Artificial gene-clusters engineered into plants using a vector system based on intron- and intein-encoded endonucleases

J. Michael Thomson, Peter R. Lafayette, Monica A. Schmidt, Wayne A. Parrott

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

The ability to create artificial gene-clusters for genetic transformation could facilitate the development of crops with multiple engineered traits, or with traits which result from the expression of multiple genes. A simple method to assemble artificial gene-clusters was developed by designing a multiple cloning site consisting of an array of homing endonuclease cleavage sites into a single vector. These enzymes are also known as intron- or intein-encoded endonucleases, and have very long recognition sequences, which makes them very rare cutters. The resulting vectors are pUGA for microprojectile-mediated transformation, and pUGA2 for Agrobacterium-mediated transformation. In addition, a series of unidirectional shuttle vectors containing various combinations of homing endonuclease restriction sites was constructed. Gene cassettes can be cloned into individual shuttles, and then transferred to either pUGA or pUGA2 to construct artificial gene-clusters. To test the feasibility of this approach, a six-gene cluster was constructed and transformed into soybean via microprojectile bombardment and into tobacco via Agrobacterium. The genes were assayed for expression in both the T0 and T1 generations for three independent transgenics. Up to five of the six genes were expressed. Additional changes to the construction of individual gene cassettes may improve the frequency with which all genes in the cluster are expressed.

Original languageEnglish (US)
Pages (from-to)537-542
Number of pages6
JournalIn Vitro Cellular and Developmental Biology - Plant
Volume38
Issue number6
DOIs
StatePublished - Nov 1 2002
Externally publishedYes

Keywords

  • Agrobacterium
  • Gene stacking
  • Metabolic engineering
  • Microprojectile bombardment
  • Soybean
  • Tobacco
  • Transformation

ASJC Scopus subject areas

  • Biotechnology
  • Plant Science

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