Team:UESTC-China/result

From 2014.igem.org

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<h1 class="SectionTitles" style="width:900px; ">Vectors Construction</h1><br/>
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<h1 class="SectionTitles" style="width:1100px; ">Vectors Construction</h1><br/>
<p style="color:#1b1b1b;">We have successfully constructed 2 backbones, <em>piGEM001</em> and <em>piGEM002</em>. And we have verified them using digestion (Fig.1) and sequencing.</p>
<p style="color:#1b1b1b;">We have successfully constructed 2 backbones, <em>piGEM001</em> and <em>piGEM002</em>. And we have verified them using digestion (Fig.1) and sequencing.</p>
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<h1 class="SectionTitles" style="width:900px; ">Plant transformation</h1><br/>
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<h1 class="SectionTitles" style="width:1100px; ">Plant transformation</h1><br/>
<p style="color:#1b1b1b;">Tobacco was transformed essentially using the leaf disk co-cultivation protocol of Horsch. This protocol includes three stages, co-cultivation (Fig.4A), screening cultivation (Fig.4B and C) and rooting cultivation (Fig.4D). We have successfully transformed each vector into babacco and got positive transgenic seedlings (Fig.5). Table 1 is the statistical result of quantity of each transgenic line. And we have got PCR positive plantlet of every transgenic line.</p>
<p style="color:#1b1b1b;">Tobacco was transformed essentially using the leaf disk co-cultivation protocol of Horsch. This protocol includes three stages, co-cultivation (Fig.4A), screening cultivation (Fig.4B and C) and rooting cultivation (Fig.4D). We have successfully transformed each vector into babacco and got positive transgenic seedlings (Fig.5). Table 1 is the statistical result of quantity of each transgenic line. And we have got PCR positive plantlet of every transgenic line.</p>
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<h1 class="SectionTitles" style="width:900px; ">Expression of four key enzymes in tobacco</h1><br/>
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<h1 class="SectionTitles" style="width:1100px; ">Expression of four key enzymes in tobacco</h1><br/>
<p style="color:#1b1b1b;">We extracted DNA from tobacco plantlets. And then we used specific primers to amplify the target gene to verify the kan-resistant seedlings (Fig.6). Next we extracted RNA from tobacco leaves which are PCR positive. We used  RT-PCR to detect whether target gene was expressed (Fig.7).</p>
<p style="color:#1b1b1b;">We extracted DNA from tobacco plantlets. And then we used specific primers to amplify the target gene to verify the kan-resistant seedlings (Fig.6). Next we extracted RNA from tobacco leaves which are PCR positive. We used  RT-PCR to detect whether target gene was expressed (Fig.7).</p>
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<h1 class="SectionTitles" style="width:900px; ">Enhanced HCHO Tolerance </h1><br/>
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<h1 class="SectionTitles" style="width:1100px; ">Enhanced HCHO Tolerance </h1><br/>
<p style="color:#1b1b1b;">The transgenic plants and wildtype, which had been grown separately in sealed boxes, were exposed to HCHO evaporated from a micro tube (0.5ml) containing HCHO solution (37%, 50ul) (Fig.8). One week later we observed the phenotype of transgeneic plants and widetype (Fig.9). We found that the transgenetic seedling is stronger than wildtype after formaldehyde exposure.This indicates that production of HPS/PHI, Faldh and FDH enhanced HCHO tolerance of transgenic seedlings to some extent.</p>
<p style="color:#1b1b1b;">The transgenic plants and wildtype, which had been grown separately in sealed boxes, were exposed to HCHO evaporated from a micro tube (0.5ml) containing HCHO solution (37%, 50ul) (Fig.8). One week later we observed the phenotype of transgeneic plants and widetype (Fig.9). We found that the transgenetic seedling is stronger than wildtype after formaldehyde exposure.This indicates that production of HPS/PHI, Faldh and FDH enhanced HCHO tolerance of transgenic seedlings to some extent.</p>
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<h1 class="SectionTitles" style="width:900px; ">Enhanced HCHO Absorbing</h1><br/>
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<h1 class="SectionTitles" style="width:1100px; ">Enhanced HCHO Absorbing</h1><br/>
<p style="color:#1b1b1b;">We detected the concentration of gaseous formaldehyde evaporated from a micro tube (0.5ml) containing HCHO solution (37%,10ul) and made standard curve (Fig.10). And we saw a linear relationship between HCHO concentration and time before formaldehyde is saturated. For quantity result, we used a HCHO detector to detect the concentration of  gaseous HCHO (Fig.11). The transgenic plants and wildtype, which had been grown separately in sealed boxes, were exposed to HCHO evaporated from a micro tube (0.5ml) containing HCHO solution (37%, 50ul) for about 2 weeks. Two weeks later, the covers of the plant boxes were removed and quickly replaced with covers equipped with HCHO dose-monitoring tubes in order to determine roughly the gaseous HCHO levels remaining in the boxes. We have not got the precise data results now, and this work is to be continued.</p>
<p style="color:#1b1b1b;">We detected the concentration of gaseous formaldehyde evaporated from a micro tube (0.5ml) containing HCHO solution (37%,10ul) and made standard curve (Fig.10). And we saw a linear relationship between HCHO concentration and time before formaldehyde is saturated. For quantity result, we used a HCHO detector to detect the concentration of  gaseous HCHO (Fig.11). The transgenic plants and wildtype, which had been grown separately in sealed boxes, were exposed to HCHO evaporated from a micro tube (0.5ml) containing HCHO solution (37%, 50ul) for about 2 weeks. Two weeks later, the covers of the plant boxes were removed and quickly replaced with covers equipped with HCHO dose-monitoring tubes in order to determine roughly the gaseous HCHO levels remaining in the boxes. We have not got the precise data results now, and this work is to be continued.</p>
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<h1 class="SectionTitles" style="width:900px; ">Transit Peptides Affect HCHO Degrading Efficiency </h1>
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<h1 class="SectionTitles" style="width:1100px; ">Transit Peptides Affect HCHO Degrading Efficiency </h1>
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<p style="color:#1b1b1b;">HPS, PHI, and FDH are located in chloroplast, while FALDH plays a role in cytoplasm. So we used transit peptides to locate the productions of these genes. We hope to know the effects of transit peptide on degrading HCHO. So we exposed transgenic tobaccos with and without transit peptides to HCHO (37%, 50ul). However, there are no obvious phenotype difference compairing different transgenic lines because time limited.</p>
<p style="color:#1b1b1b;">HPS, PHI, and FDH are located in chloroplast, while FALDH plays a role in cytoplasm. So we used transit peptides to locate the productions of these genes. We hope to know the effects of transit peptide on degrading HCHO. So we exposed transgenic tobaccos with and without transit peptides to HCHO (37%, 50ul). However, there are no obvious phenotype difference compairing different transgenic lines because time limited.</p>
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<h1 class="SectionTitles" style="width:900px; ">Different Genes Affect HCHO Degrading Efficiency </h1>
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<h1 class="SectionTitles" style="width:1100px; ">Different Genes Affect HCHO Degrading Efficiency </h1>
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<p style="color:#1b1b1b;">To test which enzymes play the most important role in pathway of metabolizing HCHO, we constructed mono-gene expression vectors to express each enzyme individually. We also constructed multi-gene expression vectors to test whether the ability of metabolizing HCHO of transgenic tobacco enhanced. Then these transgenic tobaccos were exposed to HCHO (37%, 50ul). However, we have not got obvious phenotype difference among transgenic lines, because there is not enough time</p>
<p style="color:#1b1b1b;">To test which enzymes play the most important role in pathway of metabolizing HCHO, we constructed mono-gene expression vectors to express each enzyme individually. We also constructed multi-gene expression vectors to test whether the ability of metabolizing HCHO of transgenic tobacco enhanced. Then these transgenic tobaccos were exposed to HCHO (37%, 50ul). However, we have not got obvious phenotype difference among transgenic lines, because there is not enough time</p>
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<h1 class="SectionTitles" style="width:900px; ">Tapetal expression of AdCP results in male sterility in high expression plants</h1><br/>
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<h1 class="SectionTitles" style="width:1100px; ">Tapetal expression of AdCP results in male sterility in high expression plants</h1><br/>
<p style="color:#1b1b1b;">Considering the problem of environment and safety, we use male sterility system which prevents the horizontal transgene flow. Morphological and histological analysis of AdCP transgenic plants showed ablated tapetum and complete pollen abortion. However, there is not enough time to wait until transgenic tobacco flowers.</p>
<p style="color:#1b1b1b;">Considering the problem of environment and safety, we use male sterility system which prevents the horizontal transgene flow. Morphological and histological analysis of AdCP transgenic plants showed ablated tapetum and complete pollen abortion. However, there is not enough time to wait until transgenic tobacco flowers.</p>
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Revision as of 08:21, 16 October 2014

UESTC-China