Colin R. Bird

Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes

Regulation of expression of specific genes by antisense RNA is a naturally occurring mechanism in bacteria1,2, although gene regulation by this mechanism has not yet been observed in higher eukaryotes. However, antisense RNA has been shown to reduce expression of specific genes when injected into frog oocytes3 and Drosophila embryos4. Inhibition of expression of artificially introduced genes has been demonstrated by transient expression of antisense RNA constructs in mammalian cells5,6, and plant protoplasts7, and by stable expression in transgenic plants8. Here, we report a striking inhibition of expression of the endogenous, developmentally regulated gene for polygalacturonase in stably transformed tomato expressing antisense RNA.

Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants.

SummaryTomato plants were transformed with a chimaeric polygalacturonase (PG) gene, designed to produce a truncated PG transcript constitutively. In these plants expression of the endogenous PG gene was inhibited during ripening, resulting in a substantial reduction in PG mRNA and enzyme accumulation. This inhibition was comparable to that achieved previously using antisense genes. The expression of the truncated gene in ripe fruit was substantially lower than its expression in green fruit. Thus expression of both the endogenous and truncated genes is reduced in ripe fruit in which both are active. The implication of this observation is discussed in relation to the possible mechanism whereby sense constructs inhibit gene expression.

The tomato polygalacturonase gene and ripening-specific expression in transgenic plants.

Polygalacturonase (PG) is the major cell wall degrading enzyme of tomato fruit. It is developmentally regulated and is synthesised de novo in ripening fruit. Genomic clones encoding a PG gene of tomato (Lycopersicon esculentum Mill cv. Ailsa Craig) have been isolated, mapped and sequenced. The sequence of the protein-coding region is identical to that of a PG cDNA [20]. Comparison of the cloned restriction fragments with genomic Southern data suggests that there may only be one gene for PG per haploid genome. The PG gene, which covers approximately 7 kb, is interrupted by 8 intervening sequences ranging in size from 99 bp to 953 bp. The transcription start point was identified by S1 mapping and primer extension analysis. About 1.4 kb of 5′ flanking DNA has been sequenced. This contains putative TATA and CAAT boxes and also direct repeat sequences. A transcriptional fusion has been constructed between the putative 1.4 kb promoter fragment and the chloramphenicol acetyl transferase (CAT) gene. Constructs containing this gene have been transferred to tomato using binary vectors. Regenerated transgenic plants express CAT in ripe tomato fruit, but not in unripe tomatoes, leaves, or roots.