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.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The development of ―energycane‖ varieties of sugarcane for ethanol production is underway, targeting the use of both sugar juice (first generation ethanol) and bagasse (second generation ethanol). Nevertheless, identification of the preferred varieties represents the biggest challenge to the development of energycane due to large number of samples produced during breeding. In the present study, dilute acid pretreatment, enzymatic hydrolysis and fermentation processes were used to evaluate the processability of bagasse (fibrous residue generated after juice sugar extraction) from different varieties of sugarcane to select preferred varieties with the properties of improving combined ethanol yield (ethanol from juice and bagasse) per hectare. The impact of variety selection on combined ethanol yield (ethanol from juice and bagasse) per hectare was also assessed. In the first part of this study, 115 varieties of sugarcane originated from classical breeding and precision breeding (genetic engineering) were screened based on agronomic data and experimental data from biochemical processes (dilute acid pretreatment and enzymatic hydrolysis) applied to the bagasse fraction of each variety. The results showed wide variations in the chemical composition of bagasse between the varieties. Structural carbohydrates and lignin content ranged from 66.6 to 77.6% dry matter (DM) and 14.4 to 23.1% DM, respectively. The majority of precision breeding varieties showed higher arabinoxylan, lower lignin and lower ash content than most of classical breeding varieties. Combined sugar yield from the bagasse after pretreatment and enzymatic hydrolysis also varied significantly among the varieties. Up to 27.9 g/100g (dry bagasse) difference in combined sugar yield was observed. Combined sugar yield was inversely correlated with lignin as well as ash content, but it correlated positively with structural carbohydrates content. Total potential ethanol yields per hectare, calculated based on cane yield, soluble and non-soluble sugar content also differed significantly among the varieties (8,602−18,244 L/ha). Potential ethanol from bagasse contributed approximately one third of the total potential ethanol yield. Interestingly, some of the varieties had combined properties of high potential ethanol yield per hectare and improved bagasse convertibility. Thus, six varieties (3 from each breeding technology) were selected as preferred varieties for further investigation. To enhance sugar yield from bagasse, optimisation of pretreatment was conducted on the selected varieties. Industrial bagasse was included for comparison purposes. The pretreatment optimisation was based on maximising combined sugar yield from the combined pretreatment-hydrolysis process. A central composite design (CCD) was applied to investigate the effects of temperature, acid concentration and residence time on the responses and was later used to determine the maximum combined sugar yield. Pretreatment optimisation was conducted at gram scale (22.9 ml reactor) and at bench scale (1000 ml reactor). Significant differences in sugar yields (xylose, glucose, and combined sugar) between the varieties were observed. The combined sugar yields from the best performing varieties and industrial bagasse at optimal pretreatment-hydrolysis conditions differed by up to 34.1% and 33% at gram and bench scale, respectively. A high ratio of carbohydrates to lignin and low ash contents increased the release of sugar from the substrates. At mild pretreatment conditions, the differences in bioconversion efficiency between varieties were greater than at severe conditions. This observation suggests that under less severe conditions the conversion efficiency was largely determined by the properties of the biomass. Furthermore, it was demonstrated that the pretreatment conditions with temperature ranged from 184 to 200 °C and varying residence time to provide a severity factor between 3.51 and 3.96 was observed to be the area in common where 95% of maximum combined sugar yield could be obtained. Simultaneous Saccharification and Fermentation (SSF) was performed on the unwashed pressed-slurry from bagasse pretreatment at conditions for maximum combined sugar yield at bench scale. Batch and fed-batch SSF feeding strategy at different solid loadings and enzyme dosages were used aiming to reach an ethanol concentration of at least 40 g/L. The results revealed significant improvement in overall ethanol yield after SSF for the selected varieties (84.5–85.6%) compared to industrial bagasse (74.8%). The maximum ethanol concentration from the best performing varieties was 48.6−51.3 g/l and for poor performing varieties was 37.1−38.3 g/l. Ethanol concentration in the fermentation broth was inversely correlated with lignin content and the ratio of xylose to arabinose, but it showed positive correlation with glucose yield from pretreatment-enzymatic hydrolysis. The overall assessment of the varieties showed greater improvement in combined ethanol yields per hectare (71.1–90.7%) for the best performing varieties with respect to industrial sugarcane. The performance in terms of ethanol yields of selected varieties from a number harvest years was evaluated. The results showed considerable variations in ethanol yields across harvests. The results showed that the best variety in terms combined ethanol yield was not maintained across harvests. The differences in ethanol yields were greater among the varieties than across the harvests. Prolonged severe drought significantly affected the ethnol yields of all varieties represented by lower and intermediate lignin content for cane yield compared to that which had highest lignin content. However, carbohydrates content in the bagasse and sugar yield/recovery between the harvest years did not change for the most of the varieties. In summary, the present study provides evidence of the impact of cultivar selection and pretreatment optimisation in increasing conversion efficiency of bagasse. The results demonstrate that varieties with lower lignin and ash content, as well as highly substituted xylan resulted in higher sugar and ethanol yields. These results suggest that lower process requirements can be achieved without adversely affecting juice ethanol and cane yield per hectare. Nonetheless, an attempt to reduce lignin content in the bagasse, to reduce processing requirements for ethanol production, can also target the improvement of crop tolerance toward severe drought conditions. / AFRIKAANSE OPSOMMING: Die ontwikkeling van ―energie-riet‖ rasse vir etanol produksie is goed op dreef, waar beide die sap (eerste generasie etanol) en die bagasse (tweede generasie etanol) geteiken word. Die groot aantal monsters wat tydens teling geproduseer word, bied egter die grootste uitdaging vir die identifisering van nuwe rasse ten einde energie-riet te ontwikkel. In die huidige studie is verdunde suurvoorbehandeling, ensiematiese hidrolise en fermentasie-prosesse gebruik om die verwerkbaarheid van bagasse (veselagtige residu gegenereer na sap suiker ekstraksie) van verskillende suikerrietrasse te evalueer om nuwe variëteite te selekteer wat eienskappe van verbeterde gekombineerde etanolopbrengs (etanol van sap en bagasse) per hektaar toon. Die impak van variëteit-seleksie op gekombineerde etanol opbrengs (etanol van sap en bagasse) per hektaar is ook beoordeel. In die eerste deel van hierdie studie het uit ‗n siftingsproses van 115 suikerriet rasse bestaan wat deur klassieke en presisie (geneties gemodifiseerde) teling gegenereer is. Die sifting was op agronomiese data gebaseer, asook op data van verdunde suur voorafbehandeling en ensimatiese hidrolise eksperimente wat op die bagasse fraksie van elke ras uitgevoer is. Die resultate het op groot variasie in die chemiese samestelling van die bagasse van verskillende rasse gedui. Die strukturele koolhidrate het tussen 66.6 en 77.6% droë massa (DM) gewissel, terwyl die lignien inhoud ‗n variasie van 14.4 en 23.1% DM getoon het. Verder het meeste van die presisie-teling variëteite ‗n hoër arabinoxilaan, maar ‗n laer lignien en as-inhoud as meeste van die klassieke teling rasse gehad. Die gekombineerde suikeropbrengs (GSO) van die bagasse na voorafbehandeling en ensimatiese hidrolise het ook beduidend tussen rasse gewissel, waar ‗n verskil van tot 27.9 g/100g (droë bagasse) waargeneem is. Daar was ‗n omgekeerde korrelasie tussen die gekombineerde suikeropbrengs en die lignien en as-inhoud gewees, maar die opbrengs het ‗n sterk positiewe korrelasie met die strukturele koolhidrate getoon. Die totale potensiële etanol opbrengs per hektaar wat vanaf die suikerriet se oplosbare en nie-oplosbare suikerinhoud bereken is, het ook beduidend tussen rasse verskil (8,602−18,244 L/ha), waar die potensiële etanol opbrengs van die bagasse gedeelte ongeveer een derde van die totale potensiële etanol opbrengs beslaan het. Interessante bevindinge het op sommige rasse met gekombineerde eienskappe van hoë potensiële opbrengs per hektaar asook ‗n hoë omskakelingsvermoë gedui. Derhalwe is ses variëteite (drie van elke telingstegnologie) as voorkeurvariëteite vir verdere studie gekies. Om die etanol opbrengs vanaf die bagasse te verbeter was voorafbehandeling van die voorkeurvariëteite geoptimeer, en waar industriële bagasse vir vergelykingsdoeleindes ingesluit was. Vir die optimering was dit ten doel gestel om die gekombineerde suikeropbrengs van die gekombineerde voorafbehandeling-hidrolise proses te maksimeer. ‗n Sentrale saamgestelde ontwerp (SSO) is gebruik om die effek van temperatuur, suurkonsentrasie en residensietyd op die responsveranderlikes vas te stel wat uiteindelik gebruik is om die maksimum gekombineerde suikeropbrengs te bepaal. Die optimering van die voorafbehandeling is op gram-skaal in ‗n 22.9 ml reaktor, asook op bank-skaal in ‗n 1000 ml reaktor uitgevoer. Beduidende verskille in die suikeropbrengs (xilose, glukose en gekombineerde suiker) is tussen die voorkeurrasse waargeneem. Tussen die rasse wat die beste gevaar het, asook die industriële bagasse, het die gekombineerde suikeropbrengs by optimale voorafbehandeling-hidrolise toestande onderskeidelik met tot 34.1% en 33% op gram-skaal en bank-skaal gevarieer. ‗n Hoë verhouding van koolhidrate tot lignien, asook ‗n lae as-inhoud het tot ‗n toename in die vrystelling van suiker uit die substraat gelei. By matige voorafbehandelingstoestande was die verskille in omskakelingseffektiwiteit tussen rasse groter as onder hewige toestande, wat daarop gedui het dat omskakelingseffektiwiteit grotendeels deur die eienskappe van die biomassa bepaal is. Verder is daar ook gedemonstreer dat die voorbehandelingsomstandighede met temperatuur tussen 184 en 200ºC en verandering van die residensietyd om 'n hewigheidsfaktor van tussen 3.51 en 3.96 te verskaf, 'n gemeenskaplike area gelewer het waar 95% van maksimum gekombineer suiker opbrengs (GSO) verkry kon word. Gelyktydige versuikering en fermentasie (GVF) is na voorafbehandeling op ongewaste, gepersde bagasse substraat by toestande vir die maksimum gekombineerde suikeropbrengs op bank-skaal uitgevoer. Bondel en voerbondel SSF voerstrategie by verskillende vaste ladings en ensiemdoserings is gebruik om 'n etanol konsentrasie van ten minste 40 g/L te bereik. Ná GVF was die algehele etanol opbrengs vir die voorkeurvariëteite (84.5–85.6%) beduidend beter relatief tot die industriële bagasse (74.8%). Die maksimum etanol opbrengs na SSF van die rasse met die beste prestasie was 48.6-51.3 g/L en 37.1-38.3 g/L vir rasse wat swak presteer het. Die etanol konsentrasie in die fermentasiesop was omgekeerd met lignien en die verhouding van xilose tot arabinose gekorreleer, maar was duidelik positief met die glukose opbrengs vanaf voorafbehandeling-hidrolise gekorreleer. ‗n Algemene assessering het op ‗n duidelike verbetering van die voorkeurvariëteite in terme van gekombineerde etanol opbrengs per hektaar gedui (71.1–90.7%), relatief tot die industriële suikerriet. Die prestasie in terme van etanol opbrengs van geselekteerde variëteite is oor 'n reeks oesjare ge-evalueer. Die resultate het aansienlike variasies in etanol opbrengs oor oesjare getoon. Die resultate het gewys dat die beste variëteite in terme van gekombineerde etanol opbrengs nie volhou is oor oeste nie. Die verskille in etanol opbrengste tussen variëteite was groter as die verskille oor oesjare. Verlengde ernstige droogte het die etanol opbrengs van alle variëteite met laer en intermediere lignien inhoud vir rietopbrengs aansienlik beinvloed, in vergelyking met dié wat die hoogste lignien inhoud gehad het. Die koolhidraatinhoud in die bagasse en suiker opbrengs/lewering tussen die oesjare het vir die meeste variëteite egter nie gewissel nie. Ter opsomming, die huidige studie verskaf bewyse van die impak van kultivarseleksie en voorbehandelings optimisering op die verhoging van die omskakelings-doeltreffendheid van bagasse. Die resultate wys dat variëteite met laer lignien- en asinhoud, en hoogs-gesubstitueerde xilaan hoër suiker- en etanol opbrengs gelewer het. Hierdie resultate stel voor dat verminderde voorbehandelingsvereistes bereik kan word sonder om die sap etanol en rietopbrengs per hektar te benadeel. Nieteenstaande, 'n poging om die lignien inhoud van die bagasse te verminder om die verwerkingsvereistes vir etanolproduksie te verminder, kan ook die verbetering van gewas-toleransie tov ernstige droogte-toestande teiken.

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.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Despite numerous attempts involving a variety of target genes, the successful transgenic manipulation of sucrose accumulation in sugarcane remains elusive. It is becoming increasingly apparent that enhancing sucrose storage in the culm by molecular means may depend on the modification of the activity of a novel gene target. One possible approach to identify target genes playing crucial coarse regulatory roles in sucrose accumulation is to assess gene expression during the developmental transition of the culm from active growth to maturation. This study has resulted in the successful optimisation of a mRNA hybridisation technique to characterise the expression of 90 carbohydrate metabolism-related genes in three developmentally distinct regions of sugarcane culm. A further goal of this work was to extend the limited knowledge of the regulation of sucrose metabolism in sugarcane, as well as to complement existing data from physiological and biochemical studies. Three mRNA populations derived from the different culm regions were assayed and their hybridisation intensities to the immobilised gene sequences statistically evaluated. The relative mRNA transcript abundance of 74 genes from three differing regions of culm maturity was documented. Genes exhibiting high relative expression in the culm included aldolase, hexokinase, cellulase, alcohol dehydrogenase and soluble acid invertase. Several genes (15) were demonstrated to have significantly different expression levels in the culm regions assessed. These included UDP-glucose pyrophosphorylase and UDP-glucose dehydrogenase, which were down-regulated between immature and mature internodes. Conversely, sucrose phosphate synthase, sucrose synthase and neutral invertase exhibited up-regulation in maturing internodal tissue. A variety of sugar transporters were also found to be up-regulated in mature culm, indicating a possible control point of flux into mature stem sink tissues. Combined with knowledge of the levels of key metabolites and metabolic intermediates this gene expression data will contribute to identifying key control points of sucrose accumulation in sugarcane and assist in the identification of gene targets for future manipulation by transgenic approaches. / AFRIKAANSE OPSOMMING: Ondanks verskeie pogings, waartydens verskeie gene geteiken is, is daar nog weinig sukses behaal om sukrose-akkumulering te verhoog. Toenemend wil dit voorkom asof suksesvolle genetiese manipulering van sukroseberging in die stingel van die verandering van ‘n nuwe geen afhanklik sal wees. Een van die moontlike benaderings wat gevolg kan word om potensiële teiken gene wat ‘n belangrike rol in die beheer van sukrose-opberging speel te identifiseer, is om geen uitdrukkingspatrone in die stingel tydens die omskakeling van aktiewe groei tot volwassenheid te karakteriseer. In hierdie studie is ‘n metode gebaseer op die hibridisering van mRNA geoptimiseer en suksesvol aangewend om die uitdrukkingspatrone van 90 verskillende geselekteerde gene, wat vir sleutelensieme in die beheer van koolhidraatmetabolisme kodeer, te bestudeer. Die doel met die ondersoek was om die beperkte kennis oor die regulering van koolhidraatmetabolisme uit te brei en om die bestaande inligting afgelei van fisiologiese en biochemiese-studies aan te vul. Drie verskillende mRNApopulasies, verkry uit verskillende dele van die stingel, is ontleed deur verskillende peilers te gebruik. Die gegewens is statisties ontleed en dit het afleidings oor die verandering in uitdrukking van hierdie gene moontlik gemaak. Die relatiewe konsentrasies van 74 verskillende gene is gedokumenteer. Gene wat sterk uitgedruk word het aldolase, heksokinase, sellulase, alkoholdehidrogenase en ongebonde suurinvertase ingesluit. Die uitdrukkingspatrone van 15 gene het tussen die verkillende weefsels gevarieer. Gene waarvan die uitdrukking tydens die oorgang na volwassenheid verlaag sluit in UDP-glukose pirofosforilase en UDP-glukose dehidrogenase en waarvan die uitdrukking verhoog sukrosefosfaatsintase, sukrosesintase en neutrale invertase in. Die uitdrukking van verskeie suikertransporter gene verhoog tydens volwassewording. Hierdie inligting te same met die huidige kennis oor heersende metabolietvlakke sal bydrae tot die identifisering van geenteikens vir toekomstige genetiese manupulering.