Fruit developmental regulation of the kiwifruit actinidin promoter is conserved in transgenic petunia plants

Abstract

We have examined the expression of actinidin, a cysteine protease found in kiwifruit, over the course of fruit development. Protease activity was first seen in fruit that had reached about half their final weight, and rose to high levels at harvest. The 5'-flanking region (nucleotides -1301 to +58) of a kiwifruit actinidin gene was fused to the beta-glucuronidase (GUS)-coding region, and the chimaeric gene was introduced into transgenic petunia plants. Induction of the GUS gene was observed during the later stages of seed pod development, closely resembling the pattern of actinidin induction in fruit tissues of kiwifruit. Some GUS expression was also detected in the vascular system of the receptacle, leaves, stems and roots. A shorter promoter fragment consisting of nucleotides -115 to +58 conferred similar spatial and temporal regulation in some of the transgenic plants.

Differential Constitution in Promoter Region Leads to a Phenotype with a Lower Allergic Actinidin Level in Yellow-fleshed Kiwifruit (<i>Actinidia chinensis</i>)

“…Therefore, it was more likely that the causal factor for Act1a suppression resides in the upstream promoter region. The Act1a promoter in 'Hayward' has been previously reported (Lin et al, 1993;Snowden and Gardner, 1990). Using primers based on the 'Hayward' sequence, the corresponding regions of 'Kohi' were tested by amplification (Fig.…”

“…. Two sections of the Act1a upstream region, designated (I) and (II), were amplified with genomic PCR using primers based on the sequence of 'Hayward' previously reported (L07552, Lin et al, 1993). As controls to ensure DNA quality, UbiC9 and EF1a were amplified.…”

“…Although this region did not show similarity to any annotated sequence, it was predicted that DNA deletion or insertion occurred in this region during evolution or breeding processes. Analysis of the 'Hayward' Act1a promoter using transgenic petunia plants suggested that a 1.3 kb promoter is necessary for the high level expression of Act1a while a shorter 173 b region (proximal to Act1a gene) appears to be involved in the spatial and temporal regulation (Lin et al, 1993). Thus, the sequence variation found in 'Kohi' could disrupt the original Act1a promoter functionality for strong gene expression.…”

“…To our knowledge, a TF for Act1a transcription has not yet been identified. Given that actinidin gradually accumulates as fruit mature in 'Hayward' (Lewis and Luh, 1988;Lin et al, 1993), the unknown TF would be induced as fruit develops and bind to a cis-element in the Act1a promoter to induce gene expression. Our results suggest that such TF binding is disrupted by the modified promoter sequence in 'Kohi', possibly via epigenetic regulation involving DNA methylation.…”

Applications of Biotechnology in Kiwifruit (Actinidia)

“…Another approach to gain insights into the regulation of a gene's expression has been the analysis of promoter-reporter gene fusions in transgenic systems. Lin et al (1993) fused an upstream region of an actinidin coding region to the -glucuronidase (GUS) coding region and observed GUS expression during the later stages of transgenic petunia seed pod development, resembling the induction of actinidin in Actinidia fruit tissues. Similar promoter-GUS fusions were used to analyse an A. chinensis polygalacturonase promoter, and at the breaker stage of transgenic tomato fruit development, GUS expression was observed throughout the inner and outer pericarp, the columella and seeds, and became restricted to the inner pericarp and seeds at the later stages of ripening (Z.Y.…”