Cloning and Expression of Dclcyb1 Gene

For Dclcyb1 expression, the complete Dclcyb1 coding sequence (DQ192190) was cloned into pCR®8/GW/TOPO (Invitrogen) using DcLcybF and DcLcybR (Supplementary Table S1 available at JXB online) and following the manufacturer’s instructions. Positive clones obtained by enzymatic digestion were sequenced by Macrogen Corp. (USA). Subsequently, pCR8/lcyb1 was recombined into pGWB2® (Curtis and Grossniklaus, 2003 (link)) to produce the pGWB2-Dclcyb1 expression vector (Moreno et al., 2013 (link)). Positive clones were analyzed through PCR and enzymatic digestion, and transformed into Agrobacterium tumefaciens (GV3101 strain).

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Moreno J.C., Cerda A., Simpson K., Lopez-Diaz I., Carrera E., Handford M, & Stange C. (2016). Increased Nicotiana tabacum fitness through positive regulation of carotenoid, gibberellin and chlorophyll pathways promoted by Daucus carota lycopene β-cyclase (Dclcyb1) expression. Journal of Experimental Botany, 67(8), 2325-2338.

Publication 2016

pCR®8/GW/TOPO

Agrobacterium tumefaciens Clones Coding sequence Digestion Enzymatic Strain Topo Vector

Corresponding Organization : University of Chile

Other organizations : Max Planck Institute of Molecular Plant Physiology, Instituto de Biología Molecular y Celular de Plantas, Universitat Politècnica de València

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Variable analysis

independent variables

Recombination of pCR8/lcyb1 into pGWB2® to produce the pGWB2-Dclcyb1 expression vector

dependent variables

Expression of Dclcyb1

control variables

Positive clones obtained by enzymatic digestion were sequenced by Macrogen Corp. (USA)
Positive clones were analyzed through PCR and enzymatic digestion
Transformed into Agrobacterium tumefaciens (GV3101 strain)

controls

Positive controls: Positive clones obtained by enzymatic digestion were sequenced
Negative controls: Not explicitly mentioned

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