Differential gene expression during berry ripening in Vitis vinifera (cv Chardonnay) : isolation of specific sequences through subtractive cloning

Olivier, Abraham Jacobus (2002-12)

Thesis (MSc)--Stellenbosch University, 2002.

Thesis

ENGLISH ABSTRACT: Grapevine is worldwide an agronomically important crop. Traditionally selective breeding has been used to improve existing cultivars. In the last ten years, however, the advent of biotechnology has shortened these breeding programmes by producing transgenic grapevine. Because this new technology is aimed at the possible genetic manipulation of the ripening process in grape berries, it is important to elucidate all the mechanisms that may be involved in ripening. The aim of the present study was the identification of genes that play an important role during the ripening process in grape berries. This was achieved by investigation of putative differentially expressed genes in ripening Chardonnay berries isolated through subtractive hybridisation. Two subtraction libraries, representing early and late ripening stages were constructed. Four of the ten genes analysed exhibited expression during berry ripening. One of the four genes was expressed in a tissue and stage specific manner. Further characterisation of eight of the DNA and protein sequences revealed that the putative translation products of these clones had homologues that are involved in amongst others cell wall structure in other species. These included UDP-glucose dehydrogenase, which is involved in the synthesis of hemicellulose precursors. The remaining seven clones encoded putative stress response proteins. These included two heat shock proteins, a vacuolar pyrophosphatase and a protein involved in cell division. It is suggested that specific grape mRNAs accumulate in response to stresses such as the storage of high concentrations of sugars and rapid cell expansion. These processes occur rapidly during the ripening of berries. Accumulation of specific mRNAs can be attributed to part of the normal ripening developmental programme.

AFRIKAANSE OPSOMMING: Druiwe is wêreldwyd 'n belangrike landbougewas en kultivars word tradisioneel deur middel van tydsame selektiewe teling verbeter. Die tyd wat hieraan bestee word, kan verkort word deur die implementering van biotegnologie en die produksie van transgeniese duiwe. Omdat hierdie nuwe tegnologie op die moontlike genetiese manipulering van die rypwordingsproses in druiwe gemik is, is dit belangrik dat alle meganismes betrokke by rypwording ondersoek en verstaan word. Die doel van hierdie studie was om gene wat moontlik tydens die rypwordingsproses in druiwe 'n rol kan speel, te identifiseer. Hierdie doel is bereik deurdat differensieel uitgedrukte gene uit die kultivar Chardonnay geïsoleer is met behulp van verrykingsbiblioteke vanuit jong en volwasse druiwekorrels. Vier van die tien gene wat geanaliseer is, word uitgedruk tydens die rypwordingsproses. Verder het een van die vier gene weefsel- en rypwordingstadium- spesifisiteit getoon. Volledige karakterisering van agt van die DNA- en proteïenvolgordes het aangedui dat die proteïenprodukte van hierdie gene homoloog is aan volgordes wat onder andere by selwandstruktuur betrokke is. Dit sluit UDP-glukose dehidrogenase in, wat betrokke is by die sintese van hemi-sellulose boustene. Die ander sewe gene kodeer vir moontlike spanningsproteïene. Twee hitteskokproteïene, 'n vakuolêre pirofosfatase en 'n proteïen wat betrokke is by selverdeling is geïdentifiseer. Daar word voorgestel dat druiwe mRNA versamel in reaksie op spanningsituasies soos die berging van hoë konsentrasies suikers en selvergroting. Hierdie prosesse vind baie vinnig plaas tydens rypwording. Versameling van spesifieke mRNAs kan toegeskryf word as 'n normale deel van die rypwordingsproses.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/52802
This item appears in the following collections: