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

<div class="h2_font_size">Vector construction</div>

<div class="p_font_size">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.</div>

<div class="p_font_size" style="margin-bottom:20px"><small>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 Isites. (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>

<div class="h2_font_size">Rice transformation</div>

<div class="p_font_size">The multigene construct pYLTAC380MF-BBPC was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation. Then the Agrobacterium cells were cocultured 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, <a href="https://2016.igem.org/Team:SCAU-China/Protocol#rice_transformation" text-decoration=underline>please click here!</a></div><!--(加链接到Notebook栏目下面的3.protocol的2. rice transformation)-->

<div class="p_font_size" style="margin-bottom:20px"><small>Figure 2 Agrobacterium-mediated rice calli transformation process.</small></div>