Strategies for the suppression of peroxidase gene expression in tobacco. II. In vivo suppression of peroxidase activity in transgenic tobacco using ribozyme and antisense constructs.
Several strategies involving the use of antisense and ribozyme constructs in different expression vectors were investigated as methods of suppressing gene expression in planta. We had previously identified an efficiently cleaving ribozyme (Rz), with two catalytic units and 60 nucleotide (nt) of complementary sequence, to the lignin-forming peroxidase of tobacco (TPX). This Rz was cloned behind the 35S CaMV (35S) and nopaline synthase (NOS) promoters, and into a vector utilising the tobacco tyrosine tRNA for expression. For comparison with more traditional antisense strategies, full-length TPX antisense (AS) constructs were also constructed behind the NOS and 35S promoters. Populations of transgenic tobacco containing these constructs were produced and compared to control plants transformed with the vector only. Significant suppression of peroxidase expression in the range of 40-80% was seen in the T0 and T1 populations carrying 35S-AS, 35S-Rz and tRNA-Rz constructs. Co-segregation of the suppressed peroxidase phenotype and the tRNA-Rz transgenes was demonstrated. Northern blot analysis indicated that levels of TPX mRNA were lower in the Rz plants. No evidence of mRNA cleavage was observed and thus it was unclear if the Rz constructs were acting as Rzs in vivo. Transgenic plants containing the tRNA-Rz construct had significantly lower levels of peroxidase than the other transgenic plants. There was no significant difference in levels of suppression of TPX between the short Rz in the 35S vector and the full-length AS constructs. Although peroxidase levels were significantly reduced in transgenic plants carrying 35S-AS, 35S-Rz and tRNA-Rz constructs, no significant difference in lignin levels was observed.
McIntyre
C L
CL
CSIRO Division of Tropical Crops and Pastures, St. Lucia, Australia.
Bettenay
H M
HM
Manners
J M
JM
D016960:Agrobacterium tumefaciens; D000594:Amino Acid Oxidoreductases / Q000235:genetics; D001483:Base Sequence; D005822:Genetic Vectors; D007613:Kanamycin Resistance / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010544:Peroxidases / Q000096:biosynthesis* / Q000235:genetics; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D016372:RNA, Antisense; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D012365:RNA, Transfer, Tyr / Q000096:biosynthesis; D015183:Restriction Mapping; D013489:Suppression, Genetic; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D016372:RNA, Antisense; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012365:RNA, Transfer, Tyr; D010544:Peroxidases; C042858:lignin peroxidase; C101317:lignin-forming peroxidase; D000594:Amino Acid Oxidoreductases; C021883:nopaline synthase