Doctoral Degrees (Genetics)

Permanent URI for this collection

https://scholar.sun.ac.za/handle/10019.1/551

Browse

Browsing Doctoral Degrees (Genetics) by Author "Burger, Anita L."

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    The isolation and characterisation of a developmentally-regulated gene from Vitis vinifera L. berries
    (Stellenbosch : University of Stellenbosch, 2004-12) Burger, Anita L.; Botha, F. C.; University of Stellenbosch. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.
    ENGLISH ABSTRACT: Despite increased focus on ripening-related gene transcription in grapevine, and the large number of ripening-related cDNAs identified from grapes in recent years, the molecular basis of processes involved in grape berry ripening is still poorly understood. Moreover, little is known about the mechanisms involved in the ripening-related regulation of fruit-specific genes, since the isolation and characterisation of no ripening-related, fruit-specific promoter elements has been reported to date. This study was aimed at the isolation and characterisation of a fruit-specific, ripeningregulated gene from Vitis vinifera L. In the first phase of the work, gene transcription in ripening berries of Cabernet Sauvignon (a good quality wine cultivar) and Clairette blanche (a poor quality wine cultivar) were studied by Amplified Fragment Length Polymorphism analysis of complementary DNA (cDNA-AFLP analysis). Total RNA from immature (14-weeks post flowering, wpf) and mature (18-wpf) berries was used for the analysis. A total of 1 276 cDNA fragments were visualised, of which 175 appeared to be ripening related. Average pairwise difference of the fragments amplified from immature and mature Clairette and Cabernet berries, suggested that ripening-related gene transcription in these two phenotypically different cultivars is remarkably similar. Nevertheless, it was shown that seventy percent of the 175 ripening-related cDNA fragments were cultivar-specific. It was suggested that these differences should be targeted to identify genes related to the phenotypical differences between the two cultivars, but also to identify genes possibly involved berry quality. Moreover, the analysis illustrated the usefulness of cDNA-AFLPs for the analysis of ripening-related gene transcription during grape berry ripening. In the second phase of the work, one of the ripening-related cDNAs identified by the cDNA-AFLP analysis, was selected for further characterisation. This work highlighted the limitation placed on the isolation of a single specific sequence from a cDNA-AFLP gel, indicating the presence of multiple ripening-related genes in a single band excised from a cDNA-AFLP gel. Steps to overcome this limitation of cDNA-AFLP analysis to identify and clone a specific ripening-related gene, were implemented. In short, the band corresponding to the particular ripening-related cDNA was band was excised from the cDNA-AFLP polyacrylamide gel and re-amplified. Northern blot analysis using the re-amplified, uncloned product confirmed the ripening-related transcription demonstrated by cDNA-AFLP analysis. The re-amplified, uncloned product was then cloned. Sequence analysis of two randomly selected candidate clones revealed two distinctly different sequences, of which neither hybridised to messenger RNA from ripening grape berries. Furtheranalysis revealed an additional five cDNAs with terminal sequences corresponding to the selective nucleotides of the primers used for selective amplification, in the re-amplified, uncloned product. Of these, only two were abundantly expressed in ripening grape berries, accounting for the ripeningrelated transcription visualised by cDNA-AFLP analysis. All seven cDNAs identified from the particular excised band were shown to be ripening-regulated during berry development, although most were characterised by low levels of transcription during berry ripening. One of the clones, based on the relative high levels of the transcript and the initiation of gene transcription at the onset of véraison (10- to 12-wpf), was identified for isolation and characterisation of the full length coding sequence. In the third phase of the work, it was shown that this cloned sequence corresponded to a gene encoding a proline-rich protein (PRP) associated with ripening in Merlot and Chardonnay (mrip1, Merlot ripening-induced protein 1). It was shown that the gene is specifically transcribed in the fruit tissue, seed and bunchstems of grapes, from 10-wpf (véraison) to the final stages of berry ripening. The results showed that mrip1 encodes a distinct member of the plant PRP family. Most obvious is the central region of mrip1, which is comprised of eight consecutive repeats of 19 amino acid residues each. In comparison with other grapevine PRPs, mrip1 revealed single amino acid differences and deletion of one of the 19 amino acid residues repeats, all in the central region of mrip1. In situ hybridisation studies showed that accumulation of the mrip1 transcript in the ripening berry is limited to the mesocarp and exocarp cells of the ripening grape berry. No transcript with high sequences similarity to mrip1 could be detected in ripening strawberry or tomato fruit. Based on the properties and proposed function of PRPs, and the results obtained in this study, potential applications for the use of this gene in the control of cell wall architecture in fruits, were proposed. Furthermore, as manipulation of fruit properties in grape berries would be most important in the later stages of ripening, mrip1 was proposed an ideal candidate gene for the isolation of a fruit- and late-ripening-specific promoter to achieve transgene transcription in genetically modified grapevine. The final phase of the work was dedicated to the isolation and characterisation of the mrip1 promoter element. A 5.5 kb sequence corresponding to the mrip1 5’ untranslated (UTR) flanking region was isolated and characterised by sequence analysis. In the 2.8 kb sequence directly upstream of the mrip1 transcription initiation site, several putative cis-acting regulatory elements were identified. These include a spectrum of hormone-, light-, phytochrome-, sugar-and stressresponsive elements, as well as elements implicated in tissue-specific transcription. Analysis of the sequence further upstream (3.6 – 5.5 kb) of the mrip1 transcription initiation site (TIS), revealed the presence of another proline-rich protein directly upstream of mrip1. Sequence identity of this sequence (mprp2) to the mrip1 coding sequence was 88%. This information provided the first insight into the chromosomal organisation of grapevine PRPs. For functional analysis of the mrip1 promoter element, the 2.2 kb sequence directly upstream of the mrip1 TIS, was translationally fused to the sgfpS65T reporter gene. Functionality of the mrip1:sgfpS65T fusion was verified by transient expression in green pepper pericarp tissue, before introduction into tobacco by Agrobacteriummediated transformation. In transgenic tobacco, transcription of the mrip1:sgfpS65T fusion was developmentally-regulated and specific to the ovary and nectary-tissue of the developing flower. Whilst low in immature flowers, the green fluorescent protein (GFP) rapidly accumulated to the high level of expression visualised in the flower in full-bloom, followed by a decrease in the final stages of ovary development. These observations suggested that the 2.2 kb mrip1 promoter is functional and that this promoter region harbours cis-elements necessary for tissue- and developmental-specific regulation of GFP accumulation. It furthermore suggested that the transcriptional activation of mrip1 is mediated by developmental signals present in both grapevine berries and tobacco flowers. Results presented, suggest that the use of tobacco as heterologous system for the analysis of ripening-related promoters, can be more generally applied. Evidently, characterisation of the mrip1 promoter region contributes towards a better understanding of the regulatory mechanisms involved in non-climacteric fruit ripening, and forms a basis for future experiments defining the cis-acting elements necessary for tissue- and cell-specific gene regulation in fruit, more specifically in grapevine. Moreover, the mrip1 promoter is an ideal candidate for the ripening-related, tissue-specific regulation of transgene transcription in genetically modified grapevine.