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

 
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<div class="h1_font_size">Proof</div>
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<div class="h1_font_size">Experiments</div>
  
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<div class="h2_font_size">Analysis of transgenes</div>
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<div class="h2_font_size"><font style="font-weight:bold">Vector construction</font></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" >To synthesize astaxanthin and delete antibiotic resistance selection marker gene, six gene expression cassettes were assembled into a TAC-based binary acceptor vector, designated as pYLTAC380MF-BBPC (Figure 1), by multiple rounds of gene assembly cycles using our marker-free TransGene Stacking II system, in which contained four genes (<font style="font-style:italic">CrtI, PSY, BKT </font>and <font style="font-style:italic">BHY</font>) under the control of 4 different endosperm-specific promoters for astaxanthin biosynthesis, and two genes (<font style="font-style:italic">HPT</font> and <font style="font-style:italic">Cre</font>) for marker-free deletion.</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 1</font> &nbsp;&nbsp;Structure and restriction analysis of multigene recombinant constructs. Structure and restriction analysis of recombinant constructs. (A) Structure of a construct pYLTAC380MF-BBPC containing four genes for astaxanthin biosynthesis and two genes for marker free. The numbers in parentheses indicate the order of the sequences inserting into the vector. N denotes Not I sites. (B) Restriction analysis of a series of multigene constructs containing different numbers of genes (from lanes 3 to 6). Arrows indicate the different insertion genes.</small></div>
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<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="h2_font_size"><font style="font-weight:bold">Rice transformation</font></div>
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<div class="p_font_size">The multigene construct pYLTAC380MF-BBPC was introduced into <font style="font-style:italic">Agrobacterium tumefaciens</font> strain <font style="font-style:italic">EHA105</font> by electroporation. Then the <font style="font-style:italic">Agrobacterium</font> cells were co-cultured with embryogenic calli induced from mature seeds of <font style="font-style:italic">Indica</font> rice varieties Huaguang1 (HG1) as described (Lin et al., PNAS, 2003, 100: 5962-5967). Regenerating calli were selected in the presence of 50 mg/L hygromycin and subsequently transferred to rooting medium containing hygromycin. After further culturing for 3 to 4 weeks, transformed plants were transferred to soil in a greenhouse. Figure 2 showed the <font style="font-style:italic">Agrobacterium</font>-mediated transformation process and transgenic rice cultivation. If you want to read more details, you can click here to see our <a href="https://2016.igem.org/Team:SCAU-China/Protocol#rice_transformation" text-decoration=underline>protocol</a> and <a href="https://2016.igem.org/Team:SCAU-China/Notebook" text-decoration=underline>notebook!</a></div><!--(加链接到Notebook栏目下面的3.protocol的2. rice transformation)-->
 
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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 2</font> &nbsp;&nbsp;The proccess of <font style="font-style:italic">Agrobacterium</font>-mediated transformation in rice.</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">To confirm the realization of our designed pathway, PCR amplification and semi-quantitative RT-RCR analysis were performed. The experimental results were presented in our <a href="https://2016.igem.org/Team:SCAU-China/Proof" text-decoration=underline>Proof page.</a></div><!--(超链接到 Proof).-->
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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 analyses 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 05:31, 18 October 2016

SCAU

Experiments
Vector construction
To synthesize astaxanthin and delete antibiotic resistance selection marker gene, six gene expression cassettes were assembled into a TAC-based binary acceptor vector, designated as pYLTAC380MF-BBPC (Figure 1), by multiple rounds of gene assembly cycles using our marker-free TransGene Stacking II system, in which contained four genes (CrtI, PSY, BKT and BHY) under the control of 4 different endosperm-specific promoters for astaxanthin biosynthesis, and two genes (HPT and Cre) for marker-free deletion.
image
Figure 1   Structure and restriction analysis of multigene recombinant constructs. Structure and restriction analysis of recombinant constructs. (A) Structure of a construct pYLTAC380MF-BBPC containing four genes for astaxanthin biosynthesis and two genes for marker free. The numbers in parentheses indicate the order of the sequences inserting into the vector. N denotes Not I sites. (B) Restriction analysis of a series of multigene constructs containing different numbers of genes (from lanes 3 to 6). Arrows indicate the different insertion genes.
Rice transformation
The multigene construct pYLTAC380MF-BBPC was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation. Then the Agrobacterium cells were co-cultured with embryogenic calli induced from mature seeds of Indica rice varieties Huaguang1 (HG1) as described (Lin et al., PNAS, 2003, 100: 5962-5967). Regenerating calli were selected in the presence of 50 mg/L hygromycin and subsequently transferred to rooting medium containing hygromycin. After further culturing for 3 to 4 weeks, transformed plants were transferred to soil in a greenhouse. Figure 2 showed the Agrobacterium-mediated transformation process and transgenic rice cultivation. If you want to read more details, you can click here to see our protocol and notebook!
image
Figure 2   The proccess of Agrobacterium-mediated transformation in rice.
To confirm the realization of our designed pathway, PCR amplification and semi-quantitative RT-RCR analysis were performed. The experimental results were presented in our Proof page.