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

Research output: Contribution to journalArticle

132 Citations (Scopus)

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

Fingerprint

Catalytic RNA
messenger RNA
Soybeans
Seeds
Fatty Acids
Down-Regulation
genetically modified organisms
RNA
soybeans
fatty acids
oleic acid
seeds
Oleic Acid
Genes
genes
seed storage
embryo (plant)
tobacco
Tobacco
gene expression

Keywords

  • Gene silencing
  • Glycine max
  • Nuclear retention
  • Transformation

ASJC Scopus subject areas

  • Genetics
  • Plant Science
  • Cell Biology

Cite this

Ribozyme termination of RNA transcripts down-regulate seed fatty acid genes in transgenic soybean. / Buhr, Tony; Sato, Shirley; Ebrahim, Farida; Xing, Aiqiu; Zhou, You; Mathiesen, Michelle; Schweiger, Bruce; Kinney, Anthony; Staswick, Paul; Clemente, Tom.

In: Plant Journal, Vol. 30, No. 2, 01.06.2002, p. 155-163.

Research output: Contribution to journalArticle

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, vol. 30, no. 2, pp. 155-163. https://doi.org/10.1046/j.1365-313X.2002.01283.x
Buhr, Tony ; Sato, Shirley ; Ebrahim, Farida ; Xing, Aiqiu ; Zhou, You ; Mathiesen, Michelle ; Schweiger, Bruce ; Kinney, Anthony ; Staswick, Paul ; Clemente, Tom. / Ribozyme termination of RNA transcripts down-regulate seed fatty acid genes in transgenic soybean. In: Plant Journal. 2002 ; Vol. 30, No. 2. pp. 155-163.
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AU - Buhr, Tony

AU - Sato, Shirley

AU - Ebrahim, Farida

AU - Xing, Aiqiu

AU - Zhou, You

AU - Mathiesen, Michelle

AU - Schweiger, Bruce

AU - Kinney, Anthony

AU - Staswick, Paul

AU - Clemente, Tom

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