Expression of atrial natriuretic factor in transgenic potatoes

Abstract

This thesis describes the expression of the precursor molecule of rat atrial natriuretic factor, ANF-(1-126), in transgenic potatoes (Solanum tuberosum L. cultivar Iwa). Four ANF gene fusions were constructed for extracellular expression of ANF-(1-126) either by retaining the native signal peptide of ANF or replacing it with the secretory signal of a pathogenesis-related protein from tobacco. Two of the constructs were targeted for constitutive expression by transcriptional fusion with the CaMV 35S promoter. The other two were driven by a 0.7-kb tuber-specific patatin promoter that was isolated by PCR amplification from potato cultivar Maris Piper. Enhancement of expression was attempted by fusing the 5' untranslated leader from the tobacco mosaic virus immediately downstream of the promoter but preceding the coding sequence. All chimaeric fusions were terminated by the 3' termination sequence from the transcript of gene 7 of the octopine-type Ti-plasmid, pTiA6. Two similar constructs were also made for constitutive or tuber-specific expression of cytosolic beta-glucuronidase (GUS) to confirm the functionality of the DNA regulatory sequences used.

All six chimaeric gene fusions were cloned into the binary vector, pGA643, or its derivative. These were then introduced into potato plants via leaf disc transformation using Agrobacterium tumefaciens strain LBA4404 carrying the disarmed helper Ti-plasmid, pAL4404. About 300-400 kanamycin-resistant plants were obtained per 100 leaf explants inoculated. Morphological abnormalities were observed among some regenerants but most of these were transient.

Seventy-seven kanamycin-resistant regenerants were analysed by Southern blotting of which 75% were found to contain detectable T-DNA sequences. Seventy-six percent of these were one or two copy transformants. Incomplete T-DNA transfer and rearrangements were observed among some transformants. The possible presence of endogenous ANF-homologues in potato genomic DNA was revealed by two weak hybridising bands consistently observed with both non-transformed and transformed potato plants.

Northern analysis confirmed that ANF and GUS mRNA were transcribed correctly and efficiently in potato with ANF transcripts comigrating with that from murine cardiac tissues. The activity of the 35S and patatin promoters was found to be appropriate with respect to tissue/organ specificity. Under the 35S promoter, ANF and GUS mRNA accumulated to much higher levels in leaves compared with tubers. Patatin driven expression was tuber specific but promoter activity was weaker than that of 35S. Both ANF and GUS mRNA levels generally coincided with the copy number status of the transformant. The possible presence of a partial homologue of mammalian ANF in potatoes was alluded to by the presence of weak hybridising sequences in potato RNA to rat ANF cDNA.

Histochemical analysis of GUS transformants showed strong GUS expression coinciding with the activity and tissue-specificity of the promoters used. Immunoblot experiments revealed a 16-kDa protein which may correspond to the product of transcripts from endogenous ANF-homologues deduced from Southern and Northern blot data. Western analysis of crude acid-extractable protein fractions from high ANF mRNA expressors failed to show the presence of any ANF-(1-126) in leaves or tubers. In vitro translation experiments indicated the possibility of poor translatability of ANF mRNA for failure in protein expression. This was further substantiated by analysis of codon usage which revealed incompatibilities between the co dons found in ANF and those preferred in potatoes. Reconstitution experiments with proANF from murine cardiac tissues added to ground untransformed tubers showed ANF degradation, indicating the instability of proANF in vitro and, possibly, in vivo.

It was proposed that translational or post-translational control be investigated to obtain ANF expression at the protein level. Redesign of the coding sequence of ANF to incorporate potato preferred codons without changing the primary amino acid sequence was suggested. Another option was to target proANF to an organellar compartment where it may be more stable. Targeting proANF to the chloroplast may pose certain problems with post-translational modification. Sequestration of proANF in the vacuoles or its retention in the endoplasmic reticulum was advocated.