Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: For the expression of transgenes in plant cells, appropriate promoter
sequences have to be introduced upstream of the gene to ensure efficient
transcription. While to date the maize ubiquitin (Ubi1) promoter has been the
most effective transgene promoter for sugarcane, there is a high demand for
tissue and stage specific promoters for localised transgene expression in the
mature culm. The present study sought to characterise genes preferentially
expressed in the core and peripheral tissues of the mature culm, which can
further be used as research tools for specific promoter isolation.
cDNA expression arrays containing 3840 clones from a late stage cDNA
library representative of the core and peripheral tissues of the mature culm
were prepared. The cDNA expression arrays were then differentially
screened in independent hybridisation experiments with radioactively-labeled
cDNA representations of core and peripheral tissues of internode 7, and
peripheral tissues of internode 10. Comparison of the expression profiles of
the arrayed cDNA targets in the three probes led to the identification of 60
tissue-specific, 17 stage-specific and 50 selectively expressed cDNAs within
the mature sugarcane culm.
~ESTs of 33 chosen selectively expressed cDNAs with a relatively stronger
pattern of expression in the core than in the peripheral tissues revealed
sequence homology to a diverse collection of genes in the mature culm.
These included genes associated with general cellular metabolism such as
protein synthesis, protein modification and structural protein. Also identified
were stress-responsive genes. The putative translational products of some of
these clones had homologs that are involved in cell-wall structure in other
species. These included the [acalin homolog, a lectin, hydroxyproline rich
glycoprotein and structured polyprotein C. Many of the cDNAs thought to be
involved in cell wall structure or stress related responses also accumulate in
a developmental manner in other plants. These may indicate that specific
mature culm mRNAs accumulate in response to stresses such as rapid cell
expansion or as part of the late developmental program. An unexpected observation was that only one gene associated with sucrose metabolism was
identified, namely sucrose synthase. These results confirmed that culm
maturation was not controlled by sucrose metabolism despite its distinct
physiological characteristic of storing high levels of sugars.
ESTs analysis further revealed that sequence homology was not obtained for
all the cDNAs exhibiting stage and tissue specific expression in the core and
peripheral tissues of the mature culm. These could represent novel genes not
only from sugarcane but all plants.
Northern analysis demonstrated that 9 putatively identified selectively
expressed genes tested so far accumulated specifically in the core and
peripheral tissues of the mature culm. No expression was detected in root,
leaf, leafroll and internode 3. However, their selective expression in a single
internode as observed on the arrays (i.e hybridisation signal intensity being
higher in the core than in the peripheral tissue) was not detected on the
northern blots. These showed that cDNA expression arrays were not a highcapacity
gene expression assay since they were prone to false expression
analysis. The validity of results obtained through array screening should
always be verified in an independent manner, preferably by the northern
hybridisation analysis.
Hence, the present study shows that the combination of differential
screening, northern blot and DNA sequence analysis permits the rapid
characterisation of differentially expressed genes in the core and peripheral
tissues of the mature sugarcane culm. These can further be used as
research tools for mature culm - specific promoter isolation in the sugarcane. / AFRIKAANSE OPSOMMING: Die doeltreffende uitdrukking van transgene in plantselle is afhanklik van 'n gepaste
promotorvolgorde wat stroomop van die geen ingevoeg word. Die Ubi1-promotor van
mielies was tot dusver die doeltreffendste transgeenpromotor in suikerriet, maar daar is
'n groot behoefte aan promotors wat weefsel- en ontwikkelingstadium-spesifieke
geenuitdrukking kan beheer. Hierdie studie het op die isolering en karakterisering van
gene wat selektief in die kern- of periferale stingelweefsel van suikerriet uitgedruk
word, gefokus. Hierdie gene sal verder benut kan word om promotors te isoleer.
eDNA uitdrukkingsreekse ("expression arrays") van 'n volwasse stingel eDNA
biblioteek is voorberei. Hierdie reekse, wat 3840 klone bevat het, is in onafhanklike
hibridiseringseksperimente met radioaktiefgemerkte eDNA van onderskeidelik kern- en
periferale stingelweefsel van lit 7 en periferale stingelweefsel van lit 10 afgetas. 'n
Vergelyking van die uitdrukkingsprofiele van die eDNA teikens in dié drie peilergroepe
het tot die identifisering van 60 weefsel-spesifieke-, 17 ontwikkelingstadium-spesifiekeen
50 selektief uitgedrukte eDNAs in die volwasse suikerrietstingel gelei.
Uitdrukkingsvolgordemerkers ("ESTs") van 33 geselekteerde eDNAs wat in hoër vlakke
in die kern uitgedruk is, se volgordes toon homologie aan 'n wye verskeidenheid gene
in die volwasse stingel. Hierdie groep sluit gene in wat met algemene sellulêre
..metabolisme soos proteïensintese, proteïenmodifisering en strukturele proteïene
geassosieer is. Spanningsverwante gene is ook hier geïdentifiseer. Die
transleringsprodukte van sommige klone het homoloë wat by selwandstruktuur in
ander spesies betrokke is, soos die jaealin-homoloog, 'n lektien, hidroksiprolien-ryke
glikoproteïen en gestruktureerde poliproteïen C. 'n Wye verskeidenheid eDNAs wat by
selwandstruktuur of spanningsverwante reaksies betrokke is, akkumuleer ook in 'n
ontwikkelingsafhanklike wyse in ander plante. Dit mag 'n aanduiding wees dat
spesifieke mRNAs in die volwasse stingel in reaksie op spanning wat met vinnige
seluitsetting gepaardgaan, versamel. Slegs een geen wat met sukrose metabolisme
geassosieer is, nl. sukrosesintase, is in hierdie studie geïdentifiseer. Hierdie
onverwagte waarneming het bevestig dat, ondanks suikerriet se kenmerkende vermoë
om hoë konsentrasies suiker te berg, stingelveroudering nie net met sukrose
metabolisme geassosieer kan word nie. Nie al die eDNA-fragmente wat geïsoleer is, het homologie aan ander gene in die internasionale databasisse getoon nie, wat
moontlik kan aandui dat nuwe gene suksesvol geïsoleer is.
Nege ontwikkelingstadium-spesifieke gene wat slegs in die volwasse stingelweefsels
uitgedruk word, is dmv noordelike oordraganalises geïdentifiseer. Geen transkripte van
hierdie gene is in die wortels, blaarrol, blare of jong stingel waargeneem nie. Die
weefselspesifisiteit wat met die uitdrukkingsreekse waargeneem is, kon nie mbv
noordelike orrdraganalises bevestig word nie. Dit mag 'n aanduiding wees dat die
uitdrukkingsreekse vals positiewe resultate kan oplewer en dit is raadsaam om
voortaan altyd die verkrygde profiele met ander, meer sensitiewe tegnieke, te bevestig.
Die studie het aangetoon dat 'n kombinasie van differensiële aftasting, noordelike
oordraganalise en DNA-volgordebepaling gebruik kan word om gene wat differensieel
uitgedruk word in die volwasse suikerrietstingel, te identifiseer. Hierdie geenfragmente
kan nou vir promotorisoleringsdoeleindes aangewend word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52803 |
| Date | 12 1900 |
| Creators | Rogbeer, Omeswaree |
| Contributors | Botha, F. C., Groenewald, S., Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 106 p. : ill. |
| Rights | Stellenbosch University |
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