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Molecular Breeding

, Volume 19, Issue 3, pp 241–253 | Cite as

Cloning of new rubisco promoters from Brassica rapa and determination of their activity in stably transformed Brassica napus and Nicotiana tabacum plants

  • Andrey Anisimov
  • Kimmo Koivu
  • Anne Kanerva
  • Seppo Kaijalainen
  • Kari Juntunen
  • Viktor Kuvshinov
Original Paper

Abstract

The aim of our study was to identify the highest expressing rubisco small subunit (RbcS) promoters (pRbcS) from the cotyledons of germinating seedlings of Brassica rapa var. oleifera to drive high-level and preferably stage-specific transgenic protein expression in Brassicaceae plants. We cloned four new pRbcS promoters using several approaches, including the construction of a cDNA library and use of genome walking technique. Real-time PCR analysis of RbcS mRNA expression clearly showed that two of these promoters exhibited the highest activity on the germination stage of plant development. We used gusA expression as a reporter of promoter activity in Brassica napus and Nicotiana tabacum plants that were transformed with the constructs using an Agrobacterium-mediated transformation strategy. The mRNA level of RbcS and of gusA was quantified in transformed plants. The data obtained demonstrate that the promoter most active in seedlings under native conditions was also most active in transgenic constructs at the same stage of plant development. The fine structure of the promoters is discussed herein.

Keywords

Brassica species and Nicotiana tabacum transformations Rubisco promoters Rubisco small subunit 

Abbreviations

GUS

β-d-glucuronidase, encoded by the gusA gene

pRbcS

Promoter of RbcS gene

RbcS

Rubisco small subunit

Notes

Acknowledgements

We are grateful to Dr. Nick Domansky and Ghebre Sebahtu for carrying out the Northern analysis, and Tatiana Shavkera and Svetlana Kuvshinova for their assistance in the transformation and in vitro culture work. We also thank Maritta Timonen and Dr. Eric Murphy for their generous help in preparation of the manuscript and two anonymous reviewers for their helpful comments. This Work was funded in part by the National Technology Agency of Finland (TEKES).

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Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Andrey Anisimov
    • 1
  • Kimmo Koivu
    • 1
  • Anne Kanerva
    • 1
  • Seppo Kaijalainen
    • 1
  • Kari Juntunen
    • 2
  • Viktor Kuvshinov
    • 1
  1. 1.Helsinki Business & Science ParkUniCrop Ltd.HelsinkiFinland
  2. 2.Department of Biosciences, Division of BiochemistryUniversity of HelsinkiHelsinkiFinland

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