Ribozyme termination of RNA transcripts down-regulate seed fatty acid genes in transgenic soybean.
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 beta-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 Delta-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.
Buhr
Tony
T
Department of Agronomy, Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, NE, USA.
Sato
Shirley
S
Ebrahim
Farida
F
Xing
Aiqiu
A
Zhou
You
Y
Mathiesen
Michelle
M
Schweiger
Bruce
B
Kinney
Anthony
A
Staswick
Paul
P
D020413:3' Untranslated Regions / Q000235:genetics; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000235:genetics; D015536:Down-Regulation; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D017343:Genes, Plant / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D017403:In Situ Hybridization; D018613:Microscopy, Confocal; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D013025:Glycine max / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014026:Nicotiana / Q000235:genetics; D014158:Transcription, Genetic
D020413:3' Untranslated Regions; D005227:Fatty Acids; D016337:RNA, Catalytic; D012333:RNA, Messenger; D018749:RNA, Plant; D034741:RNA, Small Interfering; D022661:RNA, Untranslated; D044943:Fatty Acid Desaturases; C040448:delta-12 fatty acid desaturase; D005966:Glucuronidase