The isolation and characterisation of a developmentally-regulated gene from Vitis vinifera L. berries

[摘要] ENGLISH ABSTRACT: Despite increased focus on ripening-related gene transcription in grapevine, and the large number ofripening-related cDNAs identified from grapes in recent years, the molecular basis of processesinvolved in grape berry ripening is still poorly understood. Moreover, little is known about themechanisms involved in the ripening-related regulation of fruit-specific genes, since the isolationand characterisation of no ripening-related, fruit-specific promoter elements has been reported todate. This study was aimed at the isolation and characterisation of a fruit-specific, ripeningregulatedgene from Vitis vinifera L.In the first phase of the work, gene transcription in ripening berries of Cabernet Sauvignon (a goodquality wine cultivar) and Clairette blanche (a poor quality wine cultivar) were studied by AmplifiedFragment Length Polymorphism analysis of complementary DNA (cDNA-AFLP analysis). TotalRNA from immature (14-weeks post flowering, wpf) and mature (18-wpf) berries was used for theanalysis. A total of 1 276 cDNA fragments were visualised, of which 175 appeared to be ripeningrelated. Average pairwise difference of the fragments amplified from immature and matureClairette and Cabernet berries, suggested that ripening-related gene transcription in these twophenotypically different cultivars is remarkably similar. Nevertheless, it was shown that seventypercent of the 175 ripening-related cDNA fragments were cultivar-specific. It was suggested thatthese differences should be targeted to identify genes related to the phenotypical differencesbetween the two cultivars, but also to identify genes possibly involved berry quality. Moreover, theanalysis illustrated the usefulness of cDNA-AFLPs for the analysis of ripening-related genetranscription during grape berry ripening.In the second phase of the work, one of the ripening-related cDNAs identified by the cDNA-AFLPanalysis, was selected for further characterisation. This work highlighted the limitation placed onthe isolation of a single specific sequence from a cDNA-AFLP gel, indicating the presence ofmultiple ripening-related genes in a single band excised from a cDNA-AFLP gel. Steps toovercome this limitation of cDNA-AFLP analysis to identify and clone a specific ripening-relatedgene, were implemented. In short, the band corresponding to the particular ripening-related cDNAwas band was excised from the cDNA-AFLP polyacrylamide gel and re-amplified. Northern blotanalysis using the re-amplified, uncloned product confirmed the ripening-related transcriptiondemonstrated by cDNA-AFLP analysis. The re-amplified, uncloned product was then cloned.Sequence analysis of two randomly selected candidate clones revealed two distinctly differentsequences, of which neither hybridised to messenger RNA from ripening grape berries. Furtheranalysis revealed an additional five cDNAs with terminal sequences corresponding to the selectivenucleotides 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 ripeningrelatedtranscription visualised by cDNA-AFLP analysis. All seven cDNAs identified from theparticular excised band were shown to be ripening-regulated during berry development, althoughmost 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 onsetof véraison (10- to 12-wpf), was identified for isolation and characterisation of the full lengthcoding sequence.In the third phase of the work, it was shown that this cloned sequence corresponded to a geneencoding 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 fruittissue, 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 isthe central region of mrip1, which is comprised of eight consecutive repeats of 19 amino acidresidues each. In comparison with other grapevine PRPs, mrip1 revealed single amino aciddifferences and deletion of one of the 19 amino acid residues repeats, all in the central region ofmrip1. In situ hybridisation studies showed that accumulation of the mrip1 transcript in the ripeningberry is limited to the mesocarp and exocarp cells of the ripening grape berry. No transcript withhigh sequences similarity to mrip1 could be detected in ripening strawberry or tomato fruit. Basedon the properties and proposed function of PRPs, and the results obtained in this study, potentialapplications 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 thelater stages of ripening, mrip1 was proposed an ideal candidate gene for the isolation of a fruit- andlate-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 mrip1promoter element. A 5.5 kb sequence corresponding to the mrip1 5' untranslated (UTR) flankingregion was isolated and characterised by sequence analysis. In the 2.8 kb sequence directlyupstream of the mrip1 transcription initiation site, several putative cis-acting regulatory elementswere identified. These include a spectrum of hormone-, light-, phytochrome-, sugar-and stressresponsiveelements, as well as elements implicated in tissue-specific transcription. Analysis of thesequence further upstream (3.6 – 5.5 kb) of the mrip1 transcription initiation site (TIS), revealed thepresence of another proline-rich protein directly upstream of mrip1. Sequence identity of thissequence (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 mrip1promoter element, the 2.2 kb sequence directly upstream of the mrip1 TIS, was translationally fusedto the sgfpS65T reporter gene. Functionality of the mrip1:sgfpS65T fusion was verified by transientexpression in green pepper pericarp tissue, before introduction into tobacco by Agrobacteriummediatedtransformation. In transgenic tobacco, transcription of the mrip1:sgfpS65T fusion wasdevelopmentally-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 thehigh level of expression visualised in the flower in full-bloom, followed by a decrease in the finalstages of ovary development. These observations suggested that the 2.2 kb mrip1 promoter isfunctional and that this promoter region harbours cis-elements necessary for tissue- anddevelopmental-specific regulation of GFP accumulation. It furthermore suggested that thetranscriptional activation of mrip1 is mediated by developmental signals present in both grapevineberries and tobacco flowers. Results presented, suggest that the use of tobacco as heterologoussystem 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 theregulatory mechanisms involved in non-climacteric fruit ripening, and forms a basis for futureexperiments defining the cis-acting elements necessary for tissue- and cell-specific gene regulationin fruit, more specifically in grapevine. Moreover, the mrip1 promoter is an ideal candidate for theripening-related, tissue-specific regulation of transgene transcription in genetically modifiedgrapevine.

[发布机构] Stellenbosch University