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

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Abstract

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.

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Abbreviations

AA:

Arachidonic acid

ALA:

α-Linolenic acid

DHA:

Docosahexaenoic acid

D8:

Δ8 Desaturase gene

D5:

Δ5 Desaturase gene

D15:

Δ15 Desaturase gene

D17:

Δ17 Desaturase gene

E9:

Δ9 Elongase gene

EDA:

Eicosadienoic acid

EPA:

Eicosapentaenoic acid

ETA:

Eicosatetraenoic acid

ETrA:

Eicosatrienoic acid

GFP:

Green fluorescent protein

GUS:

β-Glucuronidase gene

LA:

Linoleic acid

MARs:

Matrix attachment regions

MCS:

Multiple cloning site

MGT:

Multiple gene transfer

MISSA:

Multiple-round in vivo site-specific assembly

MS:

Murashige and Skoog

PEC:

Plant expression cassette

tNOS:

Nos terminator

VLCPUFAs:

Very long chain polyunsaturated fatty acids

ZFNs:

Zinc finger nucleases

35S:

CaMV 35S promoter

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Acknowledgments

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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Authors and Affiliations

  1. State Key Laboratory of Crop Biology, Shandong Agricultural University, Taian, 271000, China

    Quanxi Sun, Jiang Liu, Yaxiao Li, Qin Zhang, Xinzheng Li & Baoxiu Qi

  2. Shandong Peanut Research Institute, Qingdao, 266100, China

    Quanxi Sun & Shihua Shan

Authors
  1. Quanxi Sun
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  2. Jiang Liu
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  3. Yaxiao Li
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  4. Qin Zhang
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  5. Shihua Shan
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  6. Xinzheng Li
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  7. Baoxiu Qi
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Corresponding authors

Correspondence to Xinzheng Li or Baoxiu Qi.

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Quanxi Sun and Jiang Liu contributed equally to this work.

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Sun, Q., Liu, J., Li, Y. et al. Creation and validation of a widely applicable multiple gene transfer vector system for stable transformation in plant. Plant Mol Biol 83, 391–404 (2013). https://doi.org/10.1007/s11103-013-0096-2

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  • DOI: https://doi.org/10.1007/s11103-013-0096-2

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