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Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Soil salinity is a major limiting factor of plant and crop growth, because the absorption of water and nutrients is such a complex process while low and moderate salinity are omnipresent. Plant growth is affected negatively if a specific ion concentration exceeds its threshold and becomes toxic. The detrimental effect of soil affected by salt on crop production is increasing worldwide (Tanji, 1990). The level to which plants can tolerate high salinity levels is genetically controlled with several physiological and genetic mechanisms contributing to salt tolerance (Epstein & Rains, 1987). The most effective way of addressing the limitations of crop productivity in saline areas, is the development of salt tolerant varieties. Understanding the genetics of salt tolerance is, therefore, necessary for the development of an effective breeding strategy for salt tolerance. The department of Genetics (US) conducts a wide crosses research programme aiming to transfer genes for salt tolerance to wheat and triticale. The donor species, Thinopyrum disticum, an indigenous coastal wheat grass, adapted to high concentrations of salt, was crossed with cultivated rye (Secale cereale) in an attempt to study the genetics of salt tolerance (Marais et al., 1998). The primary goal of this study was to find molecular markers (RAPD and AFLP) which associate with chromosomes promoting salt tolerance for later attempts to transfer the genes to triticale. Seventy clones of secondary hybrids (Th disticum /4x-rye 1/2x-rye) were tested for salt tolerance and showed different levels of salt tolerance. RAPD-marker analyses were used to identify polymorphisms between salt tolerant and salt sensitive plants. Twelve RAPD primers produced clear, analyzable and repetitive polymorphic . fragments that can be used as useful markers. Different AFLP-primer combinations were tested against the genotypes of 15 clones (Marais & Marais 2001, unpublished data) and produced approximately 2000 clearly distinguishable AFLP fragments, of which 54 (3%) were polymorphic fragments. Two RAPD fragments and 4 AFLP fragments that can be used as possible markers for the presence of chromosomes that contribute to salt tolerance were identified. The interpretation of the markers was complicated by heterogeneity among plants with regard to the origin of their chromosomes and the genetic diversity of the rye genome. It is also possible that chromosome re-arrangement took place during backcrossing, which could have complicated the data. / AFRIKAANSE OPSOMMING: Versouting is een van die groot beperkende faktore op plant- en gewasgroei, omdat die opname van water en voedingstowwe so In ingewikkelde proses is en die effek van lae of matige versouting so alomteenwoordig is. Plantgroei word nadelig geaffekteer as 'n spesifieke ioonkonsentrasie sy drempelwaarde oorskry en toksies word. Die nadelige effek van soutgeaffekteerde grond op gewasproduksie, is wêreldwyd aan die toeneem (Tanji, 1990). Die vlak waartoe plante hoë konsentrasies sout kan hanteer is onder genetiese beheer met verskeie fisiologiese en genetiese meganismes wat 'n bydrae maak tot soutverdraagsaamheid (Epstein & Rains, 1987). Die mees effektiewe manier om die beperkinge op gewas produktiwiteit in versoute gebiede te oorkom, is die ontwikkeling van soutverdraagsame variëteite. Begrip van die genetika van soutverdraagsaamheid is dus noodsaaklik vir die ontwikkeling van In effektiewe telingsstrategie. Die departement Genetika (US) bedryf tans 'n wye-kruisings navorsingsprogram waarmee gepoog word om gene vir soutverdraagsaamheid na korog en koring oor te dra. Die skenkerspesie, Thinopyrum disticum, In inheemse strandkoringgras wat aangepas is by hoë konsentrasies sout, is gekruis met verboude rog (Secale cereale) in 'n poging om die oorerwing van soutverdraagsaamheid te bestudeer (Marais et al., 1998). Die hoofdoel van hierdie studie was om molekulêre merkers (RAPD en AFLP) te vind, wat assosieer met chromosome wat soutverdraagsaamheid bevorder en om nuttige merkers daar te stel vir latere pogings om die gene na korog en koring oor te dra. Ongeveer 70 klone van sekondêre hibriede (Th distichum I 4x-rog /I 2x-rog) is onderwerp aan souttoetse en het verskillende grade van soutverdraagsaamheid getoon. RAPDmerker analise is gebruik om polimorfismes te identifiseer tussen soutverdraagsame en soutsensitiewe plante. Twaalf RAPD inleiers het duidelike, ontleedbare en herhalende polimorfiese fragmente opgelewer en moontlike nuttige merkers uitgewys. Verskillende AFLP-inleier kombinasies, wat getoets is teen die genotipes van 15 klone (Marais & Marais, 2001 ongepubliseerde data) het ongeveer 2000 duidelik onderskeibare AFLP fragmente geproduseer, waarvan 54 (3%) polimorfiese fragmente was. Twee RAPD fragmente en 4 AFLP fragmente is geïdentifiseer wat as moontlike kandidaat merkers gebruik kan word vir die identifisering van chromosome wat bydra tot soutverdraagsaamheid . Die interpretasie van die merkers is bemoeilik deur heterogeniteit tussen die plante wat betref die agtergrond van chromosome wat hulle besit en die genetiese diversiteit van die rog genoom. Dit is ook moontlik dat chromosoom herrangskikking plaasgevind het tydens terugkruising, wat die data verder kon kompliseer.

Dissertation (PhD) -- University of Stellenbosch, 2004. / ENGLISH ABSTRACT: In most plants the efficiency of sucrose production and the systemic distribution thereof are the major determinants of growth, development and yield. The factors governing sugar partitioning co-ordinate its distribution in response to intrinsic and environmental signals. These factors include sugar transporters and invertases as well as metabolites, including sucrose and glucose, which function as signalling molecules to modulate gene expression. The genetic transformation of plants and the subsequent development of transgenic lines with disturbed sugar metabolism have made an unprecedented impact on the study of sugar translocation and -partitioning. For instance, the transformation of plants with a yeast-derived invertase targeted to different subcellular compartments has led to the elucidation of several key aspects of sugar metabolism, including phloem loading mechanisms, the regulation of photosynthesis by sugars, the importance of sugar-metabolism compartmentation with regards to sucrose biosynthesis, storage and distribution, as well as the role of cell-wall invertase in phloem unloading and sink strength. In this study, a similar strategy of transgenic plant analysis was employed to expand our insight into the regulation of sugar partitioning. The yeast-invertase Suc2 gene, from Saccharomyces cere visiae , was overexpressed in either the cytosol, vacuole or apoplast of transgenic tobacco plants. These transgenic lines displayed varying increases in invertase activity, altered sugar levels and consequently disturbed sink-source interactions and sugar partitioning. Transgenic lines overproducing the yeast-derived invertase in either the vacuole (Vac-Inv) or apoplast (Apo-Inv) were utilised to analyse the effect of the altered sugar levels in sink and source organs on the expression of sugar transporters, as well as the endogenous cell wall invertase and inhibitors in these plants. Transcript levels of the sucrose transporter NtSUT1 and hexose transporter NtMST1 encoding genes increased significantly in the source leaves and roots of Vac-Inv lines, whereas increased NtMst1 transcript levels were also detected in the roots of Apo-Inv lines. The increased mRNA levels could be correlated to the altered invertase activities and sugar levels in these tissues. It is concluded that NtSUT1 and NtMST1 are differentially regulated by sucrose and/or hexose content on a transcriptional level. Furthermore, the regulatory effect of the altered sugar levels on transporter expression depended on the subcellular compartment in which the yeast invertase was expressed. It would seem that the subcellular compartmentation of sugar metabolism is also fundamental to the regulation of sugar partitioning. The transcription levels of the endogenous cell wall invertase (CWt) and cell wall invertase inhibitor (Cwi-Inh) genes were examined in the various tissues of Apo-Inv and Vac-Inv lines at both the vegetative and flowering growth stages. In comparison with the control lines, the various tissues of the Apo-Inv and Vac-Inv lines displayed altered Cwi and Cwi-Inh expression levels, depending on the sink-source status and growth stage. However, no obvious correlation between the Cwi and Cwi-Inh expression levels and soluble sugar content of these tissues was found. It is suggested that the post-transcriptional and post-translation control of these proteins by sugars might play an important role in their regulation. Analysis of the Cwi:Cwi-lnh mRNA ratio and growth observations of the various tissues of control as well as Apo-Inv and Vac-Inv lines indicated that this transcription ratio could be an accurate indicator of the sink strength of sink organs. In addition, the influence of sink-source interactions on sugar partitioning was investigated. Reciprocal grafting between Apo-Inv and control lines resulted in scions with an altered sucrose metabolism in either the sink or source organs. These scions were subjected to biomass distribution, soluble sugar quantification and C4C]- radiolabelling experiments. The latter revealed an unaltered state of sugar partitioning from the above-ground tissues of the Apo/GUS scions and a significant shift in sugar partitioning towards the roots of the GUS/Apo scions in comparison to the control GUS/GUS scions. Phenotypic changes, opposite to those observed in Apo-Inv lines expressing the heterologous invertase in both sink and source organs, could initially be observed in the GUS/Apo and Apo/GUS scions. However, no significant differences in phenotype or biomass distribution could be observed between the mature GUS/Apo, Apo/GUS and GUS/GUS scions seven weeks postgrafting. This inconsistency between phenotype and sugar partitioning might be explained by an increase in the respiration rate of the tissues as supported by the soluble sugar content. These results highlight the complexity and adaptability of sucrose metabolism and sugar partitioning. In addition, it confirms that sugar partitioning can be modulated by sink-source interactions and emphasise the importance of invertases in the regulation of sugar partitioning through its ability to alter sink strength. This study forms part of the rapidly expanding initiative to unravel the control mechanisms of sugar partitioning. The results obtained in this study confirmed again that the introduction and expression of a single heterologous gene in transgenic plants could provide significant insight into the regulation of this process. It was shown here that the expression of sugar transporters is closely regulated by sugar levels and therefore fulfils a vital function in sugar sensing and consequently the regulation of sugar partitioning. The data presented in this study also demonstrated the intricate and flexible nature of the relationship that exists between sugar metabolism, partitioning and growth phenomena. / AFRIKAANSE OPSOMMING: Die doeltreffendheid van sukroseproduksie, tesame met die sistemiese verspreiding daarvan, is die vernaamste faktore wat die groei, ontwikkeling en opbrengsvermoë van die meeste plante bepaal. Die faktore wat suikerverdeling beheer, funksioneer om suikerverspreiding te koordineer in reaksie op beide inherente- en omgewingsseine. Hierdie faktore sluit suikertransporters en invertases in, asook metaboliete soos sukrose en glukose wat funksioneer as seinmolekule in die modulering van geenuitdrukking. Die genetiese transformasie van plante en die gevolglike daarstelling van transgeniese lyne met veranderde suikermetabolismes het 'n beduidende inwerking op die bestudering van suikervervoer en -verdeling gehad. Byvoorbeeld, die transformasie van plante met 'n gis-invertase geteiken na verskillende sub-sellulêre kompartemente, het tot die toeligting van verskeie aspekte van suikermetabolisme gelei, insluitende dié van floëemladingsmeganismes, die regulering van fotosintese deur suikers, die belang van kompartementalisering ten opsigte van sukrosebiosintese, -opberging en -verspreiding, en die rol van selwand-invertases in floëemontlaaiing en swelgpuntkrag. In hierdie studie is van soortgelyke transgeniese plantontledings gebruik gemaak om 'n dieper insig tot die regulering van suikerverdeling te verkry. Die gis-invertase Suc2 geen, afkomstig van Saccharomyces cerevisiae, is ooruitgedruk in óf die sitosol, vakuool óf apoplastiese ruimte van transgeniese tabakplante. Hierdie transgeniese lyne het wisselende toenames in invertase-aktiwiteite en veranderde suikervlakke getoon, asook gevolglike versteurde bron-swelgpunt interaksies en suikerverdeling. Transgeniese lyne met ooruitdrukking van die gis-invertase in óf die vakuool (Vac-Inv) óf die apoplast (Apo-Inv) is gebruik om die gevolg van die veranderde suikervlakke in bron- en swelgpuntorgane op die uitdrukking van suikertransporters, asook die endogene selwand-invertase en invertase-inhibitor in hierdie plante te bepaal. Transkripsievlakke van die sukrosetransporter NtSut1 en die heksosetransporter, NtMst1, het beduidend toegeneem in die bron-blare en wortels van die Vac-Inv lyne; 'n toename in NtMst1 transkripsievlakke is ook in die wortels van Apo-Inv lyne bevestig. Die toenames in boodskapper RNA kon gekorreleer word met die veranderde invertase-aktiwiteite en suikervlakke in hierdie weefsels. Die gevolgtrekking word gemaak dat NtSUT1 en NtMST1 differensieël gereguleer word op transkripsionele vlak deur die sukrose en/of heksose inhoud van weefsels. Meer nog, die regulerende effek van die veranderde suikervlakke op transporteruitdrukking het afgehang van die subsellulêre kompartement waarin die gis-invertase uitgedruk is. Dit wil dus voorkom dat die subsellulêre kompartementalisering van suikermetabolisme fundamenteel tot die deurgee en waarneming van suikerseine is, met In gevolglike eweneens belangrike rol in die regulering van suikerverdeling. Die transkripsievlakke van beide die endogene selwand-invertase (CWI) en die selwand-invertase-inhibitor (CWI-Inh) enkoderende gene is in verskeie weefsels van die Apo-Inv en Vac-Inv lyne, tydens beide die vegetatiewe- en blomstadia, bestudeer. Die onderskeie weefsels van die Apo-Inv en Vac-Inv lyne het, in vergelyking met die kontrole lyne, veranderde Cwi en Cwi-inh transkripsievlakke getoon wat bepaal is deur bron-swelgpunt status en groeistadium. Geen duidelike korrelasie kon tussen beide Cwi en Cwi-inh uitdrukkingsvlakke en oplosbare suiker inhoud gevind word nie. Daar word voorgestel dat post-transkripsionele en posttranslasionele beheer deur suikers 'n belangrike rol in die regulering van hierdie proteïne speel. Bestudering van die Cwi:Cwi-lnh mRNA verhouding, asook groei verskynsels van die onderskeie weefsels van kontrole en Apo-Inv en Vac-Inv lyne, dui daarop dat hierdie transkripsievlak-verhouding moontlik 'n akkurate aanwyser van die swelgpuntkrag van 'n swelgpuntorgaan kan wees. Voorts is die invloed van bron-swelgpuntorgaan interaksies op suikerverdeling ondersoek. Omgekeerde enting tussen Apo-Inv en kontrole lyne het entlote met gemodifiseerde suikermetabolisme in óf hul bron- óf hul swelgpuntorgane tot gevolg gehad. Hierdie entlote is aan biomassaverspreidings-, oplosbare suiker kwantifisering en C4C]-radiomerking eksperimente onderwerp. Hierdie resultate het gewys dat, in vergelyking met die kontrole (GUS/GUS) ente, daar geen verandering in die status van suikerverdeling vanaf die bogrondse plantdele in die Apo/GUS ente is nie, maar wel 'n beduidende verskuiwing in suikerverdeling na die wortels van die GUS/Apo ente. Fenotipiese veranderinge, wat teenoorgesteld van dié teenwoordig in die Apo- Inv lyne waar die heteroloë invertase in beide bron en swelgpuntorgane uitgedruk word, is aanvanklik in die GUS/Apo en Apo/GUS ente waargeneem. Geen verskille in fenotipe of biomassa-verspreiding kon egter sewe weke na die entings prosedures tussen die GUS/Apo, Apo/GUS and GUS/GUS ente gevind word nie. Dit mag verduidelik word deur 'n moontlike toename in respirasietempo in die betrokke weefsels; die oplosbare suikervlakke wat in die verskillende ente aangeteken is ondersteun dié moontlikheid. Hierdie resultate as geheelonderstreep die kompleksiteit en aanpasbaarheid van suikermetabolisme en -verdeling. Verder bevestig dit dat suikerverdeling beïnvloed kan word deur bron-swelgpunt interaksies, asook die belang van invertases in die regulering van suikerverdeling gegewe die vermoë om swelgpuntkrag te verander. Hierdie studie vorm deel van 'n vinnig groeiende inisiatief om die beheermeganismes van suikerverdeling te ontrafel. Die resultate verkry in hierdie studie bekragtig die belang van rekombinante DNA tegnologie in die bestudering van fundamentele plantprosesse. Die invoeging en uitdrukking van 'n geteikende gisinvertase in transgeniese plante het gelei tot veranderde suikervlakke en bronswelgpunt interaksies in hierdie lyne met die gevolglike ontginning van waardevolle inligting ten opsigte van die regulering van suikerverdeling in reaksie tot interne seine. Daar is aangetoon dat suikertransporters onlosmaakbaar gekoppel is aan die deurgee en waarneming van suikerseine, spesifiek op die vlak van transkripsionele regulering, en dus ook die regulering van suikerverdeling. Voorts wys die resultate op die komplekse en aanpasbare aard van die verhouding wat bestaan tussen suikermetabolisme, -verdeling en groeiverskynsels.

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The importance of small antimicrobial peptides in the innate immune system of plants became increasingly apparent over the past decade. Antimicrobial peptides are unique and diverse molecules that are found in many tissue types in a variety of invertebrate, plant and animal species. Many of these peptides, such as plant defensins, have been found to be ubiquitous throughout the plant kingdom and have been isolated from flowers, leaves, roots, seeds, seedlings, pods, tubers and bark. The growing relevance of antimicrobial peptides (including plant defensins) in research can be largely attributed to their broad-spectrum antifungal activity. This makes them promising potential targets, both as therapeutic agents and for their use in crop protection and disease resistance. The continuing discovery of novel antimicrobial peptides has advanced the development of strategies to overexpress these genes in plants to attempt to enhance the plant’s natural ability to resist pathogenic attack. The first grapevine antifungal peptide, Vv-AMP1, was isolated and characterized and was shown to be tissue specific and developmentally regulated, being expressed only in berries at the onset of berry ripening. The peptide showed strong antifungal activity against a number of plant pathogenic fungi in vitro. In this study, the biological role of the Vv-AMP1 peptide was further investigated, both within its native host (Vitis vinifera) and under in vitro conditions against a panel of grapevine-specific pathogens. As a first step, recombinant production of Vv-AMP1 using an existing bacterial expression system was evaluated and the heterologous production of the Vv-AMP1 peptide improved. Specific optimizations targeting both production and purification of the peptide showed to improve the yield of Vv-AMP1. Steps in the production process targeted for improvement included induction conditions of peptide production by the bacterial culture as well as a number of purification steps, such as lysate preparation, binding conditions, column washing, elution conditions and thrombin protease cleavage. The optimized purification method produced up to 3 mg of pure Vv-AMP1 peptide from 1.6 L of overnight culture. While production was markedly improved, the resultant purified Vv-AMP1 proved biologically inactive and structurally unstable. This is uncharacteristic of the peptide, suggesting that an important aspect necessary for peptide activity, such as folding or the presence of specific co-factors might not be supported in this non-host prokaryotic production system. The study also entailed the characterization and evaluation of the Vv-AMP1 peptide against a panel of grapevine-specific pathogens that are culturable to sporulating cultures using in vitro antifungal assays and microscopy analysis. Vv-AMP1 showed strong inhibitory activity against all pathogens tested, inhibiting the growth of Diplodia seriata and Cylindrocarpon liriodendri by 50% at concentrations between 4.8 μg/ml and 9.6 μg/ml. Phaemoniella chlamydospora and Phomopsis viticola proved particularly sensitive, with IC50 values of 5.5 μg/ml and 4.0 μg/ml respectively. Microscopy analysis of the effect of the Vv-AMP1 peptide on P. viticola showed a severe inhibition on fungal germination and growth. The peptide did not induce morphological changes in fungal hyphae but compromises the fungal membranes, supporting the theory that the peptide induces membrane permeabilization. Functional analysis of a transgenic V. vinifera (cv. Sultana) population overexpressing Vv-AMP1 was included in this study to provide the opportunity to study the in planta role of the peptide in its native host. The genetic characterization of the putative population included confirming gene integration and copy number through PCR and Southern blot analysis as well as gene expression through northern blot analysis. A confirmed transgenic population was evaluated for improved disease resistance against Botrytis cinerea as a first test organism in an attempt to link the overexpression of the Vv-AMP1 gene to a disease resistance phenotype. Observations of lesion type, average lesion size and further statistical analysis concluded that the transgenic population showed a definite, albeit slight, improved resistance when compared to the untransformed control lines. In conclusion, the study determined that Vv-AMP1 had a strong antifungal action against grapevine-specific pathogenic fungi when tested in vitro. A definite link could be established between the overexpression of Vv-AMP1 and a mild resistance phenotype within its native host plant. The characterized transgenic population is important for further work to evaluate the in planta activity of the peptide against more grapevine pathogens such as the stem pathogens that were proven sensitive and specifically those that cannot be cultured and are obligate pathogens, such as the downy and powdery mildews. / AFRIKAANSE OPSOMMING: Die belang van klein antimikrobiese peptiede in die ingebore immuunstelsel van plante het tydens die afgelope dekade toenemend duidelik geraak. Antimikrobiese peptide is unieke en diverse molekules wat in verskeie weefseltipes in ‘n verskeidenheid van invertebraat-, plant- en dierspesies gevind word. Baie van hierdie peptiede, soos bv. “plant defensins”, word bevind om alomteenwoordig in die plantryk te wees en is reeds geïsoleer vanuit blomme, blare, wortels, sade, saailinge, peule, knolle en bas. Die toenemende belang van antimikrobiese peptiede (insluitend “plant defensins”) in navorsing kan grootliks toegeskryf word aan hul breë-spektrum antifungiese aktiwiteit. Hierdie eienskap maak hul belowende potensiële teikens, beide as terapeutiese middels asook vir gebruik in gewasbeskerming en siekteweerstand. Die voortdurende ontdekking van nuwe antimikrobiese peptiede bevorder tans die ontwikkeling van strategieë om hierdie gene in plante uit te druk in ‘n poging om die plant se natuurlike vermoeë om patogeniese aanval teen te staan te verbeter. Die eerste wingerd antifungale peptied, Vv-AMP1, is geïsoleer en gekarakteriseer as ‘n ontwikkelings-gereguleerde peptied wat slegs uitgedruk word in korrels, tydens die aanvang van bessie rypwording. Die peptied het tydens in vitro toetse sterk antifungale aktiwiteit getoon teen ‘n verskeidenheid plant-patogeniese swamme. In hierdie studie word die biologiese rol van die Vv-AMP1 peptied verder ondersoek, beide binne sy natuurlike gasheerplant, (Vitis vinifera) asook onder in vitro kondisies teen ‘n paneel van wingerd-spesifieke patogene. As ‘n beginpunt is rekombinante produksie van Vv-AMP1 met behulp van ‘n bakteriële ekspressie sisteem evalueer en die hetereloë produksie van die Vv-AMP1 peptied stelselmatig verbeter. Spesifieke optimerings het gefokus op beide die produksie en suiwering van die peptied en het die algehele opbrengs van Vv-AMP1 verhoog. Spesifieke stappe wat in die produksieproses vir verbetering geteiken is sluit beide induksietoestande van peptiedproduksie deur die bakteriële kultuur in sowel as ‘n aantal suiweringsstappe, soos lisaatvoorbereiding, bindingskondisies, kolom wasstappe, eluasie kondisies en “thrombin” protease snyding in. Die optimale suiweringsmetode het tot 3 mg suiwer Vv-AMP1 peptied opgelewer vanaf ‘n 1.6 L oornag bakteriële kultuur. Hoewel die produksie van die peptide noemenswaardig verbeter is, was die gesuiwerde Vv-AMP1 beide onaktief en struktureel onstabiel. Dit is buitengewoon vir hierdie peptied, wat daarop dui dat belangrike aspekte benodig vir antifungiese aktiwiteit, soos korrekte vou of die teenwoordigheid van spesifieke kofaktore, moontlik ontbreek in hierdie nie-gasheer prokariotiese produksiesisteem. Die studie het ook die karakterisering en evaluering van die Vv-AMP1 peptied teen 'n paneel van wingerd-spesifieke patogene wat kultureerbaar is en sporuleer, insluitend in vitro antifungale toetse en mikroskopiese analise, behels. Vv-AMP1 toon sterk inhiberende aktiwiteit teen alle patogene getoets. Dit inhibeer die groei van Diplodia seriata en Cylindrocarpon liriodendri met 50% teen konsentrasies tussen 4.8 μg/ml en 9.6 μg/ml. Phaemoniella chlamydospora en Phomopsis viticola was besonders sensitief, met IC50 waardes van 5.5 μg/ml en 4.0 μg/ml, onderskeidelik. Mikroskopiese analise van die effek van die Vv-AMP1 peptied op P. viticola het 'n ernstige inhibisie op swam ontkieming en groei aangedui. Die peptied het geen morfologiese veranderinge in swam hifes veroorsaak nie maar het wel die swam membraan beskadig. Hierdie bevinding ondersteun die teorie dat die peptied membraan permeabilisasie induseer. Funksionele analise van ‘n transgeniese V. vinifera (cv. Sultana) populasie wat die Vv-AMP1 geen ooruitdruk is by die studie ingesluit om ‘n geleentheid te bied om die in planta rol van die peptide binne sy natuurlike gasheerplant te bestudeer. Die genetiese karakterisering van die vermeende transgeniese bevolking het die bevestiging van beide geenintegrasie en kopiegetal deur PKR en Southern-klad analise ingesluit, sowel as geenuitdrukking d.m.v. noordelike-klad analise. ‘n Bevestigde transgeniese bevolking is evalueer vir potensiële verbeterde weerstand (in vergelyking met die wilde tipe) deur infeksie met Botrytis cinerea as ‘n eerste toetsorganisme in ‘n poging om ‘n weerstandbiedende fenotipe met die ooruitdrukking van Vv-AMP1 te assosieer. Waarnemings van letsel tipe, letsel grootte en verdere statistiese analise het tot die gevolgtrekking gelei dat die transgeniese bevolking ‘n definitiewe (dog geringe) verbeterde weerstand toon in vergelyking met die ongetransformeerde lyne. Ten slotte bepaal die studie dat Vv-AMP1 ‘n sterk antifungale effek teen wingerdspesifieke patogene toon tydens in vitro toetse. ‘n Definitiewe korrelasie is vasgestel tussen die ooruitdrukking van Vv-AMP1 in wingerd en ‘n weerstandsfenotipe in die transgeniese bevolking. Die gekarakteriseerde transgeniese bevolking is uiteraard belangrik vir toekomstige werk om die in planta aktiwiteit van die peptied te evalueer teen verdere wingerdpatogene soos bv. die stampatogene wat sensitief getoets het teen die peptide, asook patogene wat nie kultureerbaar is nie, insluitend verpligte patogene soos dons- en poeierskimmel.

Orientador: Ivan de Godoy Maia / Banca: Paulo Eduardo Martins Ribolla / Banca: Marcelo Menossi Teixeira / Resumo: As proteínas desacopladoras pertencem à família de carreadores aniônicos mitocondriais. De maneira geral, as proteínas desacopladoras dissipam o gradiente eletroquímico de prótons gerados na respiração na forma de calor, sendo dependentes de ácidos graxos e sensíveis aos nucleotídeos purínicos. O presente estudo visou investigar o comportamento de plantas transgênicas de tabaco que expressam de forma constitutiva o gene AtUCP1, frente aos estresses osmótico e salino, bem como analisar a atividade das regiões promotoras dos genes AtUCP1 e AtUCP2 de Arabidopsis thaliana, em resposta aos estresses osmótico e de baixa temperatura, e ao ácido abscísico. Numa primeira abordagem foram utilizadas sementes selvagens e de duas linhagens transgênicas, germinadas em meio MS adicionados ou não de NaCl e Manitol. O teste de germinação revelou que as linhagens transgênicas apresentam uma maior tolerância aos referidos estresses. Quando o crescimento radicular foi analisado, uma maior inibição foi constatada no controle não transgênico em relação às duas linhagens transgênicas testadas. Adicionalmente, quando submetidas aos estresses, uma maior acumulação de ânion superóxido foi verificada nas folhas de plântulas não transgênicas em relação às plântulas das linhagens transgênicas. Quanto à quantificação de GUS nas plantas transformadas com os promotores dos genes AtUCP1 e AtUCP2, nenhuma alteração significativa foi observada em nenhum dos tratamentos testados / Abstract: The uncoupling proteins belong to the mitochondrial anion carrier family. In general, the uncoupling proteins dissipate the proton electrochemical gradient generated in respiration as heat, being dependent on fatty acids and sensitive to purine nucleotides. In the present study, we investigated the behavior of transgenic tobacco plants that overexpress the AtUCP1 gene when subjected to osmotic and saline stress, as well as the activity of the promoters of the AtUCP1 and AtUCP2 genes of Arabidopsis thaliana, in response to osmotic and cold stress, and abscisic acid. In the first approach, seeds from wild type and two transgenic lines were germinated in MS medium containing (or not) NaCl and mannitol. The germination test showed that the transgenic lines have a higher stress tolerance. When root growth was analyzed, a greater inhibition was observed in non-transgenic control seedlings as compared to seedlings of the two transgenic lines tested. Additionally, when subjected to stress, a greater superoxide anion accumulation was detected in leaves of non-transgenic seedlings as compared to seedlings of transgenic lines. Quantification of GUS activity in the plants transformed with the tested promoters, revealed no treatmentspecific differences / Mestre

Desde seu descobrimento, o fenômeno de silenciamento gênico por RNA (RNAi) rapidamente se tornou uma técnica amplamente estudada e utilizada nos mais diversos aspectos da biologia molecular. Uma destas possibilidades é sua aplicação no campo da entomologia agrícola, mais especificamente para o controle de insetos-praga como uma alternativa de alta eficiência, especificidade e com impacto ambiental reduzido. Por meio da geração de plantas transgênicas expressando RNAi para genes essenciais de insetos-praga específicos, a ingestão destas moléculas de RNAi pelo inseto mediante herbivoria pode resultar no silenciamento do respectivo gene, resultando em fenótipos que podem variar entre perda de apetite, infertilidade ou até a morte. Neste contexto, o presente trabalho teve como objetivo provar a viabilidade de aplicação desta técnica para a interação Tomateiro x Tuta absoluta, cultura de grande expressão econômica e social no mercado nacional e internacional e que é amplamente atacada por esta praga, com prejuizos que podem alcançar a ordem dos 100% da produção. Por meio da clonagem de genes ortólogos essenciais descritos na literatura e de genes altamente expressos nos primeiros estádios larvais, após a caracterização transcriptômica em escala do inseto, foram realizados ensaios de alimentação contendo moléculas de dsRNAs que possuíam estes genes como alvo. Também, foi realizado a transformação genética de tomateiro cultivar \"Micro-Tom\" com dois destes genes (V-ATPase A e Arginina kinase) para a realização de ensaios de herbivoria. Com os resultados obtidos nestes experimentos, foram mostradas sólidas evidências da viabilidade da técnica de RNAi para o controle de Tuta absoluta, evidenciado pelo silenciamento gênico específico observado no inseto e consequentemente os efeitos nocivos deste silenciamento. / Since their discovery, the phenomenon of gene silencing by RNA ( RNAi ) has rapidly become a widely studied and used technique in the molecular biological field. One of these possible applications is in the entomology field, more specifically for the control of insect pests, as a high efficiency, specificity and with reduced environmental impact alternative. Through the generation of transgenic plants expressing dsRNA targeting essential insect genes, their ingestion by the insect and consequently the uptake of the silencing RNA, may result in specific gene silencing, resulting in a variety of phenotypes that can range from loss of appetite, infertility to death. In this context, this study aimed to prove the feasibility of this technique to control tomato leaf miner (Tuta absoluta) in tomatoes plant, a major crop worldwide and seriously attacked by this pest, with losses that can reach 100%. For the present work, orthologous genes from successfully cases of insect gene silence described in the literature, was selected together with highly expressed genes in the early larval stages of T. absoluta, chosen after the insect molecular characterization and used in feeding assays with dsRNAs molecules to targeted these genes. Also, genetic transformation of the \"Micro-Tom\" tomato cultivar with two of these genes (V-ATPase and Arginine kinase) was conducted for testing in an herbivore assay. With these two approaches was possible to get solid evidences of the feasibility of the RNAi technique to control this insect, evidenced by specific gene silencing observed and its consequent effect on pest phenotype.

Cheung Ming-yan. / Thesis submitted in 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 169-185). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vi / General abbreviations --- p.viii / Abbreviations of chemicals --- p.x / List of figures --- p.xii / List of tables --- p.xv / Table of Contents --- p.xvii / Chapter Chapter 1 --- General Introduction - Using transgenic plants as bioreactor --- p.1 / Chapter 1.1 --- Plant as Bioreactor --- p.1 / Chapter 1.1.1 --- Plant transformation historical milestones --- p.1 / Chapter 1.1.2 --- Applications of transgenic plants --- p.5 / Chapter 1.1.2.1 --- Examples of in situ Application --- p.5 / Chapter 1.1.2.2 --- Examples of ex situ application of transgenic plant --- p.9 / Chapter 1.2 --- Plant Hosts for Transformation: Arabidopsis thaliana and Glycine max --- p.18 / Chapter 1.2.1 --- Essential components for plant transformation --- p.18 / Chapter 1.2.1.1 --- Marker genes --- p.18 / Chapter 1.2.1.2 --- Promoters --- p.18 / Chapter 1.2.2 --- Arabidopsis thaliana --- p.20 / Chapter 1.2.2.1 --- Agrobacterium-mediated transformation --- p.20 / Chapter 1.2.2.2 --- Transformation methods for A. thaliana --- p.21 / Chapter 1.2.3 --- Glycine max (soybean) --- p.22 / Chapter 1.2.3.1 --- Soybean cultivars for transformation --- p.23 / Chapter 1.2.3.2 --- Soybean regeneration systems --- p.24 / Chapter 1.2.3.3 --- Soybean transformation systems --- p.26 / Chapter 1.3 --- Target Pharmaceutical and Agricultural Proteins: Lymphocytic choriomeningitis virus and Goldfish Growth hormones I and II --- p.29 / Chapter 1.3.1 --- Production of pharmaceutical proteins --- p.29 / Chapter 1.3.1.1 --- Lymphocytic choriomeningitis virus --- p.30 / Chapter 1.3.1.2 --- Nucleoprotein of LCMV --- p.33 / Chapter 1.3.2 --- Agricultural protein category --- p.34 / Chapter 1.3.2.1 --- Carassius auratus --- p.34 / Chapter 1.3.2.2 --- Growth hormones I and II --- p.35 / Chapter 1.4 --- Hypothesis and Objectives --- p.37 / Chapter Chapter 2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.38 / Chapter 2.1.1 --- "Plants, bacterial strains and vectors" --- p.38 / Chapter 2.1.2 --- Chemicals and Regents --- p.43 / Chapter 2.1.3 --- Commercial kits --- p.44 / Chapter 2.1.4 --- Primers and Adaptors --- p.45 / Chapter 2.1.5 --- Equipments and Facilities used --- p.47 / Chapter 2.1.6 --- "Buffer, solution and medium" --- p.47 / Chapter 2.2 --- Methods --- p.48 / Chapter 2.2.1 --- Molecular Techniques --- p.48 / Chapter 2.2.1.1 --- Bacterial cultures for recombinant DNA and plant transformation --- p.48 / Chapter 2.2.1.2 --- Recombinant DNA techniques --- p.48 / Chapter 2.2.1.3 --- "Preparation and transformation of DH5a, DE3 and Agrobacterium competent cells" --- p.49 / Chapter 2.2.1.4 --- Gel electrophoresis --- p.52 / Chapter 2.2.1.5 --- "DNA, RNA and protein extractions" --- p.53 / Chapter 2.2.1.6 --- Generation of cRNA probes for Southern and Northern blot analyses --- p.56 / Chapter 2.2.1.7 --- Southern blot analysis --- p.56 / Chapter 2.2.1.8 --- Northern blot analysis --- p.57 / Chapter 2.2.1.9 --- Expression of Lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) in bacterial system --- p.58 / Chapter 2.2.1.10 --- Western blot analysis for LCMV NP --- p.59 / Chapter 2.2.1.11 --- Protein dot blot for detecting the presence of recombinant LCMV-NP generated from transgenic plants --- p.62 / Chapter 2.2.1.12 --- PCR techniques --- p.62 / Chapter 2.2.1.13 --- Sequencing --- p.63 / Chapter 2.2.2 --- Plant tissue culture and transformation --- p.64 / Chapter 2.2.2.1 --- Arabidopsis thaliana --- p.64 / Chapter 2.2.2.2 --- Soybean --- p.65 / Chapter 2.2.3 --- In vitro transcription and translation of target genes in rabbit reticulocyte and wheat germ systems --- p.68 / Chapter 2.2.3.1 --- In vitro transcription of target genes with with Ribomix large scale RNA production systems-T7 and SP6 (Promega) --- p.68 / Chapter 2.2.3.2 --- In vitro translation with rabbit reticulocyte lysate and wheat germ extract --- p.69 / Chapter Chapter 3 --- Results --- p.71 / Chapter 3.1 --- Expression of Lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) and goldfish growth hormones I and II (GHI and GHII) in transgenic Arabidopsis thaliana --- p.71 / Chapter 3.1.1 --- Expression of LCMV-NP in transgenic Arabidopsis thaliana --- p.71 / Chapter 3.1.1.1 --- Cloning of the gene encoding LCMV NP into the binary vector system W104 --- p.71 / Chapter 3.1.1.2 --- Transformation of W104-LCMV-NP into the Agrobacterium GV3101/pMP90 --- p.78 / Chapter 3.1.1.3 --- Transformation of LCMV-NP cDNA into Arabidopsis thaliana --- p.80 / Chapter 3.1.1.4 --- Southern blot and Northern blot analyses of transgenic plant containing the LCMV-NP cDNA --- p.83 / Chapter 3.1.1.5 --- Production of recombinant LCMV-NP protein in DE3 cells --- p.90 / Chapter 3.1.1.6 --- Detection of recombinant LCMV-NP protein in transgenic A.thaliana --- p.98 / Chapter 3.1.2 --- Expression of goldfish growth hormones I and II (GHI and GHII) in transgenic Arabidopsis thaliana --- p.105 / Chapter 3.1.2.1 --- "Screening of homozygous lines of goldfish, Carassius auratus, growth hormones transgenic Arabidopsis thaliana" --- p.105 / Chapter 3.1.2.2 --- Southern blot and Northern blot analyses of transgenic plant containing the LCMV-NP cDNA --- p.109 / Chapter 3.1.2.3 --- Detection of recombinant GHI and GHII from transgenic plant --- p.112 / Chapter 3.2 --- In vitro transcription and translation of target genes in rabbit reticulocyte and wheat germ systems --- p.117 / Chapter 3.2.1 --- Subcloning of target genes in pGEM-3Zf(+) vector --- p.117 / Chapter 3.2.1.1 --- Subcloning of LCMV-NP fragment into pGEM-3Zf(+) vector --- p.117 / Chapter 3.2.1.2 --- Subcloning of goldfish GHI and GHII fragments into pGEM-3Zf(+) vector --- p.120 / Chapter 3.2.2 --- In vitro transcription of target genes with Ribomix large scale RNA production systems-T7 and SP6 --- p.125 / Chapter 3.2.3 --- In vitro translation with rabbit reticulocyte lysate and wheat germ extract systems --- p.128 / Chapter 3.3 --- Establishment of Glycine max regeneration and transformation systems --- p.130 / Chapter 3.3.1 --- The Establishment of soybean regeneration system --- p.130 / Chapter 3.3.2 --- Establishment of soybean transformation system --- p.133 / Chapter 3.3.2.1 --- Definition of transformation efficiency --- p.133 / Chapter 3.3.2.2 --- Effects of plant hosts --- p.136 / Chapter 3.3.2.3 --- Effects of Agrobacterium strains --- p.138 / Chapter 3.3.2.4 --- The application of vacuum infiltration --- p.139 / Chapter 3.3.2.5 --- Effect of kanamycin --- p.140 / Chapter 3.3.2.6 --- Effect of cocultivation duration and light/ dark treatment during germination --- p.141 / Chapter 3.3.2.7 --- Application of the detergent Silwet-77 --- p.142 / Chapter 3.3.3 --- Verification of transformation results by PCR screening --- p.143 / Chapter Chapter 4 --- Discussion --- p.147 / Chapter 4.1 --- "Expression of LCMV-NP, GHI and GHII in A. thaliana" --- p.148 / Chapter 4.2 --- Establishing a soybean transformation system --- p.157 / Chapter 4.2.1 --- Plant hosts and explants --- p.158 / Chapter 4.2.2 --- Regeneration of explants --- p.159 / Chapter 4.2.3 --- Agrobacterium strains --- p.161 / Chapter 4.2.4 --- Bacteria-plant interaction --- p.161 / Chapter 4.2.5 --- Transient versus stable transformation --- p.165 / Chapter 4.3 --- Conclusion and perspective --- p.167 / References --- p.169 / Appendix --- p.186

Piu Wong. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 126-146). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.viii / General abbreviations --- p.xi / Abbreviations of chemicals --- p.xiii / List of figures --- p.xv / List of Tables --- p.xvii / Table of contents --- p.xviii / Chapter 1 --- Literature review / Chapter 1.1 --- Overviews --- p.1 / Chapter 1.1.1 --- Nutritional and economical significance of aspartate family amino acidsin human and animal nutrition --- p.1 / Chapter 1.1.2 --- Synthesis of aspartate family amino acids in plants --- p.2 / Chapter 1.2 --- Regulation of aspartate family amino acids between sink and source organs --- p.6 / Chapter 1.2.1 --- Co-ordination of genes/enzymes involved in amide amino acid metabolism to channel aspartate for aspartate family amino acid synthesis --- p.6 / Chapter 1.2.2 --- Sink-source regulation as a general mechanism in plants --- p.9 / Chapter 1.3 --- Source regulation at free amino acid level --- p.11 / Chapter 1.3.1 --- Regulation of free methionine synthesis --- p.11 / Chapter 1.3.1.1 --- Competition for OPHS between TS and CGS --- p.11 / Chapter 1.3.1.2 --- Turnover of CGS mRNA --- p.12 / Chapter 1.3.1.3 --- Post-translational regulation of CGS enzyme --- p.13 / Chapter 1.3.2 --- Regulation of lysine synthesis and catabolism --- p.15 / Chapter 1.3.2.1 --- Feedback regulation loop --- p.15 / Chapter 1.3.2.2 --- Possible intracellular compartmentalization of enzymes and metabolitesin regulating lysine level --- p.21 / Chapter 1.3.2.3 --- Co-ordination of gene/enzyme in aspartate kinase pathway in regulating flux to Lys --- p.21 / Chapter 1.3.3 --- Significance of lysine catabolism in mammals and plants --- p.24 / Chapter 1.3.3.1 --- Complex developmental regulation and stress response of LKR/SDH gene expression --- p.28 / Chapter 1.3.3.2 --- Regulation through a novel composite locus LKR-SDH --- p.28 / Chapter 1.3.3.3 --- Post-translational control of LKR-SDH activity --- p.31 / Chapter 1.3.3.4 --- Implication of two metabolic flux in Lys catabolism --- p.34 / Chapter 1.4 --- Source (free lysine) enhancement in transgenic plants --- p.36 / Chapter 1.4.1 --- Expression of feedback insensitive enzyme in transgenic plants to enhance free lysine supply in transgenic plant --- p.36 / Chapter 1.4.2 --- Reducing or eliminating lysine catabolism to enhance free lysine poolin transgenic plants --- p.40 / Chapter 1.5 --- Sink regulation --- p.41 / Chapter 1.5.1 --- Engineering transgenic plants through expression of seed storage protein (sink) --- p.41 / Chapter 1.5.2 --- "Dynamic relationship between sink protein, nitrogen metabolism and sulphur metabolism" --- p.45 / Chapter 1.6 --- Transgenic plants with improved source or enhanced sinks related to aspartate family amino acids available for our research --- p.47 / Chapter 1.6.1 --- Enhanced source: ASN1 over-expressers --- p.47 / Chapter 1.6.2 --- Enhanced source: metL transgenic plants --- p.47 / Chapter 1.6.3 --- Altered source: RNAi line --- p.47 / Chapter 1.6.4 --- Effective sink: LRP transgenic plants --- p.48 / Chapter 1.7 --- Overall concept of this study --- p.48 / Chapter 2 --- Materials and methods --- p.50 / Chapter 2.1 --- Materials and growth conditions --- p.50 / Chapter 2.1.1 --- "Plants, bacterial strains and vectors" --- p.50 / Chapter 2.1.2 --- Chemicals and reagents used --- p.53 / Chapter 2.1.3 --- Solutions used --- p.53 / Chapter 2.1.4 --- Commercial kits used --- p.53 / Chapter 2.1.5 --- Equipment and facilities used --- p.53 / Chapter 2.1.6 --- Growth condition --- p.53 / Chapter 2.1.7 --- Tagging of A. thaliana siliques of different developmental stage --- p.54 / Chapter 2.2 --- Methods --- p.55 / Chapter 2.2.1 --- Expression pattern analysis --- p.55 / Chapter 2.2.1.1 --- RNA extraction --- p.55 / Chapter 2.2.1.2 --- Generation of single-stranded DIG-labelled ASN1 DNA probes --- p.55 / Chapter 2.2.1.3 --- Testing the concentration of DIG-labelled probes --- p.56 / Chapter 2.2.1.4 --- Northern blot --- p.57 / Chapter 2.2.1.5 --- Hybridization --- p.58 / Chapter 2.2.1.6 --- Stringency washes --- p.58 / Chapter 2.2.1.7 --- Chemiluminescent detection --- p.58 / Chapter 2.2.2 --- Amino acid analysis and nitrogen determination --- p.60 / Chapter 2.2.2.1 --- Free amino acids in A. thaliana --- p.60 / Chapter 2.2.2.2 --- Phloem exudates collection from A. thaliana --- p.60 / Chapter 2.2.2.3 --- Soluble Protein quantitation --- p.61 / Chapter 2.2.2.4 --- Extraction of salt and water soluble protein from A. thaliana seeds --- p.61 / Chapter 2.2.2.5 --- Purification and amino acid analysis of protein extracts from A. thaliana seeds --- p.62 / Chapter 2.2.2.6 --- Total amino acid determination in mature dry seeds --- p.63 / Chapter 2.2.3 --- Generation of crossing progenies --- p.64 / Chapter 2.2.3.1 --- Artificial crossing of A. thaliana --- p.64 / Chapter 2.2.3.2 --- CTAB extraction of genomic DNA --- p.64 / Chapter 2.2.3.3 --- PCR screening for successful crossing --- p.65 / Chapter 2.2.4 --- Generation of transgenic plants --- p.67 / Chapter 2.2.4.1 --- Cloning of E.coli dapA gene --- p.67 / Chapter 2.2.4.2 --- Preparation of recombinant plasmid --- p.68 / Chapter 2.2.4.3 --- Gene sequencing --- p.68 / Chapter 2.2.4.4 --- Homology search of differentially expressed genes --- p.69 / Chapter 2.2.4.5 --- Construction of chimeric dapA genes (TP-Phas-dapA) --- p.69 / Chapter 2.2.4.6 --- Transformation of electro-competent Agrobacterium cell --- p.73 / Chapter 2.2.4.7 --- Transformation of A. thaliana through vacuum infiltration --- p.73 / Chapter 2.2.4.8 --- Selection of hemizygous and homozygous transgenic plants --- p.74 / Chapter 2.2.4.9 --- Expression analysis of homozygous LRP/dapA transgenic plants --- p.75 / Chapter 3 --- Results --- p.77 / Chapter 3.1 --- Characterization of ASN1 over-expressers --- p.77 / Chapter 3.1.1 --- Overexpression of the ASN1 gene enhances the sink-source relationship of asparagine transport under regular daylight cycle --- p.88 / Chapter 3.1.2 --- Spatial distribution of total free amino acids under normal daylight cycle --- p.88 / Chapter 3.1.3 --- Over-expression of the ASN1 gene affects free amino acid level quantitatively under normal daylight cycle --- p.89 / Chapter 3.1.4 --- Over-expression of the ASN1 gene affects composition of total amino acid under normal daylight cycle --- p.89 / Chapter 3.2 --- Construction of dapA transgenic Arabidopsis --- p.91 / Chapter 3.2.1 --- Construction of chimeric gene for expression of the dapA gene --- p.91 / Chapter 3.2.2 --- Transformation of p1300/Phas-dapA into Arabidopsis and selection of homozygous progenies --- p.91 / Chapter 3.3 --- Generation of transgenic plants with altered sink-source relationship through crossing and in-planta transformation --- p.96 / Chapter 3.3.1 --- Rationale in methods for generating transgenic plants with different combination of sources and sinks --- p.96 / Chapter 3.3.2 --- Screening for double homozygous progenies through crossing --- p.98 / Chapter 3.3.3 --- Screening for F1 progenies of successful crossing --- p.100 / Chapter 3.3.4 --- Selection of homozygous crossing progenies --- p.102 / Chapter 3.3.5 --- Screening for homozygous dapA/LRP transgenic plants --- p.104 / Chapter 3.4 --- Amino acid composition analysis --- p.109 / Chapter 3.4.1 --- The change of aspartate family amino acids in mature seeds of transgenic plants with altered sources --- p.113 / Chapter 3.4.2 --- The change of aspartate family amino acids in mature seeds of transgenic plants with improved sink --- p.114 / Chapter 3.4.3 --- The change of aspartate family amino acids in mature seeds of transgenic plants with improved sink --- p.115 / Chapter 4. --- Discussion / Chapter 4.1 --- Characterization of ASN1 over-expressers --- p.116 / Chapter 4.1.1 --- Possible regulation of ASN1 mRNA stability through level of asparagine --- p.117 / Chapter 4.1.2 --- Over-expression of ASN1 gene may improve nitrogen remobilisation from source to sink tissues --- p.118 / Chapter 4.1.3 --- Over-expression of ASN1 gene has modified the composition of amino acidsin sink organs --- p.119 / Chapter 4.2 --- ASN1 RNAi transgenic plants increases the relative contents of lysine in the seeds --- p.122 / Chapter 4.2.1 --- Role of ASN1 in supplying or competing aspartate in developing seeds --- p.122 / Chapter 4.2.2 --- Possible role of glutamate receptor --- p.123 / Chapter 4.3 --- Lysine catabolism may strictly control the level of lysine --- p.123 / Chapter 4.3.1 --- Possible role of lysine-tRNA in protein synthesis --- p.124 / Chapter 5. --- Conclusion and prospective --- p.125 / References --- p.126 / Appendix --- p.147