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Plant Molecular Biology

, Volume 65, Issue 3, pp 329–341 | Cite as

A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants

  • Zhongsen LiEmail author
  • Aiqiu Xing
  • Bryan P. Moon
  • Susan A. Burgoyne
  • Anthony D. Guida
  • Huiling Liang
  • Catharina Lee
  • Cheryl S. Caster
  • Joanne E. Barton
  • Theodore M. Klein
  • Saverio C. Falco
Article

Abstract

Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either β-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.

Keywords

Cre/loxP recombination system Self-activating gene excision Marker gene-free transgenic soybean 

Abbreviations

35S

Cauliflower mosaic virus 35S promoter

Als

Acetolactate synthase

app1

An Arabidopsis embryo-specific promoter

Cre

Cre recombinase

Gat

Glyphosate acetyltransferase

Gus

β-Glucuronidase

Hpt

Hygromycin β-phosphotransferase

loxP

Cre recombinase recognition site of phage P1

nos3

Nopaline synthase transcription terminator

pinii

Potato proteinase inhibitor II protein transcription terminator

scp1

A synthetic plant constitutive promoter

T0 plants

The first generation of transgenic soybean plants regenerated from tissue culture

Notes

Acknowledgments

We are grateful to Dr. Lucy Liu from Pioneer Hi-Bred Int’l, Johnston, Iowa, USA for providing the anti-Gat antibody serum and E. coli expressed Gat protein; to Dr. Barbara J. Mazur from DuPont Agriculture & Nutrition, Wilmington, Delaware, USA for reviewing the manuscript.

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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Zhongsen Li
    • 1
    Email author
  • Aiqiu Xing
    • 1
  • Bryan P. Moon
    • 1
  • Susan A. Burgoyne
    • 1
  • Anthony D. Guida
    • 1
  • Huiling Liang
    • 1
  • Catharina Lee
    • 1
  • Cheryl S. Caster
    • 1
  • Joanne E. Barton
    • 1
  • Theodore M. Klein
    • 1
  • Saverio C. Falco
    • 1
  1. 1.DuPont Agriculture & Nutrition, Experimental StationWilmingtonUSA

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