Ribozyme termination of RNA transcripts down-regulate seed fatty acid genes in transgenic soybean

Tony Buhr, Shirley Sato, Farida Ebrahim, Aiqiu Xing, You Zhou, Michelle Mathiesen, Bruce Schweiger, Anthony Kinney, Paul Staswick, Tom Clemente

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137 Scopus citations

Abstract

We investigated whether termination of transcripts with a self-cleaving ribozyme can enhance nuclear retention and serve as a tool to decrease specific plant gene expression. Nuclear retention was first monitored in tobacco using the β-glucuronidase gene terminated with either the 35S CaMV 3′ untranslated sequence (UTR) or a cis-acting ribozyme. Northern blot analysis of nuclear RNA and total RNA, and in situ hybridizations showed that the ribozyme-terminated transcripts were preferentially retained in the nucleus of transgenic tobacco. Ribozyme-terminated transcripts were subsequently tested as a gene down-regulation strategy in soybean. The embryo-specific Δ-12 fatty acid desaturase FAD2-1 gene was targeted because its down-regulation elevates oleic acid content of seed storage lipids. Both ribozyme-terminated antisense and standard antisense constructs were capable of gene down-regulation, producing over 57% oleic acid compared with less than 18% in wild-type seed. Ribozyme termination cassettes were also constructed to evaluate sense transcripts for single gene down-regulation and the simultaneous down-regulation of two embryo-specific genes in soybean using a single promoter. Eight independent soybean transformants were screened that harboured standard plus sense or ribozyme terminated FAD2-1 cassette. Two of the eight ribozyme terminated transformants displayed oleic acids levels in the seed storage lipids of over 75%, while none of the standard plus sense FAD2-1 lines showed elevated oleic acid phenotypes. The dual constructs targeted FAD2-1 and the FatB gene encoding a palmitoyl-thioesterase. Five transgenic soybean lines harbouring the dual constructs had oleic acid levels, greater than 85%, and saturated fatty acids levels, less than 6%. Thus, ribozyme termination of transcripts can be utilized to specifically down-regulate endogenous gene expression in soybean.

Original languageEnglish (US)
Pages (from-to)155-163
Number of pages9
JournalPlant Journal
Volume30
Issue number2
DOIs
StatePublished - Jun 1 2002

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Keywords

  • Gene silencing
  • Glycine max
  • Nuclear retention
  • Transformation

ASJC Scopus subject areas

  • Genetics
  • Plant Science
  • Cell Biology

Cite this

Buhr, T., Sato, S., Ebrahim, F., Xing, A., Zhou, Y., Mathiesen, M., Schweiger, B., Kinney, A., Staswick, P., & Clemente, T. (2002). Ribozyme termination of RNA transcripts down-regulate seed fatty acid genes in transgenic soybean. Plant Journal, 30(2), 155-163. https://doi.org/10.1046/j.1365-313X.2002.01283.x