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

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<div class="h1_font_size">Protocol</div>

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

<div class="p_font_size" style="text-indent:0em">To construct the multigene vector for astaxanthin biosynthesis, we used a modified multigene vector system, TransGene Stacking II (TGSII) (Zhu et al., unpublished). This novel system is based on our previous studies on the transformation-competent artificial chromosome (TAC) for the larger DNA fragments transformation (Liu et al., PNAS, 1999, 96: 6535-6540) and the early version of the multigene assembly and transformation vector system (Lin et al., PNAS, 2003, 100: 5962-5967). This system consists of a transformation-competent artificial chromosome (TAC)-based binary acceptor vector (pYLTAC380GW), together with two donor vectors (pYL322-d1/ pYL322-d2). By using the Cre/loxP recombination system and two pairs of mutant loxP sites, multiple rounds of gene assembly cycles were carried out with alternative use of the donor vectors, and multiple genes were sequentially delivered into the TAC vector (Zhu et al., unpublished).</div>

<div class="p_font_size">To sequentially assemble them, the four endosperm-specific promoters drove four genes (CrtI, PSY, BKT and BHY) expression cassettes were constructed into two donor vectors, respectively. And these obtained donors, (I) d1-CrtI, (II) d2-PSY, (Ⅲ) d1-BKT and (Ⅳ) d2-BHY, were sequentially assembled into the 380GW acceptor by four rounds of gene assembly cycles, according to I\II\Ⅲ\Ⅳorder. Finally, after completing four-gene assembly, the marker-free element, containing a HPT selectable resistance gene expression cassette and a Cre-induced gene expression cassette by an anther-specific promoter, was integrated into the four-gene acceptor by Gateway BP reaction. The obtained six-gene marker-free multigene vector, named 380MF-BBPC, was transferred into Agrobacterium tumefaciens strain EHA105 for further rice callus transformation. </div>

<div class="p_font_size"><font style="font-weight:bold">Round I:</font> Cre/loxP reversible recombination of the d1-CrtI plasmid into the TAC-based acceptor plasmid pYLTAC380GW (i), followed by release of the d1 backbone by Cre-mediated irreversible recombination between one pair of mutant loxP sites (ii).The first CrtI gene expression cassette was assembled into the acceptor vector to form <font style="font-weight:bold">380GW-C</font>. </div>

 

<table class="table">

<th>Component</th>

<th>Dosage</th>

<td>10×N6 macroelement solution</td>

<td>50 mL</td>

<td>1000×B5 microelement solution</td>

<td>0.5 mL</td>

<tr>

<td>100×B5 vitamin solution</td>

<td>5 mL</td>

<tr>

<td>100×Ferric salt solution</td>

<td>5 mL</td>

<td>0.5 mg/mL 2,4-D</td>

<td colspan="2">Add distilled water until the medium volume reach 500 mL</td>

</tr>

<td colspan="2">Blend with magnetic stirrer</td>

<tr>

<td colspan="2">Adjust medium pH to 5.8 with 1 mol/L NaOH</td>

<tr>

<td>Phytagel</td>

<td>1.5 g</td>

</div>                                          

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