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

Summary 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.