Difference between revisions of "Team:SCAU-China/Proof"

 
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<div class="h2_font_size">Analysis of transgenes</div>
 
<div class="h2_font_size">Analysis of transgenes</div>
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<div class="p_font_size">After agrobacterium-mediated rice calli transformation, several hygromycin resistant rice lines were obtained, and their further molecular analyses were done. The genomic DNA of T0 transformants were extracted to do PCR amplifying HPT and Cre. Then PCR positive lines were selected to detect four foreign genes (PSY, CrtI, BHY and BKT). The PCR results of some lines showed the expected bands of four genes were detected (Figure 3).</div>
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<div class="p_font_size"  style="text-indent:0em" >After <font style="font-style:italic">Agrobacterium</font>-mediated rice calli transformation, several hygromycin resistant rice lines were obtained, and their further molecular analysis were done. The genomic DNA of T<SUB>0</SUB> transformants were extracted to do PCR amplifying HPT and Cre. Then PCR positive lines were selected to detect four foreign genes <font style="font-style:italic">(PSY, CrtI, BHY </font>and<font style="font-style:italic"> BKT)</font>. The PCR results of some lines showed the expected bands of four genes were detected (Figure 3).</div>
 
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<div class="p_font_size" style="margin-bottom:20px"><small>Figure 3 PCR assays of several transgenic rice lines. M, Marker 2K plus. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negetive control (wild-type rice cultivar HG1).</small></div>
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<div class="p_font_size" style="margin-bottom:20px"><small> <font style="font-weight:bold">Figure 3</font> &nbsp;&nbsp;PCR assays of several transgenic rice lines. M, Marker 2K plus. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negative control (wild-type rice cultivar HG1).</small></div>
 
<div class="p_font_size">To detect expression levels of these four foreign genes involved in astaxanthin biosynthesis, total RNA of transgenic rice seeds were extracted and their cDNA was synthesized from 1 μg of DNase-treated RNA. The results of RT-PCR showed the expected bands of the four key genes for synthesizing astaxanthin in transgenic rice seeds (Figure 4).</div>
 
<div class="p_font_size">To detect expression levels of these four foreign genes involved in astaxanthin biosynthesis, total RNA of transgenic rice seeds were extracted and their cDNA was synthesized from 1 μg of DNase-treated RNA. The results of RT-PCR showed the expected bands of the four key genes for synthesizing astaxanthin in transgenic rice seeds (Figure 4).</div>
 
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<div class="p_font_size" style="margin-bottom:20px"><small>Figure 4 RT-PCR analyses of expression levels of PSY, CrtI, BKY, and BHY genes in several transgenic rice. Rice OsActin1 was as an internal control. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negetive control (wild-type rice cultivar HG1).</small></div>
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<div class="p_font_size" style="margin-bottom:20px"><small> <font style="font-weight:bold">Figure 4</font> &nbsp;&nbsp;RT-PCR analysis of expression levels of <font style="font-style:italic">PSY, CrtI, BKY, </font>and <font style="font-style:italic">BHY</font> genes in several transgenic rice. Rice <font style="font-style:italic">OsActin1</font> was as an internal control. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negative control (wild-type rice cultivar HG1).</small></div>
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<div class="p_font_size">These results of PCR and RT-PCR demonstrated that we successfully obtained transgenic rice with all of four stacking genes (PSY, CrtI, BHY and BKT). </div>
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<div class="p_font_size">These results of PCR and RT-PCR demonstrated that we successfully obtained transgenic rice with all of four stacking genes <font style="font-style:italic">(PSY, CrtI, BHY </font>and<font style="font-style:italic"> BKT). </font></div>
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<div class="p_font_size"><font style="font-style:italic">In summary, we successfully obtained transformants with all of transgenes in lab and detected obvious transcriptional activity in endosperm. These results indicated that our designed pathway was realizable in rice endosperm. To further confirm astaxanthin production, several analyses were carried out. The results were shown in Demonstrate page.</font></div>
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<p>&nbsp;</p>
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<div class="p_font_size">In summary, we successfully obtained transformants with all of transgenes in lab and detected obvious transcriptional activity in endosperm. These results indicated that our designed pathway was realizable in rice endosperm. To further confirm astaxanthin production, several analysis were carried out. The results were shown in <a href="https://2016.igem.org/Team:SCAU-China/Demonstrate" text-decoration=underline>Demonstrate page</a>.</div>
 
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Latest revision as of 05:33, 18 October 2016

SCAU

Proof
Analysis of transgenes
After Agrobacterium-mediated rice calli transformation, several hygromycin resistant rice lines were obtained, and their further molecular analysis were done. The genomic DNA of T0 transformants were extracted to do PCR amplifying HPT and Cre. Then PCR positive lines were selected to detect four foreign genes (PSY, CrtI, BHY and BKT). The PCR results of some lines showed the expected bands of four genes were detected (Figure 3).
image
Figure 3   PCR assays of several transgenic rice lines. M, Marker 2K plus. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negative control (wild-type rice cultivar HG1).
To detect expression levels of these four foreign genes involved in astaxanthin biosynthesis, total RNA of transgenic rice seeds were extracted and their cDNA was synthesized from 1 μg of DNase-treated RNA. The results of RT-PCR showed the expected bands of the four key genes for synthesizing astaxanthin in transgenic rice seeds (Figure 4).
image
Figure 4   RT-PCR analysis of expression levels of PSY, CrtI, BKY, and BHY genes in several transgenic rice. Rice OsActin1 was as an internal control. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negative control (wild-type rice cultivar HG1).
These results of PCR and RT-PCR demonstrated that we successfully obtained transgenic rice with all of four stacking genes (PSY, CrtI, BHY and BKT).

 

In summary, we successfully obtained transformants with all of transgenes in lab and detected obvious transcriptional activity in endosperm. These results indicated that our designed pathway was realizable in rice endosperm. To further confirm astaxanthin production, several analysis were carried out. The results were shown in Demonstrate page.