDH). These genes are transformed into plants and will promote formaldehyde metabolism. For security reasons, we also induce <i>AdCP</i> gene into our plans because of its capability to lead to pollen abortion. At the same time, chloroplast transformation is taken into consideration to avoid gene flow and improve gene expression.
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The ribulose monophosphate (RuMP) pathway is one of the formaldehyde-fixation pathways found in microorganisms called methylotrophs, which utilize one-carbon compounds as the sole carbon source. The key enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS), which fixes formaldehyde to D-ribulose-5-phosphate (Ru5P) to produce D-arabino-3-hexulose-6-phosphate (Hu6P), and 6-phospho-3-hexuloisomerase (PHI), which converts Hu6P to fructose 6-phosphate (F6P).The two key enzymes work in chloroplast both.We will use fusion expression to conduct heterologous expression in tobacco <i>(Chen et al., 2010)</i>.  
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The ribulose monophosphate (RuMP) pathway is one of the formaldehyde-fixation pathways found in microorganisms called methylotrophs, which utilize one-carbon compounds as the sole carbon source. The key enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS), which fixes formaldehyde to D-ribulose-5-phosphate (Ru5P) to produce D-arabino-3-hexulose-6-phosphate (Hu6P), and 6-phospho-3-hexuloisomerase (PHI), which converts Hu6P to fructose 6-phosphate (F6P). The two key enzymes work in chloroplast both. We will use fusion expression method to conduct heterologous expression in tobacco (<i>Chen et al., 2010</i>).  
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Formate dehydrogenase is a mitochondrial-localized NAD-requiring enzyme while the HCOOH is getting into the mitochondrial,FDH will oxidize the formic acid into CO2, and reduce NAD+ to NADH with a high degree of specificity.In our project, the heterologous expression of <i>FDH</i> from arabidopsis thaliana in tobacco was completed.
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Formate dehydrogenase is a mitochondrial-localized NAD-requiring enzyme while the HCOOH is getting into the mitochondrial, FDH will oxidize the formic acid into CO2, and reduce NAD+ to NADH with a high degree of specificity. In our project, the heterologous expression of Arabidopsis <i>FDH</i> gene in tobacco was completed.
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<strong>Fig.3</strong> The abbreviations are as follows: FALDH:glutathione-dependent formaldehyde dehydrogenase; FDH: Formate dehydrogenase; HM-GSH: S-Hydroxymethyl glutathione; Forml-GSH: Formyl glutathione; SMM cycle: Methionine cycle.
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<strong>Fig.3</strong> Folate-independent pathway. The abbreviations are as follows: FALDH:glutathione-dependent formaldehyde dehydrogenase; FDH: Formate dehydrogenase; HM-GSH: S-Hydroxymethyl glutathione; Forml-GSH: Formyl glutathione; SMM cycle: Methionine cycle.
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Stomata are microscopic pores surrounded by two guard cells and play an important role in the uptake of CO2 for photosynthesis. Recent researches revealed that light-induced stomatal opening is mediated by at least three key components: blue light receptor phototropin, plasma membrane H+-ATPase, and plasma membrane inward-rectifying K+ channels. However, Wang et al (2014) showed that only increasing the amount of H+-ATPase in guard cells had a significant effect on light-induced stomatal opening (Fig. 4). Transgenic Arabidopsis plants by overexpressing H+-ATPase in guard cells exhibited enhanced photosynthesis activity and plant growth. Therefore,in order to strengthen the ability of absorbing formaldehyde, we overexpressed H+-ATPase (AtAHA2) in transgenic tobacco guard cells,resulting in a significant effect on light-induced stomatal opening.
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Stomata are microscopic pores surrounded by two guard cells and play an important role in the uptake of CO2 for photosynthesis. Recent researches revealed that light-induced stomatal opening is mediated by at least three key components: blue light receptor phototropin, plasma membrane H+-ATPase, and plasma membrane inward-rectifying K+ channels. However, Wang et al (2014) showed that only increasing the amount of H+-ATPase in guard cells had a significant effect on light-induced stomatal opening (Fig. 4). Transgenic Arabidopsis plants by overexpressing H+-ATPase in guard cells exhibited enhanced photosynthesis activity and plant growth. Therefore, in order to strengthen the ability of absorbing formaldehyde, we overexpressed H+-ATPase (<i>AtAHA2</i>) in transgenic tobacco guard cells, resulting in a significant effect on light-induced stomatal opening.
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<strong>Fig.5</strong> Pollen germination of untransformed control plant and sterile transgenic plantsin vitro. Pollen grains were germinated on sucrose-boric acid medium and over 500 pollen grains were observed. a. Untansformed control plant pollen, b. Sterile pollen.Scale bar 25 μm (<i>Shukla et al., 2014</i>).  
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<strong>Fig.5</strong> Pollen germination of untransformed control plant and sterile transgenic plants <i>in vitro</i>. Pollen grains were germinated on sucrose-boric acid medium and over 500 pollen grains were observed. a. Untansformed control plant pollen, b. Sterile pollen. Scale bar 25 μm (<i>Shukla et al., 2014</i>).  
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<strong>Fig.7</strong> Schematic of vectors we constructed
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<strong>Fig.7</strong> Schematic of vectors construction.
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Latest revision as of 02:27, 18 October 2014

UESTC-China