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Plants as bioreactors: expression of toxoplasma gondii surface antigen P30 in transgenic tobacco plants.January 2001 (has links)
by Yu Wing Sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 119-126). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.vi / 摘要 --- p.viii / Table of Contents --- p.x / List of Tables --- p.xvi / List of Figures --- p.xvii / List of Abbreviations --- p.xx / Chapter CHAPTER 1 --- General Introduction --- p.1 / Chapter CHAPTER 2 --- Literature Review --- p.3 / Chapter 2.1 --- Toxoplasma gondii --- p.3 / Chapter 2.1.1 --- Morphology and Life Cycle of T. gondii --- p.3 / Chapter 2.1.2 --- Routes of Transmission --- p.7 / Chapter 2.2 --- Toxoplasmosis --- p.8 / Chapter 2.2.1 --- Influences and Symptoms --- p.8 / Chapter 2.2.2 --- Treatment of Toxoplasmosis --- p.10 / Chapter 2.2.2.1 --- Antitoxoplasma Drugs --- p.10 / Chapter 2.2.2.2 --- Toxoplasma Vaccines --- p.12 / Chapter 2.3 --- Major T. gondii Surface Antigen - P30 --- p.16 / Chapter 2.4 --- Plants as Bioreactors --- p.19 / Chapter 2.4.1 --- Advantages of Plant Bioreactors --- p.19 / Chapter 2.4.2 --- Plant-based Vaccines --- p.20 / Chapter 2.4.2.1 --- VP2 Capsid Protein of Mink Enteritis Virus --- p.21 / Chapter 2.4.2.2 --- Hepatitis B Surface Antigen --- p.21 / Chapter 2.4.2.3 --- Norwalk Virus Capsid Protein --- p.22 / Chapter 2.5 --- Tobacco Expression System --- p.23 / Chapter 2.5.1 --- Transformation Methods --- p.23 / Chapter 2.5.1.1 --- Agrobacterium-mediated Transformation --- p.23 / Chapter 2.5.1.2 --- Direct DNA Uptake --- p.24 / Chapter 2.6 --- Phaseolin and Its Regulatory Sequences --- p.26 / Chapter CHAPTER 3 --- Expression of P30 in Transgenic Tobacco --- p.28 / Chapter 3.1 --- Introduction --- p.28 / Chapter 3.2 --- Materials and Methods --- p.29 / Chapter 3.2.1 --- Chemicals --- p.29 / Chapter 3.2.2 --- Oligos: Primers and Adapters --- p.29 / Chapter 3.2.3 --- Plant Materials --- p.31 / Chapter 3.2.4 --- Bacterial Strains --- p.31 / Chapter 3.2.5 --- Construction of Chimeric Genes --- p.31 / Chapter 3.2.5.1 --- Modification of pET-ASP30ΔPI --- p.32 / Chapter 3.2.5.2 --- Cloning of P30 into Vectors with Different Promoters --- p.38 / Chapter 3.2.5.2.1 --- Cloning ofP30 into Vector with CaMV 35S Promoter --- p.38 / Chapter 3.2.5.2.2 --- Cloning of P30 into Vector with Maize Ubiquitin 1 Promoter --- p.38 / Chapter 3.2.5.2.3 --- Cloning of P30 into Vector with Phaseolin Promoter --- p.38 / Chapter 3.2.5.2.4 --- Cloning of P30 into Vector with Phaseolin Promoter and Phaseolin SP --- p.39 / Chapter 3.2.5.3 --- Cloning of P30 into Agrobacterium Binary Vector pBI121 --- p.44 / Chapter 3.2.6 --- Transformation of Agrobacterium by Electroporation --- p.49 / Chapter 3.2.7 --- "Transformation, Selection and Regeneration of Tobacco " --- p.50 / Chapter 3.2.8 --- GUS Assay --- p.51 / Chapter 3.2.9 --- Synthesis of Single-stranded DIG-labeled DNA Probe --- p.51 / Chapter 3.2.10 --- Extraction of Genomic DNA from Leaves --- p.52 / Chapter 3.2.11 --- PCR of Genomic DNA with P30 Specific Primers --- p.53 / Chapter 3.2.12 --- Southern Blot Analysis of Genomic DNA --- p.53 / Chapter 3.2.13 --- Extraction of Total RNA from Leaves or Developing Seeds --- p.54 / Chapter 3.2.14 --- Reverse Transcription-Polymerase Chain Reaction of Total RNA --- p.55 / Chapter 3.2.15 --- Sequencing of RT-PCR Product --- p.56 / Chapter 3.2.16 --- Northern Blot Analysis of Total RNA --- p.56 / Chapter 3.2.17 --- Extraction of Total Protein from Leaves or Mature Seeds --- p.57 / Chapter 3.2.18 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.58 / Chapter 3.2.19 --- Purification of 6xHis-tagged Proteins --- p.58 / Chapter 3.2.20 --- Western Blot Analysis of Total Protein --- p.59 / Chapter 3.2.21 --- In vitro Transcription and Translation --- p.60 / Chapter 3.2.21.1 --- Construction of Transcription Vector Containing Chimeric P30 Gene --- p.60 / Chapter 3.2.21.2 --- In vitro Transcription --- p.60 / Chapter 3.2.21.3 --- In vitro Translation --- p.60 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- Construction of Chimeric P30 Genes --- p.65 / Chapter 3.3.2 --- "Tobacco Transformation, Selection and Regeneration " --- p.65 / Chapter 3.3.3 --- Detection of GUS Activity --- p.67 / Chapter 3.3.4 --- Detection of P30 Gene in Transgenic Plants --- p.69 / Chapter 3.3.4.1 --- PCR of Genomic DNA --- p.69 / Chapter 3.3.4.2 --- Southern Blot Analysis --- p.72 / Chapter 3.3.5 --- Detection of P30 Transcript in Transgenic Plants --- p.75 / Chapter 3.3.5.1 --- RT-PCR --- p.75 / Chapter 3.3.5.2 --- Sequencing of RT-PCR Product --- p.79 / Chapter 3.3.5.3 --- Northern Blot Analysis --- p.79 / Chapter 3.3.6 --- Detection of P30 Protein in Transgenic Plants --- p.83 / Chapter 3.3.6.1 --- Western Blot Analysis of Total Protein and Ni-NTA Purified Proteins --- p.83 / Chapter 3.3.7 --- In vitro Transcription and Translation --- p.92 / Chapter 3.3.7.1 --- In vitro Transcription --- p.92 / Chapter 3.3.7.2 --- In vitro Translation --- p.92 / Chapter CHAPTER 4 --- Discussion --- p.97 / Chapter 4.1 --- General Conclusion --- p.97 / Chapter 4.2 --- Further Speculations and Investigations --- p.100 / Chapter 4.2.1 --- Other Protein Detection Procedures --- p.100 / Chapter 4.2.2 --- In vitro Transcription and Translation --- p.100 / Chapter 4.2.3 --- Gene Silencing at Transcription and/or Post-transcription Levels --- p.101 / Chapter 4.2.4 --- Gene Silencing at Translation and/or Post-translation Levels --- p.102 / Chapter (A) --- AUG Context Sequence --- p.102 / Chapter (B) --- Codon Usage --- p.103 / Chapter (C) --- N-end Rule --- p.107 / Chapter (D) --- Phaseolin Sorting Signal --- p.107 / Chapter CHAPTER 5 --- Future Perspectives --- p.109 / Chapter 5.1 --- Codon Modification of the P30 Gene --- p.110 / Chapter 5.2 --- Fusion of the P30 Gene with the LRP Gene --- p.117 / Chapter CHAPTER 6 --- Conclusion --- p.118 / References --- p.119
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The functional analysis of Vitaceae polygalacturonase-inhibiting protein (PGIP) encoding genes overexpressed in tobaccoVenter, Alida 03 1900 (has links)
Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Agriculture worldwide is under great pressure to produce enough food in order to sustain the
ever-growing world population. Among the many challenges faced by food producers, crop
losses and damage caused by fungal plant pathogens is a major problem. The study of fungal
pathogens and the interaction between plants and fungi is therefore essential, and has been
carried out for many years. Much has been learned in this time, but the full mechanisms of the
various modes of fungal attack and plant defence have still not been elucidated.
Many fungi rely on the action of cell-wall degrading enzymes (CWDEs) to breach the
plant cell wall and facilitate access to the nutrients within. CWDEs are among the very first
enzymes to be secreted at the start of fungal attack, and many of them are considered to be
essential pathogenesis factors. Endopolygalacturonases (ePGs) are CWDEs that cleave the
homogalacturonan stretches of the plant cell wall and are vital virulence factors for a number of
fungi, including Botrytis cinerea. An important defence mechanism of plants involves the
inhibition of CWDEs in order to halt or slow down the fungal attack. Plant polygalacturonaseinhibiting
proteins (PGIPs) are cell wall associated CWDE-inhibiting proteins that specifically act
on fungal ePGs. Many different PGIPs from a number of diverse plant species have been
described to date. They are known to have differential inhibition capabilities that often result
from only a few key amino acid changes within the leucine-rich repeat (LRR) active domains.
Previously, the first grapevine PGIP was isolated and characterised from Vitis vinifera
cultivar Pinotage (Vvpgip1). This Vvpgip1 gene was overexpressed in the tobacco species
Nicotiana tabacum, and was shown to be very effective in reducing the susceptibility of tobacco
towards B. cinerea. The combined results confirmed transgene overexpression, increased PGIP
activity and a strong resistance response against Botrytis, leading to the characterisation of
these lines as having PGIP-specific resistance phenotypes. In a subsequent transcriptomic
analysis of these lines it was found that they display differential expression of cell wall
metabolism genes and biochemical characteristics that might indicate possible cell wall
strengthening compared to wild-type tobacco under uninfecting conditions.
The V. vinifera cultivars are all very susceptible to fungal attack, whereas other
grapevine species, specifically the North American Vitis species, are known for their strong
resistance and even immunity against many fungal pathogens. Thirty seven PGIPs have
previously been isolated from these more resistant species. The amino acid sequences of the
active domains of these PGIPs were previously aligned with that of VvPGIP1, and the proteins
were found to be highly homologous with each other and with VvPGIP1. The different nonvinifera
PGIPs separated into 14 subgroups based on their active domain sequences. For this
study, one PGIP from each group was selected for functional analysis in tobacco.
The selected PGIP-encoding genes were transformed into tobacco by means of
Agrobacterium tumefaciens. Analyses of the putatively transformed plantlets were performed to
test for transgene presence, transgene expression, and PGIP activity: final transgenic tobacco
populations consisting of three to twelve individually transformed lines of nine different nonvinifera
PGIPs were obtained. A subset of the resultant transgenic lines was infected with B.
cinerea in two independent whole plant infections over 11-14 days in order to investigate the
disease resistance afforded by the various PGIPs towards this fungus. A line from the
previously characterised VvPGIP1 population was included as reference; all the infections were
contrasted to the WT tobacco. All the infected lines overexpressing the non-vinifera PGIPs
displayed very strong disease reduction in comparison to the WT control: after initial primary
lesion formation, the spread of fungal infection was contained and halted in these lines, while
wild-type tobacco plants were severely affected. Although the VvPGIP1 line displayed the characteristic PGIP-defense response, the non-vinifera PGIP plants displayed smaller lesions,
indicating very strong resistance phenotypes.
The characterised non-vinifera PGIP overexpressing lines, together with the VvPGIP1
line and the WT control were also used to further evaluate the previous observation that
overexpression might lead to changes in expression of cell wall genes. Analysis of the
expression of a xyloglucan endotransglycosylase (xth) gene in the transgenic population
showed that this gene was down-regulated in healthy uninfected tissue from all the transgenic
lines tested. This confirmed previous results and have confirmed in all grapevine PGIP
overexpressing lines tested so far that this gene is downregulated. XTH is typically involved in
cell wall metabolism and specifically in controlling the strength and elasticity of the plant cell
wall. From previous work it is known that downregulation of this gene leads to strengthening of
the wall.
The results obtained in this study showed that the PGIP-specific resistance phenotype
seen for VvPGIP1-overexpressing tobacco could be confirmed in transgenic tobacco
overexpressing non-vinifera PGIPs from more resistant grapevine species as well. The fact that
these PGIPs lines all performed even better than the VvPGIP1 lines in conferring resistance
towards B. cinerea provides an interesting angle for further investigation into the structural
differences between the non-vinifera PGIPs and VvPGIP1. The transgenic lines are also
excellent material to study the in vivo functions of PGIPs further in the context of plant-pathogen
interactions. / AFRIKAANSE OPSOMMING: Die landboubedryf is wêreldwyd onder groot druk om genoeg voedsel te produseer vir die
groeiende wêreldbevolking. Een van die grootste probleme wat die bedryf ondervind, is die
groot skade wat aan gewasse aangerig word deur patogeniese swamme. Dit is dus noodsaaklik
om swamme en die interaksie tussen plante en swamme te bestudeer, en dit word al vir jare
gedoen. Hoewel daar al baie geleer is in hierdie tydperk, is die volle meganismes van die
verskeie maniere hoe swamme aanval en hoe plante hulleself verdedig, nog nie bekend nie.
Verskeie swamme maak staat op die aktiwiteit van selwand-afbrekende ensieme
(SWAEe) om deur die plantselwand te breek en sodoende toegang tot voedingstowwe in die
plantsel te fasiliteer. SWAEe is van die eerste ensieme wat tydens die begin van patogeniese
aanval deur swamme afgeskei word en verskeie SWAEe word as noodsaaklike patogeniese
faktore beskou. Endopoligalakturonases (ePGs) is SWAEe wat die homogalakturoniese dele
van die plantselwand verteer en is noodsaaklike virulensie faktore vir ‘n aantal swamme, onder
andere Botrytis cinerea. ‘n Belangrike weerstandsmeganisme van plante behels die inhibering
van swam SWAEe om sodoende die patogeen-aanval te stop of te vertraag. Die
poligalakturonase-inhiberende proteïne (PGIPs) van plante is selwand-geassosieerde SWAEinhiberende
proteïne wat spesifiek teen swam ePGs optree. Verskeie verskillende PGIPs vanuit
verskillende plantspesies is tot dusver beskryf. Dit is bekend dat hulle differensiële inhiberende
vermoëns het wat dikwels toegeskryf kan word aan slegs ‘n paar belangrike
aminosuurvolgordeverskille in die leusien-ryke herhalende (LRH) aktiewe areas.
Die eerste wingerd PGIP is vantevore geïsoleer vanuit Vitis vinifera kultivar Pinotage
(Vvpgip1) en gekarakteriseer. Hierdie Vvpgip1 geen is ooruitgedruk in die tabakspesie
Nicotiana tabacum en was baie effektief om die weerstand van tabak teen die swam Botrytis
cinerea te verhoog. Die ooruitdrukking van die transgeen, verhoogde PGIP aktiwiteit en goeie
weerstand teen Botrytis cinerea is bevestig, en het gelei daartoe dat die transgeniese VvPGIP1
plantlyne geklassifiseer is as lyne met PGIP-spesifieke weerstandsfenotipes. ‘n
Daaropvolgende transkriptomiese analise van die plantlyne het gewys dat hulle differensiële
uitdrukking van selwand-geassosieerde gene het, asook biochemiese eienskappe, wat ‘n
moontlike selwandversterking aandui in vergelyking met wilde-tipe tabak in die afwesigheid van
infeksie.
Die V. vinifera kultivars is hoogs vatbaar vir swamme, terwyl ander wingerdspesies,
spesifiek die Noord-Amerikaanse spesies, bekend is vir hoë weerstand en selfs immuniteit
teenoor verskeie patogeniese swamme. Sewe-en-dertig PGIPs is vantevore geïsoleer vanuit
hierdie meer weerstandbiedende spesies. Die aminosuurvolgordes van die aktiewe areas van
hierdie PGIPs is vantevore vergelyk met die van VvPGIP1 en dit is gevind dat hierdie proteïne
hoogs homoloog is aan mekaar, sowel as aan VvPGIP1. Die verskillende nie-vinifera PGIPs het
in 14 groepe verdeel na aanleiding van die homologie van hulle aktiewe areas. Vir hierdie studie
is een PGIP vanuit elkeen van hierdie groepe gekies vir verdere funksionele analise in tabak.
Die 14 nie-vinifera PGIP-koderende gene is stabiel oorgedra na tabak deur middel van
Agrobacterium tumefaciens. Die vermeende transgeniese plante is geanaliseer vir die
teenwoordigheid van die transgeen, die uitdrukking daarvan en PGIP aktiwiteit: bevestigde
transgeniese tabak populasies wat wissel van drie tot 12 individuele getransformeerde lyne kon
verkry word vir nege van die verskillende nie-vinifera PGIPs. ‘n Aantal van die transgeniese lyne
is geïnfekteer met B. cinerea in twee onafhanklike heelplantinfeksies vir 11-14 dae om die
siekteweerstand van hierdie PGIPs teenoor die swam te evalueer. ‘n Plantlyn van die
VvPGIP1-populasie is as ‘n verwysing ingesluit en al die infeksies is vergelyk met die wilde-tipe
tabak. Al die geïnfekteerde lyne wat die nie-vinifera PGIPs ooruitdruk het ‘n baie sterk afname in siektesimptome getoon in vergelyking met die wilde-tipe kontrole: na aanvanklikle primêre
lesies gevorm het, is die verspreiding van die infeksie ingeperk en gestop in hierdie lyne, terwyl
die wilde-tipe plante baie erg geaffekteer is. Terwyl die VvPGIP1 lyn ook die tipiese PGIPweerstandsrespons
getoon het, het die nie-vinifera PGIPe kleiner lesies ontwikkel, wat dui op
baie sterk weerstandsfenotipes.
Die gekarakteriseerde nie-vinifera PGIP ooruitdrukkende lyne, asook die VvPGIP1 lyn
en die wilde-tipe kontrole, is gebruik om die vorige waarneming dat die ooruitdrukking kan lei tot
veranderinge in selwandgeen-uitdrukking verder te ondersoek. Analise van die uitdrukking van
‘n xiloglukaan-endotransglikosilase (xth) geen in die transgeniese populasie het getoon dat
hierdie geen afgereguleer is in gesonde, oninfekteerde weefsel van al die transgeniese lyne wat
getoets is. Dit het vorige resultate bevestig en het ook bevestig dat hierdie geen afgereguleer is
in alle wingerd PGIP-ooruitdrukkende lyne wat tot dusver getoets is. XTH is tipies betrokke by
selwandmetabolisme, spesifiek by die beheer van selwandsterkte en selwandelastisiteit. Dit is
uit vorige werk bekend dat die afregulering van hierdie geen lei tot versterking van die
plantselwand.
Die resultate verkry tydens hierdie studie het gewys dat die PGIP-spesifieke weerstand
fenotipe van VvPGIP1-ooruitdrukkende tabak ook bevestig kon word in transgeniese tabak wat
nie-vinifera PGIPs vanuit meer weerstandbiedende wingerdspesies ooruitdruk. Die feit dat
hierdie PGIP lyne almal selfs beter weerstand teen B. cinerea bied as VvPGIP1 lyne is ‘n
interessante invalshoek vir opvolgende ondersoeke na die belang van strukturele verskille
tussen die nie-vinifera PGIPs en VvPGIP1. Hierdie transgeniese lyne is ook uitstekende
hulpbronne om die in vivo funksies van PGIPs verder te bestudeer in die konteks van plantpatogeen
interaksies.
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Functional Characterization of PtaRHE1, a gene that encodes a RING-H2 type protein in poplar / Caractérisation fonctionnelle de PtaRHE1, un gène qui code pour une protéine de type RING-H2 chez le peuplierMukoko Bopopi, Johnny 14 January 2011 (has links)
PtaRHE1 is a poplar (Populus tremula x P. alba) gene encoding a REALLY INTERESTING NEW GENE (RING) domain-containing protein. RING proteins are largely represented in plants and play important roles in the regulation of many developmental processes as well as in plant-environment interactions. In this thesis, we present a functional characterization of PtaRHE1. To gain further insight into the role of this gene, molecular and genetic alteration approaches were used. The results of in vitro ubiquitination assays indicate that PtaRHE1 protein is a functional E3 ligase and this activity was shown to be specific with the human UbCH5a, among the tested ubiquitin-conjugating enzymes. Histochemical GUS stainings showed that the PtaRHE1 promoter is induced by plant pathogens and by elicitors such as salicylic acid and cellulase and is also developmentally regulated. In silico predictions and the transient expression of PtaRHE1-GFP fusion protein in N. tabacum epidermal cells revealed that PtaRHE1 is localized both in the plasma membrane and in the nucleus. The localization of expression of PtaRHE1 in poplar stem by in situ hybridization indicated that PtaRHE1 transcripts are localized within the cambial zone mainly in ray cells, suggesting a role of this gene in vascular tissue development and/or functioning. The overexpression of PtaRHE1 in tobacco resulted in a pleiotropic phenotype characterized by a curling of leaves, the formation of necrotic lesions on leaf blades, growth retardation as well as a delay in flower transition. Plant genes expression responses to PtaRHE1 overexpression provided evidence for the up-regulation of defence and/or programmed cell death (PCD) related genes. Moreover, genes coding for WRKY transcription factors as well as for MAPK, such as WIPK, were also found to be induced in the transgenic lines as compared to the wild type (WT). Taken together, our results suggest that the E3 ligase PtaRHE1 plays a role in the signal transduction pathways leading to defence responses against biotic and abiotic stresses. Identification of PtaRHE1 target(s) is required in order to fully assess the role of this E3 ligase in the ubiquitination-mediated regulation of defence response./<p>RÉSUMÉ<p><p><p>PtaRHE1 est un gène qui code pour une protéine possédant un domaine RING (REALLY INTERESTING NEW GENE) chez le peuplier (Populus tremula x P. alba). Les protéines de type RING sont très répandues chez les végétaux où elles jouent de rôles importants dans la régulation de plusieurs processus de développement et également dans les interactions plantes-environnement. Dans le cadre de ce travail, nous avons procédé à la caractérisation fonctionnelle du gène PtaRHE1. Dans le but de découvrir la fonction de ce gène, nous avons adopté une stratégie faisant usage d’approches moléculaires ainsi que de l’altération de l’expression génique. Les résultats obtenus montrent que la protéine PtaRHE1 est une E3 ligase et que cette activité enzymatique est spécifique à l’Ubiquitin-Conjugating enzym humaine UbCH5a. Les résultats du test histochimique GUS ont montré que le promoteur du gène PtaRHE1 est induit par des pathogènes et aussi par l’acide salicylique et la cellulase. Par ailleurs, ce promoteur est aussi régulé au cours du développement végétal. Les prédictions in silico et l’expression transitoire d’une fusion traductionnelle GFP-PtaRHE1, au niveau de l’épiderme des feuilles du tabac N. tabacum, ont révélé que la protéine PtaRHE1 se situe tant au niveau de la membrane cytoplasmique qu’au niveau du noyau. La localisation de l’expression du gène PtaRHE1, par les techniques d’hybridation in situ, montre que les transcrits de ce gène se retrouvent principalement au niveau des cellules de rayon, dans la zone cambiale, suggérant que ce gène pourrait jouer un rôle dans le développement ou la formation du tissu vasculaire. La surexpression du gène PtaRHE1 chez le tabac a conduit à l’obtention d’un phénotype pléiotropique caractérisé par un recroquevillement (incurvation) des feuilles, la formation des lésions nécrotiques sur le limbe, un retard de croissance ainsi qu’un retard dans la transition florale. L’analyse de la réponse de l’expression de différents gènes à la surexpression de PtaRHE1 a mis en évidence l’induction des gènes liés à la défense et ou à la mort cellulaire programmée. En outre, l’expression des gènes codant pour des facteurs de transcription WRKY et aussi des MAPKs, tel que WIPK, était aussi plus élevée chez les plantes transgéniques comparées au type sauvage. Les résultats de ce travail suggèrent que PtaRHE1, comme E3 ligase, pourrait jouer un rôle dans la transduction des signaux cellulaires conduisant aux réactions de défense contre les stress biotiques et abiotiques. L’identification de la (des) cible(s) de PtaRHE1 est indispensable pour la compréhension du rôle de cette protéine dans la régulation des réponses de défense par l’intermédiaire de l’ubiquitination.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished
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Functional analysis of a lignin biosynthetic gene in transgenic tobaccoMbewana, Sandiswa 03 1900 (has links)
Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Necrotrophic fungi infect many economically important crop plants. This results in great losses
in the agricultural sector world-wide. Understanding the nature by which plants respond to
pathogens is imperative for genetically enhancing disease resistance in plants. Research tools
have significantly contributed to our understanding of how the plant responds to pathogen
attack, identifying an array of defence mechanisms used by plants upon attack.
Many fungal pathogens secrete endopolygalacturonases (endoPGs) when infecting
plants. These hydrolytic enzymes are inhibited by polygalacturonase-inhibiting proteins (PGIPs)
associated with plant cell walls. PGIPs are well characterised and their current known functions
are all linked to endoPG inhibition and the subsequent upregulation of plant defence pathways.
Work on grapevine PGIPs have shown that apart from being efficient antifungal proteins,
leading to protection of the plant against Botrytis cinerea when overexpressed, PGIPs might
also have additional functions linked to cell wall strengthening. This working hypothesis formed
the motivation of this study where a cinnamyl alcohol dehydrogenase (CAD) (1.1.1.195) gene
was targeted for functional analysis in tobacco (Nicotiana tabacum). Some previous work and
genetic resources obtained is relevant to this study, specifically previously characterized
transgenic tobacco lines overexpressing the Vitis vinifera pgip1 (Vvpgip1) gene. These lines
have confirmed PGIP-specific resistance phenotypes against B. cinerea, as well as increased
levels of CAD transcripts in healthy plants. Moreover, preliminary evaluations indicated
increased lignin levels as well as differential expression of several other cell wall genes in these
overexpressing lines (in the absence of infections).
In this study we generated a transgenic tobacco population, overexpressing the native
CAD14 gene, via Agrobacterium transformations. The transgene was overexpressed with the
Cauliflower Mosaic Virus promoter (CaMV 35Sp). The CAD transgenic population was analyzed
for transgene integration and expression and showed active transcription, even from leaves that
normally don’t express CAD to high levels. These lines, together with the untransformed control,
and a representative transgenic VvPGIP1 tobacco line previously characterized with elevated
expression of CAD were used for all further analyses, specifically CAD activity assays of stems
and leaves, as well as whole plant infections with B. cinerea. CAD enzyme activity assays were
performed on healthy uninfected plant lines, without inducing native CAD expression or
resistance phenotypes (i.e. without Botrytis infection). CAD activity was detected in leaves and
stems, but a statistically sound separation between the CAD population and the untransformed
control was only observed in the stems. The CAD assays also confirmed previous results that
indicated that CAD transcription was upregulated in the PGIP line in the absence of infection.
Overall, in all plant lines the stems exhibited 10-fold higher levels of CAD activity than the
leaves, but the transgenic VvPGIP1 line showed a further 2-3-fold increase in CAD activity in the stems, when compared to the untransformed control and the majority of the CAD
overexpressing lines.
Disease assessment by whole plant infections with B. cinerea of the CAD transgenic
plants revealed reduced disease susceptibility towards this pathogen. A reduction in disease
susceptibility of 20 – 40% (based on lesion sizes) was observed for a homologous group of
transgenic lines that was statistically clearly separated from the untransformed control plants
following infection with Botrytis over an 11-day-period. The VvPGIP1 transgenic line displayed
the strongest resistance phenotype, with reduction in susceptibility of 47%. The reduction in
plant tissue maceration and lesion expansion was most pronounced in the VvPGIP1 line
compared to the CAD transgenic plants, while the CAD transgenic plants showed more
reduction than the untransformed control. In combination, the data confirms that CAD
upregulation could lead to resistance phenotypes. Relating this data back to the previously
observed upregulation of CAD in the VvPGIP1-overexpressing lines, the findings from this study
corroborates that increased CAD activity contributes to the observed resistance phenotypes,
possibility by strengthening the cell wall.
In conclusion, this study yielded a characterized transgenic population overexpressing
the CAD14 gene; this overexpression contributed to increased RNA transcription compared to
the untransformed control plant, increased CAD activity (most notably in the stems) and a
disease resistance phenotype against Botrytis. These findings corroborates the current working
hypothesis in our group that PGIPs might have a role in preparing the plant cell for attack by
contributing to specific cell wall changes. The exact mechanisms are still currently unknown and
under investigation. The transgenic lines generated in this study will be invaluable in the
subsequent analyses where these various phenotypes will be subjected to profiling and
accurate cell wall analyses. / AFRIKAANSE OPSOMMING: Nekrotrofiese swamme infekteer en beskadig verskeie ekonomies belangrike gewasse. Dit lei
wêreldwyd tot groot verliese vir die landbousektor. Dit is noodsaaklik om te verstaan hoe plante
reageer teenoor patogene, sodat die siekteweerstand van plante verbeter kan word.
Navorsingshulpbronne het beduidend bygedra tot die kennis van plantreaksies tydens
patogeniese aanvalle, en het sodoende ‘n reeks van weerstandmeganismes, wat die plant
inspan tydens ‘n aanval, geïdentifiseer.
Verskeie patogeniese swamme skei endopoligalakturonases (endoPGs) af tydens plantinfeksie.
Hierdie hidrolitiese ensieme word geïnhibeer deur poligalakturonase-inhiberende
proteïene (PGIPs) wat met die plantselwand geassosieerd is. PGIPs is goed gekarakteriseerd
en al hulle huidiglik bekende funksies is gekoppel aan endoPG inhibisie en die daaropvolgende
opregulering van plant weerstandspaaie. Navorsing op wingerd PGIPs het gewys dat, afgesien
van die feit dat PGIPs goeie antifungiese proteïene is wat lei tot beskerming van die plant teen
Botrytis cinerea wanneer dit ooruitgedruk word, PGIPs ook moontlik addisionele funksies verrig
wat verwant is aan selwandversterking. Hierdie werkshipotese vorm die motivering vir hierdie
studie waarin ‘n sinnamiel alkohol dehidrogenase (SAD) (1.1.1.195) geen geteiken is vir
funksionele analise in tabak (Nicotiana tabacum). Vorige navorsing en genetiese hulpbronne
daardeur verkry is belangrik vir hierdie studie, spesifiek die gekarakteriseerde transgeniese
tabaklyne wat die Vitis vinifera pgip1 (Vvpgip1) geen ooruitdruk. Hierdie lyne besit bevestigde
PGIP-spesifieke weerstandsfenotipes teen B. cinerea, sowel as hoër vlakke van SAD
transkripte in gesonde plante. Voorlopige analises het ook aangedui dat hierdie ooruitdrukkende
lyne hoër lignien vlakke het, asook differensiële uitdrukking van verskeie ander selwandgene (in
die afwesigheid van infeksie).
In hierdie studie is ‘n transgeniese tabakpopulasie gegenereer wat die natuurlike tabak
SAD14 geen ooruitdruk, deur middel van Agrobacterium transformasie. Die transgeen is
ooruitgedruk deur die Blomkool Mosaïek Virus promoter (CaMV 35Sp). Die SAD transgeniese
populasie is geanaliseer vir transgeen integrasie en uitdrukking en het aktiewe transkriptering
getoon, selfs in blare waar daar normaalweg nie hoë vlakke van SAD uitgedruk word nie.
Hierdie lyne, die ongetransformeerde wilde-tipe kontrole sowel as ’n verteenwoordigende
transgeniese VvPGIP1 tabaklyn wat vooraf gekarakteriseerd was met hoë SAD uitdrukking, is
gebruik vir alle verdere analises, spesifiek SAD aktiwiteitstoetse in stingels en blare, asook
heelplantinfeksies met B. cinerea. Aktiwiteitsanalises van die SAD ensiem is gedoen op
gesonde ongeinfekteerde plantlyne, sonder om natuurlike tabak SAD uitdrukking of
weerstandsfenotipes te induseer (dus sonder Botrytis infeksie). SAD aktiwiteit is waargeneem in
beide die blare en stingels, maar ‘n statisties betekenisvolle skeiding is slegs gevind tussen die
SAD populasie en die ongetransformeerde kontrole in die stingels. Die SAD toetse het ook vorige resultate bevestig wat aangedui het dat SAD transkripsie opgereguleer word in die PGIP
lyn in die afwesigheid van infeksie. Die stingels het oor die algemeen ‘n 10-voudige
vermeerdering in SAD aktiwiteitsvlakke getoon in vergelyking met die blare, maar die
transgeniese VvPGIP1 lyn het ‘n verdere 2-3-voudige verhoging in SAD aktiwiteit gehad in die
stingels ,in vergelyking met die ongetransformeerde kontrole en die meerderheid van die SADooruitdrukkende
lyne.
Siekteweerstand ondersoeke deur middel van heelplantinfeksies met B. cinerea van die
SAD transgeniese plante, het verminderde vatbaarheid aangedui ten opsigte van hierdie
patogeen. ‘n Afname in siekte-vatbaarheid van 20 – 40% (gebaseer op wondgroottes) is
waargeneem vir ‘n homoloë groep transgeniese lyne wat statisties betekenisvol geskei kon
word van die ongetransformeerde kontrole plante na infeksie met Botrytis in ‘n infeksietoets wat
11 dae geduur het. Die VvPGIP1 transgeniese lyn het die mees weerstandbiedende fenotipe
gehad, met ‘n afname in siekte-vatbaarheid van 47%. Die afname in plantweefselafbreking en
wondgrootte was die duidelikste in die VvPGIP1 lyn in vergelyking met die SAD transgeniese
plante, terwyl die SAD transgeniese plante ‘n groter afname aangedui het as die
ongetransformeerde kontrole. In kombinasie het die data bevestig dat SAD opregulasie kan lei
tot weerstandbiedende fenotipes. Hierdie data, in vergelyking met die vorige bevinding van
opregulasie van SAD in die VvPGIP1-ooruitdrukkende lyne, bevestig dat hoër SAD aktiwiteit
bydra tot die waargenome weerstandbiedende fenotipes, moontlik deur versterking van die
plantselwand.
Ter afsluiting, hierdie studie het ‘n gekarakteriseerde transgeniese populasie wat die
SAD14 geen ooruitdruk gelewer; hierdie ooruitdrukking het bygedra tot hoër RNA transkripsie in
vergelyking met die kontrole, verhoogde SAD aktiwiteit (veral in die stingels) en siekteweerstandbiedende
fenotipes teenoor Botrytis. Hierdie bevindinge ondersteun die huidige
werkshipotese in ons groep dat PGIPs moontlik ‘n rol speel in die voorbereiding van die plantsel
teen infeksie deur spesifieke selwandveranderinge te veroorsaak. Die spesifieke meganismes is
steeds onbekend en word verder ondersoek. Die transgeniese lyne wat tydens hierdie studie
gegenereer is, sal baie belangrik wees in opvolgende analises waar hierdie verskillende
fenotipes gebruik kan word om die profiel van selwandkomponente, maar ook die akkurate
selwandsamestelling te bestudeer.
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Synthetic Gene Complementation to Determine off-Target SilencingKumar, Dhirendra R. 01 January 2015 (has links)
RNA interference (RNAi) is a conserved mechanism in a wide range of eukaryotes. Introduction of synthetic dsRNA could specifically target suppression of a gene or could result in off-target silencing of another gene due to sequence similarity. To verify if the observed phenotype in an RNAi transgenic line is due to silencing of a specific gene or if it is due to another nontarget gene, a synthetic gene complementation approach could be used. Synthetic gene complementation described in this method uses the technology of synthesizing a variant of a native gene (used in RNAi silencing) to maximize the difference in DNA sequences while coding for the exact same amino acids as the original native gene. This is achieved through the use of alternate codons. The new variant gene is expressed in the original RNAi transgenic lines and analyzed for complementation of the RNAi phenotype. Complementation of the RNAi-induced phenotype will indicate gene-specific silencing and not off-target silencing.
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