Abstract
Gateway technology has been used to facilitate the generation of a large number of constructs for the modification of plants for research purposes. However, many of the currently available vectors only allow the integration of a single cDNA of interest into an expression clone. The ability to over-express multiple genes in combination is essential for the study of plant development where several transcripts have a role to play in one or more metabolic processes. The tools to carry out such studies are limited, and in many cases rely on the incorporation of cDNA into expression systems via conventional cloning, which can be both time consuming and laborious. To our knowledge, this study reports on the first development of a vector allowing the simultaneous integration of two independent cDNAs via a single LR-clonase reaction. This vector “pGEMINI” represents a powerful molecular tool offering the ability to study the role of multi-cDNA constructs on plant development, and opens up the process of gene stacking and the study of gene combinations through transient or stable transformation procedures.
Highlights
Gateway technology has become the cornerstone in the facilitation of the generation of a large number of constructs for the modification of plants for research purposes, and due to this, a wide array of gateway-adapted plant expression vectors have been developed over the last few years [1,2,3,4,5,6].Modern studies of metabolic processes, such as photosynthesis, focus on the modification of multiple enzymatic steps in a given pathway to modify metabolic flux or yields
The backbone of the pGEMINI vector was constructed in pGWB2 that had been previously generated from a modified pBI vector containing the hygromycin B phosphotransferase and the Neomycin phosphotransferase III genes [2] (AB289765); pGEMINI carries both hygromycin- and kanamycin-resistant cassettes. pGWB2 [2] (AB289764) was cut with HindIII (AAGCTT)
Position 1 is under the constitutive control of the Figwart Mosaic Virus (FMV) promoter and Position 2 is under the constitutive control of the 35S promoter followed by the nos 30 terminator. pGEMINI carries flanking resistance cassettes for kanamycin and hygromycin
Summary
Gateway technology has become the cornerstone in the facilitation of the generation of a large number of constructs for the modification of plants for research purposes, and due to this, a wide array of gateway-adapted plant expression vectors have been developed over the last few years [1,2,3,4,5,6].Modern studies of metabolic processes, such as photosynthesis, focus on the modification of multiple enzymatic steps in a given pathway to modify metabolic flux or yields. The initial goal of gene stacking has been accomplished in two ways: (1) Crossing lines carrying independent transgenes and the identification of plants carrying both transcripts of interest; and (2) Retransformation of plants already carrying a transgene. This can be accomplished using available vectors with different selection criteria (e.g., antibiotics). A third option has presented itself: Transformation of plants with two or more genes of interest integrated into a single construct This system has the advantage of allowing the integration of two or more genes through a unique transformation event.
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