Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Sugarcane is a tropical perennial grass species belonging to the Poaceae (true grasses) family.
Mature sugarcane is comprised mostly of sugarcane stalks, which accumulate high amounts
of sucrose, a fact that has led to its wide cultivation of sugarcane for sucrose production.
Sugar yields from sugarcane have been improved in the past by either creating transgenic
sugarcane or through using traditional breeding methods. Increasing sugar yields in sugarcane
is still of interest and new cisgenic strategies are being considered to alleviate consumer
concerns over transgenic plants.
This thesis consists of two parts. The first was aimed at understanding the relation between
trehalose-6-phosphate (T6P) synthesis and sucrose accumulation in sugarcane. In this study
the E. coli genes involved in trehalose synthesis, otsA and otsB, were overexpressed in
sugarcane in order to observe their effects on soluble sugar accumulation. Nine otsA and two
otsB overexpressing lines were created, confirmed by gDNA insertion PCRs, sq-RT-PCR and
immuno detection of encoded enzymes. Preliminary measurements of soluble sugars showed
that four out of the nine otsA lines had significantly decreased and one line significantly
increased sucrose concentrations. Correlating sq-RT-PCR results with soluble sugar
measurements suggest that trehalose-6-phosphate synthase (TPS) expression affects sucrose
levels in sugarcane, but further research of TPS activity is required before a conclusion can be
reached. Further analysis of mature cane material in regard to relevant enzyme levels,
carbohydrate levels and gene expression should contribute to more conclusive results.
Three novel sugarcane TPS encoding sequences were isolated and proven to be functional
through complementation of the growth defect in tps1Δ yeast grown on glucose as a carbon
source. Sugarcane TPS isoforms named SoTPSa, SoTPSb and SoTPSc, were isolated by
successful application of 5‟ RACE alongside standard PCR using primers based on other
monocotyledonous TPS sequences. The encoded SoTPSa contains a 25 amino acid insertion
within the partial TPP domain. The encoded SoTPSc contains a 126 amino acid long N
terminal truncation, which removes one of the thirteen amino acids found within the active
site of the TPS domain. Future characterization of the encoded enzymes will determine the
effects of these modifications on TPS activity.
The second part of this thesis describes initial efforts made in attempting to develop a
cisgenic in vitro selectable marker system for sugarcane, S. officinarum callus, which uses a
diphenylether type (DPE) herbicide as a selection agent and a sugarcane protoporphyrinogen
oxidase (PPO) gene as a selection marker. Firstly the plastid targeted PPO from tobacco
(NtPPO-1) was isolated and mutagenized, to mimic the double mutated Arabidopsis PPO,
used by Li et al., (2003) in maize. However, sugarcane calli transformed with the double
mutated NtPPO-1 and grown on media containing fomesafen herbicide, were incapable of
regenerating. Future efforts will utilize a N-terminal sequence that is targeted to the plastid
organelle, so as to ensure translocation of the enzyme to that subcellular location. Also,
random mutations were induced in the NtPPO-1 gene to screen for mutations that confer DPE
herbicide resistance, however this work is currently on hold until a heme deficient E. coli can
be obtained. Secondly, attempts were made to isolate a putative sugarcane plastid targeted
PPO gene, so as to eventually use this in developing a cisgenic strategy. 5‟ RACE was
successful in revealing additional nucleotide sequence adding 1006 bp to the already known
partial sugarcane PPO sequence. However the fragment isolated was still a partial sequence. / AFRIKAANSE OPSOMMING: Suikerriet is 'n tropiese meerjarige gras spesie wat deel is van die Poaceae (ware grasse)
familie. Volwasse suikerriet bestaan hoofsaaklik uit suikerrietstamme, wat hoë hoeveelhede
sukrose akkumuleer, 'n feit wat gelei het tot die wye verbouing van suikerriet vir sukrose
produksie. In die verlede is suikeropbrengste vanuit suikerriet verbeter deur die skep van
transgeniese suikerriet óf die gebruik van tradisionele teelmetodes. Toenemende suiker
opbrengste in suikerriet is steeds van belang en nuwe cisgeniese strategieë word oorweeg om
verbruikerskommer oor transgeniese plante te akkommodeer.
Hierdie tesis bestaan uit twee dele. Die eerste deel is daarop gemik om die begrip van die
verhouding tussen trehalose-6-fosfaat (T6P) sintese en sukrose ophoping in suikerriet te
verstaan. In hierdie studie is die E. coli gene wat betrokke is in trehalose sintese, otsA en otsB,
ooruitgedruk in suikerriet ten einde die uitwerking daarvan in die opgaar van oplosbare suiker
te bestudeer. Nege otsA en twee otsB verhoogte uitdrukkings lyne is geskep, bevestig deur
gDNA bygevoegde PKR, sq-RT-PKR en immuno opsporing van geïnkripteerde ensieme.
Voorlopige metings van oplosbare suikers toon dat vier van die nege otsA lyne ʼn beduidende
afname in sukrose vlakke en een lyn „n beduidende toegeneem in sukrose vlakke getoon het.
Korrelerende sq-RT-PKR resultate met oplosbare suikermetings dui daarop dat trehalose-6-
fosfaat sintese (TPS) geenuitdrukking sukrose vlakke sal affekteer, maar verdere navorsing
van TPS aktiwiteit is nodig voordat 'n gevolgtrekking gemaak kan word. Verdere ontleding
van volwasse riet materiaal met betrekking tot relevante ensiem vlakke, koolhidrate vlakke en
geenuitdrukking, behoort by te dra tot meer volledige resultate.
In hierdie studie is drie nuwe suikerriet TPS gene geïsoleer en dit is bewys as funksioneel
deur die komplimentering van die groeidefek van tps1Δ gis, gegroei op glukose as 'n koolstof
bron. Suikerriet TPS isoforme, genoem SoTPSa, SoTPSb en SoTPSc, is geïsoleer deur die
suksesvolle toepassing van 5 'RACE, in kombinasie met standaard PKR, deur van spesiaal
ontwerpte primers, gebaseer op ander eensaadlobbige TPS gene, gebruik te maak. Die
gekodeerde SoTPSa bevat 'n 25 aminosuur invoeging binne-in die gedeeltelike TPP domein.
Die gekodeerde SoTPSc bevat 'n 126 aminosuur lange N terminaal afkapping, wat een van
die dertien aminosure binne die aktiewe terrein van die TPS domein verwyder. Toekomstige
karakterisering van hierdie geïnkripteerde ensiemes sal die effek van hierdie veranderinge op
TPS aktiwiteit bepaal.
Die tweede deel van hierdie tesis beskryf die aanvanklike probeerslae wat gemaak is in 'n
poging om „n cisgeniese in vitro selekteerbare merker vir suikerriet, S. officinarum kallus te
ontwikkel. Hierin word gebruik gemaak van 'n difenylether tipe (DPE) onkruiddoder as 'n
seleksie agent, en 'n suikerriet protoporphyrinogen oksidase (PPO) geen as 'n seleksie merker.
In 'n poging om dit te bewerkstellig is daar eerstens plastied geteikende PPO van tabak
(NtPPO-1) geïsoleer en geteikende mutagenese suksesvol daarop uitgevoer. Mutasies wat
geinduseer is, is gegrond op die dubbele gemuteerde Arabidopsis PPO, wat gebruik was in
mielies deur Li et al., (2003). Alhoewel die suikerriet kallus getransformeer is met die
dubbele gemuteerde NtPPO-1 konstruk en geselekteer is op media wat fomesafen
onkruiddoder bevat, was die kallus nie in staat om te regenereer nie. In toekomstige pogings
sal probeer word om 'n N-terminale volgorde, geteiken op „n plastied organel, te benut sodat
translokasie van die ensiem aan die plastied organel verseker kan word. So ook is toevallige
mutasies veroorsaak in die NtPPO-1 gene om te soek vir nuwe mutasies wat DPE
onkruiddoderweerstand verleen, maar hierdie werk is tans gestop totdat 'n heem gebrekkige E.
coli mutant verkry kan word. Tweedens, is pogings aangewend om 'n vermeende suikerriet
plastied geteikende PPO gene te isoleer, om uiteindelik te gebruik in die ontwikkeling van 'n
cisgeniese strategie in suikeriet. 5 'RACE was suksesvol in die onthulling van bykomende
nukleotiede volgorde deur 1006 bp by te voeg by die reeds bekende gedeeltelike suikerriet
PPO fragment. Nie teenstaande is die fragment wat nuut geïsoleer is, steeds slegs 'n
gedeeltelike volgorde volgens vergelykings met ander bekende plant PPO gene.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/96696 |
| Date | 04 1900 |
| Creators | Fernhout, Jean-Jacque |
| Contributors | Van der Vyver, Christell, Lloyd, James, Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB). |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xxiii, 125 pages : illustrations |
| Rights | Stellenbosch University |
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