Identification and expression analysis of flavonoid biosynthetic genes in the genus Clivia
| dc.contributor.advisor | Spies, J. J. | |
| dc.contributor.advisor | Viljoen, C. D. | |
| dc.contributor.author | Snyman, Marius Christian | |
| dc.date.accessioned | 2015-11-25T06:59:26Z | |
| dc.date.available | 2015-11-25T06:59:26Z | |
| dc.date.copyright | 2010 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010 | |
| dc.description.abstract | English: Anthocyanins belong to a large group of secondary plant metabolites, the flavonoids, and fulfil a range of biological functions that include the cyanic pigmentation they provide to flowers, fruits, vegetables and leaves. The anthocyanin biosynthetic pathway has been well elucidated and much effort has been made by researchers to modify some of the catalytic steps, thereby changing the colour of some ornamental and cut flower species. The genus, Clivia, is an ornamental monocot indigenous to South Africa and there has been a growing interest among local and international Clivia breeders to introduce novel flower colour varieties into the market. Transgene technology holds new possibilities to ensure modification of Clivia flower colour. However, the genetics and biochemistry of the Clivia anthocyanin biosynthetic pathway must first be investigated before any attempts regarding biotechnology can be made. The current study is the first to deal with the identification and expression analysis of flavonoid biosynthetic genes in the genus Clivia, specifically those involved in anthocyanin biosynthesis, thus identifying future prospects and motivating research in unexplored territory. A previous study concerning an HPLC analysis of Clivia anthocyanin content confirmed the presence of cyanidin and pelargonidin derivatives as the main pigments in the tepals and fruits. This enabled the establishment of a putative Clivia anthocyanin biosynthetic pathway illustrating each enzymatic event. Conventional PCR with degenerate primers and a tepal cDNA template was used to isolate four different target sequences. Consensus cDNA fragments of 586 bp, 326 bp, 510 bp and 225 bp confirmed the existence of Clivia orthologues for Chalcone synthase (CHS), Chalcone isomerase (CHI), Flavanone 3-hydroxylase (F3H), and Dihydroflavonol 4-reductase (DFR), respectively. The deduced amino acid sequences of CHS, DFR and F3H harboured important conserved residues that confirmed the existence of functional enzymes. Furthermore, nucleotide sequence analyses between each new Clivia cDNA fragment and the corresponding fragments of other higher plants, regarding similarity/identity and phylogeny demonstrated closer homologies and evolutionary relatedness to other monocot species. The identification of the Clivia flavonoid biosynthetic genes enabled the expression analyses of CHS and DFR. These structural genes encode enzymes responsible for two important controlling steps necessary to determine the nature of the final end-product(s) of the pathway. Real-time quantitative RT-PCR involving SYBR® Green chemistry was used to investigate the temporal expression of the two genes in the tepal, stamen and carpel tissues during five flower developmental stages of an orange and yellow variety of Clivia miniata. Statistical analyses were used to support any findings where possible. Each respective tissue type revealed its own trend in expression for both CHS (an early biosynthetic gene) and DFR (a late biosynthetic gene) throughout flower development except in the stamens of the yellow flowers. These findings suggested the co-ordinate regulation of the Clivia miniata anthocyanin biosynthetic genes as a single module, a model of transcriptional regulation that is often found in certain monocot species (Dooner et al., 1991; Meldgaard, 1992; Martin and Gerats, 1993). To understand the regulatory system that confers flowers colouration, genes that encode transcription factors should be isolated and their spatial and temporal expression investigated. The „parallelism‟ between anthocyanin biosynthetic gene expression and anthocyanin production in the tepals of the orange and yellow Clivia miniata varieties was also investigated. UV-visible spectrophotometry at A530nm was used to quantify total anthocyanins at each developmental stage after extraction. At full bloom the orange flowers had almost 16 times more anthocyanins, which support orange colour development, than the yellow flowers. It was confirmed by the outcomes of statistical analyses that the trends in expression of CHS and DFR and anthocyanin production were similar. Methods such as HPLC are recommended for more precise qualitative and quantitative determination of total monomeric anthocyanins. | en_ZA |
| dc.description.abstract | Afrikaans: Antosianiene behoort aan ‟n groot groep sekondêre metaboliete bekend as die flavonoïede en is verantwoordelik vir verskeie biologiese funksies wat hul rol in die pigmentasie van blomme, vrugte, groente en blare insluit. Die uitleg van die antosianien-biosintese-weg is goed bekend en navorsers het daarin geslaag om sekere ensiematiese stappe te manipuleer om sodoende die kleure van sommige ornamentele en snyblomspesies te verander. Die genus Clivia is ‟n ornamentele monokotiel inheems aan Suid-Afrika en daar is tans ‟n groeiende belangstelling by telers om nuwe kleure in die plant se blomme tot stand te bring. In vergelyking met gewone teling, hou transgeentegnologie baie potensiaal vir nuwe moontlikhede in dié verband in, maar ‟n goeie begrip van die genetika en biochemie van Clivia-antosianien-biosintese word vooraf vereis. Die bestaande studie is die eerste navorsingsprojek wat die identifisering en uitdrukkingsanalise van flavonoïedgene (meer spesifiek die antosianiengene) in die genus, Clivia, ondersoek en skep nuwe geleenthede en motiverings vir verdere diepgaande studies in hierdie veld. ‟n Vorige HPLC-analise van die antosianieninhoud in Clivias het die teenwoordigheid van sianidien- en pelargonidien-verbindings in die blomblare bevestig wat tot die bekendstelling van ‟n voorlopige antosianien-biosintese-weg kon lei. Gewone polimerase-kettingreaksies met priemstukmengsels en ‟n blomblaar „cDNA‟-templaat is gebruik om vier teikenvolgordes te isoleer. Hiervolgens is konsensus „cDNA‟-fragmente van 586 bp, 326 bp, 510 bp en 225 bp onderskeidelik geïdentifiseer vir Kalkoon-sintase, Kalkoon-isomerase, Flavanoon 3-hidroksilase en Dihidroflavonol 4-reduktase. Besigtiging van die afgeleide aminosuurvolgordes vir Kalkoon-sintase, Flavanoon 3-hidroksilase en Dihidroflavonol 4-reduktase het ook tot die uitwysing van gekonserveerde residue gelei wat die funksionaliteit van hierdie ensieme in Clivias kon beklemtoon. Nukleotiedvolgorde-analises tussen elke Clivia „cDNA‟-fragment en die soortgelyke „cDNA‟-fragmente van ander plante ten opsigte van eendersheid/identiteit en filogenie is ook uitgeoefen en kon so die verwantskap aan ander monokotiele op grond van homologie en evolusionêre groepering vasstel. Hoofstuk 7 Nadat die Clivia flavonoïedgene geïdentifiseer is, kon die volgende stap van hierdie studie onderneem word, nl. die uitdrukkingsanalise van Kalkoon-sintase en Dihidroflavonol 4-reduktase. Hierdie strukturele gene speel ‟n belangrike rol in die voorkoms van die finale eindproduk(te), aangesien hulle kodeer vir ensieme wat betrokke is by belangrike kontroleringstappe in die biosintese-weg. „Real-time quantitative RT-PCR‟ met „SYBR® Green‟ chemie is ingespan om die uitdrukking van die twee gene oor ‟n periode van vyf blomontwikkelingstadiums te verken. Blomblaar-, stuifmeeldraad-, en vrugbeginselweefsel van ‟n oranje variëteit en ‟n geel variëteit van Clivia miniata was tydens die ondersoek gebruik. Die verloop van uitdrukking van Kalkoon-sintase (‟n vroeë biosintese-ensiem) en Dihidroflavonol 4-reduktase (‟n laat biosintese-ensiem) tydens blomontwikkeling was meestal soortgelyk in ‟n spesifieke weefseltipe, maar het verskil tussen die verskillende weefseltipes. Hierdie bevindings het die gelyke regulering van Clivia miniata antosianiengene tentatief as ‟n enkele eenheid voorgestel. Hierdie transkripsionele reguleringsmodel word dikwels by monokotiele gevind (Dooner et al., 1991; Meldgaard, 1992; Martin and Gerats, 1993). Die gene wat die betrokke transkripsiefaktore enkodeer, moet eers geïsoleer word en hul uitdrukkingspatrone verken word, voordat die onderliggende reguleringsmeganismes, ten opsigte van blomkeur, verstaan kan word. Die ewewydigheid tussen antosianiengeenuitdrukking en antosianienproduksie tydens geel en oranje blomblaarontwikkeling van Clivia miniata is ook ondersoek. Na ekstraksie is totale antosianiene in elke blomontwikkelingstadium deur middel van UV-visuele spektrofotometrie by A530nm gekwantifiseer. Tydens die volleblomstadium het die oranje blomme, in vergelyking met die geel blomme, ongeveer 16 maal meer antosianiene bevat. Dit kon sodoende oranjekleurontwikkeling ondersteun. Ewewydigheid van die uitdrukking van Kalkoon-sintase én Dihidroflavonol 4-reduktase, aan antosianienproduksie is d.m.v. statistiese analises bevestig. Daar is ook ander metodes soos HPLC beskikbaar wat aanbeveel word vir meer akkurate kwalitatiewe en kwantitatiewe bepaling van die total monomeriese antosianiene. | af |
| dc.description.sponsorship | National Research Foundation (NRF) | en_ZA |
| dc.description.sponsorship | National Research Foundation (NRF) | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11660/1891 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Spectrophotometry | en_ZA |
| dc.subject | Real-time quantitative RT-PCR | en_ZA |
| dc.subject | PCR | en_ZA |
| dc.subject | Nucleotide sequence analyses | en_ZA |
| dc.subject | Homology | en_ZA |
| dc.subject | Flowers | en_ZA |
| dc.subject | Flavonoid biosynthetic genes | en_ZA |
| dc.subject | Clivia | en_ZA |
| dc.subject | cDNA | en_ZA |
| dc.subject | Anthocyanins | en_ZA |
| dc.subject | Clivia -- South Africa -- Genetics | en_ZA |
| dc.subject | Clivia -- Classification | en_ZA |
| dc.subject | Amaryllidaceae | en_ZA |
| dc.subject | Flavonoids | en_ZA |
| dc.subject | Anthocyanins | en_ZA |
| dc.subject | Nucleotide sequence | en_ZA |
| dc.subject | Dissertation (M.Sc. (Genetics))--University of the Free State, 2010 | en_ZA |
| dc.title | Identification and expression analysis of flavonoid biosynthetic genes in the genus Clivia | en_ZA |
| dc.type | Dissertation | en_ZA |