Plant Molecular Biology

, Volume 83, Issue 4–5, pp 391–404 | Cite as

Creation and validation of a widely applicable multiple gene transfer vector system for stable transformation in plant



Multiple gene transfer (MGT) technology has become a powerful tool for basic and applied plant biology research in recent years. Despite some notable successes in obtaining plant lines harbouring multiple transgenes, these methods are still generally unwieldy and costly. We report here a straightforward and cost effective strategy, utilizing commonly available restriction enzymes for the transfer of multiple genes into plants, hence greatly widening the accessibility of MGT. This methodology exploits the specific ‘nested’ arrangement of a pair of isocaudomer restriction enzymes (for example XbaI—AvrII–XbaI) so that through the alternate use of these two enzymes in a reiterative fashion multiple genes/constructs (up to five in this study) could be ‘stacked’ together with ease. In a proof-of-concept experiment, we constructed a plant transformation vector containing three reporter gene expression cassettes flanked by two matrix attachment region sequences. The expression of all three genes was confirmed in transgenic Arabidopsis thaliana. The usefulness of this technology was further validated by the construction of a plant transformation vector containing five transgenes for the production of eicosapentaenoic acid (EPA, C20∆5,8,11,14,17), a polyunsaturated essential fatty acid found in fish oils that is beneficial for health. In addition, we constructed four more vectors, incorporating one seed specific and three promoters conferring constitutive expression. These expression cassettes are flanked by a different isocaudomer pair (AvrII—SpeI–AvrII) and four other unique restriction sites, allowing the exchange of promoters and terminators of choice.


Multiple gene transfer Isocaudomer Transgenic plant EPA 



Arachidonic acid


α-Linolenic acid


Docosahexaenoic acid


Δ8 Desaturase gene


Δ5 Desaturase gene


Δ15 Desaturase gene


Δ17 Desaturase gene


Δ9 Elongase gene


Eicosadienoic acid


Eicosapentaenoic acid


Eicosatetraenoic acid


Eicosatrienoic acid


Green fluorescent protein


β-Glucuronidase gene


Linoleic acid


Matrix attachment regions


Multiple cloning site


Multiple gene transfer


Multiple-round in vivo site-specific assembly


Murashige and Skoog


Plant expression cassette


Nos terminator


Very long chain polyunsaturated fatty acids


Zinc finger nucleases


CaMV 35S promoter



This work was supported by the National Natural Science Foundation of China (Grant No. 30970222) and genetically modified organisms breeding major projects of China (Grant No. 009ZX08005-024B).

Conflict of interest

The authors declare competing interest for the pXAX vectors which are subjected to the patent law of China (Patent No. ZL200810160116.6). pXAX1GW and pXAX2GW are also subjected to the GATEWAY® patent law. However, all the vectors could be provided to the scientific community for research purposes only.


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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.State Key Laboratory of Crop BiologyShandong Agricultural UniversityTaianChina
  2. 2.Shandong Peanut Research InstituteQingdaoChina

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