Difference between revisions of "Team:SCAU-China/Experiments"
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| − | <div class="p_font_size" style="margin-bottom:20px"><small> <font style="font-weight:bold">Figure 2.</font><font style="font-style:italic">Agrobacterium</font>-mediated rice calli transformation process.</small></div> | + | <div class="p_font_size" style="margin-bottom:20px"><small> <font style="font-weight:bold">Figure 2. </font><font style="font-style:italic">Agrobacterium</font>-mediated rice calli transformation process.</small></div> |
<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).--> | <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).--> | ||
Revision as of 11:35, 15 October 2016
Experiement
Vector construction
To synthesize astaxanthin and delete selective resistance 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.
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. You can read more details, please click here!
Figure 2. Agrobacterium-mediated rice calli transformation process.
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.
