Dissertation (PhD) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Sugarcane (Saccharum spp. hybrids) is a commercial crop plant capable of storing up
to 20% sucrose on a fresh mass basis in the culm. Knowledge about gene expression
during sugarcane growth and maturation is limited. The aim of this study was to assess
whether an Expressed Sequence Tag (EST)-based approach towards analysis of
sugarcane would reveal new information about gene expression and metabolic
processes associated with sugarcane growth and development. The specific objectives
were two-fold: firstly, to develop an EST database for sugarcane and secondly, to
identify and analyse genes that are expressed in different sugarcane tissue types and
developmental stages, with a specific focus on leaf and culm.
An EST database for sugarcane was initiated to obtain information on sugarcane gene
sequences. A total cDNA library was constructed from sugarcane immature leaf (leaf
roll: meristematic region) tissue and 250 clones randomly selected and subjected to
single-pass DNA sequence analysis. Sugarcane ESTs were identified by sequence
similarity searches against gene sequences in international databases. Of the 250 leaf
roll clones, 26% exhibited similarity to known plant genes, 50% to non-plant genes
while 24% represented new gene sequences. Analysis of the identified clones indicated
sequence similarity to a broad diversity of genes. A significant proportion of genes
identified in the leaf roll were involved in processes related to protein synthesis and
protein modification, as would be expected in meristematic tissues. Submission of 495
sugarcane gene sequences to the dbEST database represented the first sugarcane ESTs
released into the public domain.
Two subtracted cDNA libraries were constructed by reciprocal subtractive
hybridisation between sugarcane immature and maturing internodal tissue. To explore
gene expression during sugarcane culm maturation, partial sequence analysis of
random clones from maturing culm total and subtracted cDNA libraries was
performed. Database comparisons revealed that of the 337 cDNA sequences analysed,
167 showed sequence homology to gene products in the protein databases while 111
matched uncharacterised plant ESTs only. The remaining cDNAs showed no database
match and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous to
various stress response genes were also well represented. Analysis of ESTs from the
subtracted library identified genes that may be preferentially expressed during culm
maturation.
The expression patterns of sugarcane genes were examined in different tissue sources
and developmental stages to identify differentially expressed genes. cDNA arrays
containing 1000 random clones from immature leaf and maturing culm cDNA libraries
were hybridised with poly (At RNA from immature leaf, mature leaf, immature culm
and maturing culm. All cDNAs examined hybridised to all four probes, but differences
in signal intensity were observed for individual cDNAs between hybridisation events.
No cDNAs displaying tissue- or developmental-stage specific expression were
detected. Comparisons between hybridisation patterns identified 61 cDNAs that were
more abundantly expressed in immature and mature leaf than the culm. Likewise, 25
cDNAs preferentially expressed in immature and maturing culm were detected. ESTs
established for the differentially expressed cDNAs revealed sequence homology to a
diverse collection of genes in both the leaf and the culm. These included genes
associated with general cellular metabolism, transport, regulation and a variety of
stress responses. None of the differentially expressed genes identified in the culm were
homologous to genes known to be associated with sucrose accumulation.
To examme differences at the level of gene transcription between low sucroseaccumulating
and high sucrose-accumulating tissues, subtracted cDNA libraries were
utilised. To isolate cDNAs differentially expressed during culm maturation, cDNA
arrays containing 400 random clones (200 from each library) were screened with total
cDNA probes prepared from immature and maturing culm poly (At RNA. Results
indicated that 36% and 30% of the total number of cDNAs analysed were
preferentially expressed in the immature and maturing culm, respectively. Northern
analysis of selected clones confirmed culm developmental stage-preferential
expression for most of the clones tested. ESTs generated for the 132 differentially
expressed clones isolated exhibited homology to genes associated with cell wall
metabolism, carbohydrate metabolism, stress responses and regulation, where the
specific ESTs identified in the immature and maturing culm were distinct from each other. No developmentally regulated ESTs directly associated with sucrose metabolism
were detected.
These results suggest that growth and maturation of the sugarcane culm is associated
with the expression of genes for a wide variety of metabolic processes. In addition,
genes encoding enzymes directly involved with sucrose accumulation do not appear to
be abundantly expressed in the culm. / AFRIKAANSE OPSOMMING: Kommersiële suikerriet variëteite (Saccharum spp. hibriede) is in staat om tot 20%
sukrose op 'n vars massa basis in die stingel op te berg. Kennis oor geenuitdrukking
tydens groei en rypwording is beperk. Die doel van die huidige studie was om vas te
stelof 'n grootskaalse karatersisering van die geenvolgordes wat uitgedruk word
"Expressed Sequence Tag (EST)-based approach" tot nuwe inligting aangaande die
aard en omvang van metabolisme tydens groei en ontwikkeling van suikerriet sal lei.
'n Tweeledige benadering is in hierdie studie gevolg. Eerstens is 'n data basis oor die
gene wat uitgedruk word "EST" databasis opgestel. Tweedens is gene geïdentifiseer en
gekarakteriseer wat spesifiek op verskillende stadiums van ontwikkeling en in
spesifiek weefsel uitgedruk word.
Vir die opstel van die EST-databasis is 250 klone uit 'n totale cDNA biblioteek vanaf
RNA uit suikerrietblaarweefsel (blaarrol:meristematiese streek) op 'n lukraak basis
gekies en aan 'n enkel eenrigting DNA volgorde analise onderwerp. Suikerrriet EST's
is geïdentifiseer deur middel van homologie soektogte teen geenvolgordes in
internasionale databasisse. Uit die 250 blaarrol klone het 26% ooreenkomste met
bekende plant gene en, 50% met nie-plant gene getoon. Ongeveer 24% het nuwe
geenvolgordes verteenwoordig. Analise van die geïdentifeseerde klone het
ooreenkomste met 'n breë diversiteit van gene getoon. 'n Betekenisvolle gedeelte van
gene wat in die blaarrol geïdentifiseer is, is by proteïensintese en proteïenmodifikasies
betrokke. Dit is in ooreenstemming met wat van meristematiese weefsel verwag kan
word. Die 495 suikerriet geenvolgordes wat in die internasionale dbEST databasis
gestort is, is die eerste sodanige inligting in die publieke domein.
Twee spesifieke cDNA biblioteke (subtraction libraries) wat volgordes spesifiek aan
onvolwasse suikerriet en rypwordende internodale weefsel bevat is voorberei.
Geenuitdrukking gedurende die rypwordingsproses van die suikerrietstingel is
bestudeer deur geenvolgorde analises van onwillekeurige geselekteerde klone van die
twee eDNA biblioteke te doen. Van die 337 geenvolgordes wat geanaliseer is het 167
homologie met bekende gene en net 111ooreenkomste met ongekarakteriseerde plant
gene getoon. Die oorblywende geenvolgordes het geen ooreenkomste met bekende gene getoon nie en daar kan dus aanvaar word dat hulle nuwe gene verteenwoordig.
Die meerderheid ESTs het ooreenkomste met verskeie gene wat met sellulêre
metabolisme geassosieer word getoon. ESTs wat homoloog was aan verskeie
spannings geassosieerde gene was ook goed verteenwoordig. Die analise het gene wat
by voorkeur tydens stringelrypwording uitgedruk word geidentifiseer.
Die geenuitdrukkingspatrone van suikerriet in weefsels van verskillende oorsprong en
ontwikkelingstadia is ondersoek om differensieel uitgedrukte gene te identifiseer.
Reekse wat 1000 lukrake eDNA klone van onvolwasse en rypwordende stingel eDNA
biblioteke is met poli-(A)-RNA van onvolwasse blaar, volwasse blaar, onvolwasse
stingel en volwasse stingel gehibridiseer. Al die eDNA klone wat ondersoek is het met
al vier die peilers gehibridiseer. Die intensiteit van die seine het egter grootliks
gevarieer. Die analise het gelei tot die identifisering van 61 eDNA klone wat teen hoër
vlakke in onvolwasse en volwasse blaar as in die stingel uitgedruk word. Daar is ook
25 eDNA klone wat by voorkeur in onvolwasse en rypwordende stingel uitgedruk
word gevind. Gene wat geassosieer word met gewone sel metabolisme, vervoer
prosesse, regulering en verskeie spannings-geassosieerde reaksies, is in die twee
groepe teenwoordig. Geeneen van die volgordes wat selektief uitgedruk word kan met
gene wat direk met sukrose akkumulering verband hou geassosieer word nie.
Ten einde eDNA klone wat differensieel tydens rypwording van die stingel uitgedruk
word te isoleer, is 400 eDNA klone (200 van elke biblioteek) lukraak geselekteer en
met totale eDNA peilers, wat uit onvolwasse en rypwordende stingel poli-(A)-RNA
voorberei is, gesif. Resultate het aangetoon dat 36% en 30% van die totale getal eDNA
klonewat geanaliseer is, by voorkeur in die onvolwasse en rypwordende stingel
uitgedruk word. RNA kladanalises van geselekteerde klone het getoon dat die meeste
ontwikkelingstadium spesifieke uirtdrukkingspatrone het. Daar is gevind dat 132 van
die EST klone homologie met gene geassosieerd met selwand- en
koolhidraatmetabolisme, spannings geassosieerde- en reguleringsreaksies, toon. Die
spesifieke ESTs wat in die onvolwasse en rypwordende stingel geïdentifiseer is het van
mekaar verskil. Nie een van die ESTs wat geïdentifiseer is kan direk met sukrose
metabolisme geassosieer word nie. Hierdie werk toon baie duidelik aan dat groei en rypwording van die suikerrietstingel
met die uitdrukking van gene geassosieerd is wat by 'n hele aantal metaboliese
prosesse betrokke is. Die resultate toon ook dat die gene wat vir ensieme kodeer wat
direk by sukrose akkumulering betrokke is, nie teen hoë vlakke in die stingel
uitgedruk word nie.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52860 |
| Date | 12 1900 |
| Creators | Carson, Deborah L. (Deborah Lee) |
| Contributors | Botha, F., Huckett, B., Stellenbosch University. Faculty of AcriSciences. Dept. of Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 129 p. : ill. |
| Rights | Stellenbosch University |
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