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Descobertas associadas aos mecanismos de defesa antiviral baseados em silenciamento por RNA em Glycine max e Nicotiana Benthamiana / Findings related to the antiviral defense mechanism based on RNA silencing in glycine max and Nicotiana BenthamianaFonseca, Guilherme Cordenonsi da January 2015 (has links)
O principal mecanismo de defesa das plantas frente a uma infecção viral é baseado no fenômeno chamado interferência por RNA (RNAi). Por meio da ação coordenada de proteínas como Argonautas, Dicers, RNA polimerases dependentes de RNA e proteínas de ligação a RNA de dupla fita (DRBs), o RNA viral é reconhecido e clivado a pequenos RNAs de interferência derivados de vírus (vsiRNAs). Os vsiRNAs, acoplados ao complexo proteíco de indução ao silenciamento, atuam sobre sequências de RNA ou DNA virais, podendo promover a clivagem, inibição da tradução ou metilação de seus alvos (para vírus de DNA). Neste trabalho foram realizados dois estudos que abordam mecanismos de defesa baseados em RNAi em plantas. No primeiro capítulo é descrito a integração do RNA1 do Cumcumber mosaic virus (CMV) no genoma de Glycine max. Através da análise de bibliotecas de sequenciamento de alta eficiência de RNA mensageiros (mRNAs), pequenos RNAs e DNAs de diferentes cultivares e diferentes tecidos de soja foi possível identificar que o evento de integração envolveu duas moléculas do RNA1 do CMV, o RNA de um retrotransposon e um mRNA de um gene endógeno. No locus aonde ocorreu esta integração as duas sequências do RNA1 estão em sentidos opostos. Os pequenos RNAs (sRNAs) das nossas bibliotecas alinham majoritariamente na região do RNA1 do CMV e são praticamente ausentes nas outras regiões da sequência integrada, sugerindo fortemente a formação de um grampo aonde hibridizam ambas as sequências do CMV. A presença desses sRNAs derivados do CMV em todos os tecidos estudados sugere uma provável função antiviral dessa sequencia que foi integrada em soja. No segundo capítulo, por microscopia confocal, foi estudada a interação entre as proteínas DRBs e o Potato virus X (PVX) durante a infecção viral em Nicotiana benthamiana. É demonstrado que as DRBs 2, 3 e 5 se realocam de sua posição original e se concentram em estruturas chamadas complexos de replicação viral durante a infecção por PVX. Esse fenômeno é um indicativo que essas proteínas podem estar atuando nos primeiros estágios de defesa da planta frente ao vírus. / The main defense mechanism of plants facing a viral infection is based on the phenomenon called RNA interference (RNAi). Through the coordinated action of proteins such as Argonaut, Dicers, RNA dependent RNA polymerases and double-stranded RNAbinding proteins (DRBs), the viral RNA is recognized and cleaved to virus-derived interfering small RNAs (vsiRNAs). The vsiRNAs, coupled to a protein complex that induce the silencing, act on DNA or RNA viral sequences promoting cleavage, translational inhibition or methylation of their targets (for DNA viruses). This work described two studies that address new defense mechanisms based on RNAi in plants. In the first chapter of this thesis is described the integration of the cucumber mosaic virus (CMV) RNA1 in the genome of Glycine max. Through the analysis of deep sequencing libraries of messenger RNA, small RNAs and DNA from different cultivars and different soybean tissues it was possible to identified that the integration event involved two molecules of CMV RNA1, the RNA of a retrotransposon and the mRNA of an endogenous gene. In the locus where the integration occurred the two RNA1 sequences are in opposite directions. Small RNAs (sRNAs) from our libraries mostly aligned in the region of CMV RNA1 and are practically absent in other regions of the integrated sequence, strongly suggesting the formation of a hairpin where both CMV sequences hybridize. The presence of these CMV-derived sRNAs in all surveyed tissues suggests a probable antiviral function for the sequence that was integrated into soybeans. In the second chapter, the interaction between the DRB proteins and the Potato virus X (PVX) during viral infection in Nicotiana benthamiana is assessed by confocal microscopy. It is shown that the DRBs 2, 3 and 5 relocate from its original position and concentrated in structures called viral replication complexes during infection by PVX. This is an indication that these proteins can act in the early stages of plant defense against the virus.
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Molekularbiologische Untersuchungen zur subzellulären Lokalisierung des putativen Transportproteins - P19,5k - des beet western yellows virus (BWYV) und Erarbeitung der Grundlagen für eine gentechnisch zu erzeugende Resistenz gegen das BWYVDieterich, Guido. January 2000 (has links) (PDF)
Braunschweig, Techn. Universiẗat, Diss., 2000.
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Uso de Nicotiana benthamiana para produção do fragmento scFvBap1, um anticorpo antagonista a metaloproteinases de serpentes do gênero BothropsGomes, Marinna 21 February 2018 (has links)
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Previous issue date: 2018-02-21 / Acidentes ofídicos representam um grave problema de saúde pública devido a sua alta incidência e a gravidade dos seus efeitos. No Brasil, o gênero Bothrops é responsável por 85% dos casos de acidentes. A principal forma de tratamento para tais acidentes é feita através da soroterapia, utilizando-se anticorpos eqüinos, porém os mesmos não são capazes de neutralizar os efeitos sistêmicos do envenenamento e podem causar reações adversas. Uma nova perspectiva para o tratamento de acidentes ofídicos é a utilização de anticorpos recombinantes, mais precisamente a porção scFv. O fragmento scFvBap1 é oriundo de um anticorpo monoclonal, produzido em Escherichia coli foi capaz de neutralizar os efeitos hemorrágicos do envenenamento. Atualmente as plantas vêm sendo amplamente utilizadas na produção de proteínas recombinantes de interesse farmacêutico. Neste estudo, objetivou-se a expressão do fragmento scFv em Nicotiana benthamiana, a fim de avaliar seu potencial para ser utilizado como soro antiofídico. Foram avaliadas a expressão transiente do fragmento scFvBap1 em folhas de N. benthamiana, bem como a expressão estável na mesma planta. Os fragmentos produzidos foram capazes de reconhecer e neutralizar as toxinas BaP1 de B. asper, BnP1 de B. newidae e ATX de B. atrox, demonstrando seu potencial para ser utilizado na terapia contra envenenamentos ofídicos. O sistema de produção estável apresentou-se maior rendimento de proteínas (270 μg/g) quando comparado ao sistema de produção transiente (43 μg/g). Por fim, o sistema mostrou-se uma ótima alternativa para produção do fragmento de anticorpo quando comparado ao sistema bacteriano. / Ophidian accidents are a global health problem due to its high incidence and gravity of its effects. In Brazil, the genus Bothrops is responsible for 85% of the cases of accidents. The main form of treatment for snake envenoming is serum therapy using equine antibodies, however, they are not able to neutralize the local effects of the toxin and may also cause adverse reactions. A new perspective for the treatment of snakebite envenoming has emerged with the use of recombinant antibodies, particularly the Fv fragment (scFv). The scFvBap1, is an efficient antibody capable of neutralizing the hemorrhagic effects and proteolytic activity caused by metalloproteinases. Currently the plants have been widely used in the production of recombinant proteins of pharmaceutical interest. In this study, we aimed the expression of the scFv fragment in Nicotiana benthamiana, in order to evaluate its potential as an antiophiidic serum. The transient expression of the scFvBap1 fragment in leaves of N. benthamiana, as well as the stable expression in the same plant, were evaluated.The fragments produced were able to recognize and neutralize the toxins BaP1 from B. asper, BnP1 from B. newidae and ATX from B. atrox, demonstrating their potential to be used in therapy against ophidian poisoning. The stable production system presented higher protein yield (270 μg / g) when compared to the transient production system (43 μg / g). Finally, the system proved to be a good alternative for producing the antibody fragment when compared to the bacterial system.
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Impact de la lumière sur la production de protéines recombinantes chez Nicotiana benthamianaGagné, Marielle 24 April 2018 (has links)
Tableau d'honneur de la Faculté des études supérieures et postdorales, 2015-2016 / La moléculture végétale est une approche prometteuse pour la production de protéines d’intérêt médical ou industriel. Considérant les variations de rendement possibles dans une plante soumise à différentes conditions culturales, nos objectifs étaient : (i) de cartographier l’accumulation d’un antigène viral d’intérêt clinique dans les feuilles du tabac sauvage Nicotiana benthamiana utilisé comme bio-usine, et (ii) d’évaluer l’impact de la lumière en période de croissance sur le rendement total en antigène. Nous avons étudié les relations entre l’âge foliaire, le régime lumineux, l’expression du transgène et le rendement final en antigène dans les feuilles. Nos données confirment l’influence de l’âge sur les variations de rendement d’une feuille à l’autre, et l’impact positif de l’intensité lumineuse sur le rendement par plante. Elles mettent aussi en relief l’importance des tiges secondaires sur le rendement et le rôle clé de la transcription du transgène sur la teneur en antigène à l’échelle cellulaire. / Plant molecular farming is a promising approach to produce proteins of medical or industrial interest. Considering possible variations of protein yield in plants exposed to different cultural conditions, our goals were : (i) to map the accumulation of a clinically useful viral antigen in leaves of the tobacco relative Nicotiana benthamiana used as protein biofactory, and (ii) to evaluate the impact of light conditions during plant growth on total antigen yield. We looked at eventual relations in planta between leaf age, light intensity, light quality, transgene expression and final protein yield in leaves. Our data confirmed the central influence of leaf age on antigen yield variation from one leaf to another, and the positive impact of light intensity on total yield per plant. They also highlight the importance of secondary stem growth on total yield, and the key role of transgene transcription on antigen content at the cell scale.
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Expression de protéines du rotavirus humain chez les plantes et leur utilisation pour une stimulation immunitaire chez la sourisBergeron Sandoval, Louis-Philippe January 2009 (has links) (PDF)
Au niveau de la santé publique, l'impact meurtrier des infections au rotavirus humain (RVH) et les limites d'application des vaccins disponibles déterminent l'urgence et la nécessité de développer de nouvelles approches pour immuniser une grande partie de la population mondiale sans effets secondaires néfastes. Pour pallier à l'énorme demande en médicaments et en vaccins, plusieurs groupes de recherche tentent de développer de nouvelles méthodes de production à base de plantes transgéniques. La production de médicaments et vaccins chez les végétaux possèdent des avantages inhérents dont une absence de pathogènes humain, une inoculation facile, de faibles coûts de production à grande échelle et la production d'inocula bruts stockables. Le succès des systèmes d'expression à base de plantes justifie le développement de plants qui produisent beaucoup de biomasse et se transforment génétiquement de façon courante. Le travail présenté ici démontre le potentiel du système d'expression transitoire par agroinfiltration de Nicotiana benthamiana pour produire les antigènes VP7 et VP4∆ du RVH ainsi que l'adjuvant protéinique fljB de Salmonella typhimurium. Le clonage des séquences codantes des antigènes VP7, VP4∆ et fljB a permis de générer de multiples constructions simple, double et triple. Toutes les contructions ont été exprimées dans les feuilles agroinfiltrées à l'exception de VP4∆::VP7 qui n'a pas été détectée par immunobuvardage. Le rendement de protéines d'intérêt dans les extraits végétaux se situe entre 0,85 et 31,97 µg de protéine recombinante par gramme de feuilles fraîches. Plusieurs facteurs dont la toxicité et la stabilité des différentes constructions dans les plantes peuvent expliquer cet écart. Dans nos conditions expérimentales, toutes les constructions contenant le fragment fljB ont permis la production d'anticorps spécifiques à fljB dans les souris immunisées avec les extraits végétaux. Par contre, aucune construction n'as permis la production d'anticorps spécifiques à VP7 ou VP4. Ces résultats montrent que l'expression transitoire dans Nicotiana benthaminana permet de produire rapidement de multiples antigènes et que les protéines fortement immunogéniques dans les extraits végétaux induisent une réponse humorale chez les souris. Plusieurs applications de cette technologie sont envisageables dans la cadre du contrôle endémique du RVH ou de Salmonella typhimurium. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Rotavirus humain, VP7, VP4, Salmonella typhimurium, fljB, Expression transitoire, Vaccins à base de plantes, Nicotiana benthaminana.
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Production of Recombinant Human Butyrylcholinesterase in Nicotiana benthamianaHayward, Robin L. 03 October 2012 (has links)
Nerve agents (NAs) inhibit the essential enzyme acetylcholinesterase. Classified as chemical weapons, NAs are considered a threat to soldiers on the frontlines of warzones. Current treatments can prevent death from NA poisoning, but are not effective in preventing convulsions, seizures, or subsequent brain damage.
Butyrylcholinesterase (BChE) binds to NAs, rendering the chemicals harmless to acetylcholinesterase.. Two hundred mg of BChE is the putative prophylactic dose for adult humans, but is difficult to obtain in large quantities from expired human serum. Although recombinant BChE has been expressed in several organisms, the yields are still low.
Nicotiana benthamiana is an attractive plant for transient protein production due to its quick growth rate, abundance of tissue, and history of successful recombinant protein production. For this research, N. benthamiana was infiltrated with viral based vectors as well as binary vectors containing the human BChE gene. Multiple assays indicated that binary vector BChE-105-1 + P19 enabled the best expression, producing 26 mg BChE/kg tissue. / Canada Research Chair Program, OMAFRA,
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COMPARISON OF PLANT‐ADAPTED RHABDOVIRUS PROTEIN LOCALIZATION AND INTERACTIONSMartin, Kathleen Marie 01 January 2011 (has links)
Sonchus yellow net virus (SYNV), Potato yellow dwarf virus (PYDV) and Lettuce Necrotic yellows virus (LNYV) are members of the Rhabdoviridae family that infect plants. SYNV and PYDV are Nucleorhabdoviruses that replicate in the nuclei of infected cells and LNYV is a Cytorhabdovirus that replicates in the cytoplasm. LNYV and SYNV share a similar genome organization with a gene order of Nucleoprotein (N), Phosphoprotein (P), putative movement protein (Mv), Matrix protein (M), Glycoprotein (G) and Polymerase protein (L). PYDV contains an additional predicted gene between N and P, denoted as X, that has an unknown function. In order to gain insight into the associations of viral proteins and the mechanisms by which they may function, we constructed protein localization and interaction maps using novel plant expression vectors. Sub‐cellular localization was determined by expressing the viral proteins fused to green fluorescent protein in leaf epidermal cells of Nicotiana benthamiana. Protein interactions were tested in planta using bimolecular fluorescence complementation (BiFC). All three viruses showed Mv to be localized to the cell periphery and the G protein to be membrane associated. Comparing the interaction maps revealed that only the N‐P and M‐M interactions are common to all three viruses. Associations unique to only one virus include G‐Mv for SYNV, M‐Mv, M‐G, and N‐M for PYDV and P‐M for LNYV. The cognate N‐P proteins of all three viruses exhibit changes in localization when co‐expressed. To complement the mapping data, we also mapped the functional domains in the glycoproteins of SYNV and LNYV. The truncation of the carboxy terminus has no effect on localization compared to the full‐length protein; the nuclear localization signals (NLSs) present in SYNV‐G do not interact with known importins. These data suggest that although the interactions of the three viruses differ, each protein may have similar functional domains.
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Transformation of Nicotiana benthamiana with different BWYV (Beet western yellows virus) sequences to test for virus resistanceValenzuela Aguila, Sofia. Unknown Date (has links)
Techn. University, Diss., 2000--Braunschweig.
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Desarrollo de vectores virales basados en el virus del manchado foliar de los cítricos (CLBV)Agüero González, Jesús 09 December 2013 (has links)
Los cítricos son el cultivo frutal económicamente más importante tanto en
España como en el resto de los países productores. La clave para mantener la
competitividad de este sector consiste en obtener material vegetal de alta calidad,
para lo cual son indispensables los programas de mejora. La mejora de cítricos por
métodos clásicos es muy complicada, por lo que hay que recurrir a las nuevas
tecnologías para intentar acelerar y optimizar el procedimiento. La reciente
secuenciación del genoma de dos especies de cítricos ha permitido identificar una
larga lista de genes candidatos a participar en determinados procesos biológicos. Sin
embargo, son necesarios nuevos análisis para asociar cada gen a un fenotipo
específico o función biológica.
El empleo de vectores virales para determinar la función de genes mediante
silenciamiento génico inducido por virus (VIGS) ha demostrado ser una herramienta
muy útil para los estudios de genética reversa realizados en plantas. Este sistema
presenta ventajas respecto a los métodos tradicionales para estudiar la función de
genes como son la mutagénesis o la transformación genética, ya que permite
ensayar la función de numerosos genes en un corto periodo de tiempo. Esto es
especialmente crítico en el caso de los cítricos, que poseen largos periodos juveniles
de entre 6 y 8 años y donde la transformación de plantas adultas es muy difícil.
Además, permite estudiar la función de genes que son esenciales para el crecimiento
o el desarrollo de la planta y cuyo análisis es inviable con los métodos tradicionales.
Al comienzo de la tesis se había desarrollado un vector viral para cítricos
basado en el virus de la tristeza de los cítricos (CTV) con el que se pueden expresar
proteínas pero que no se ha ensayado para estudiar la función de genes mediante
VIGS. En el laboratorio disponíamos de un clon infeccioso de cDNA del genoma
completo del virus del manchado foliar de los cítricos (CLBV), un virus que infecta a
todas las especies y variedades de cítricos ensayadas y es asintomático en la mayoría
de ellas. Este clon infeccioso se ha modificado para obtener vectores virales basados
en el genoma de CLBV que pueden servir tanto para expresar proteínas como para
silenciar mediante VIGS genes de cítricos para la mejora genética de este cultivo.
Para ello, se ha introducido un punto de corte único PmlI en dos zonas del genoma
de CLBV: en el extremo 3¿ no traducible (vector clbv3¿) o en la zona intergénica
localizada entre los genes de las proteínas de movimiento y cápsida (CP) (vector
clbvIN). Para la expresión de secuencias foráneas mediante la formación de un
nuevo RNA subgenómico (sgRNA) se delimitó la secuencia mínima promotora del sgRNA CP mediante clonación de fragmentos de distinta longitud en torno al origen
de transcripción de dicho sgRNA en el vector clbv3'. El fragmento de 92 bases
localizado entre los nt -42 y +50 respecto al inicio de transcripción del sgRNA CP
contenía todos los elementos necesarios para la promoción de un nuevo sgRNA in
vivo. Esta secuencia mínima promotora se clonó en los 2 vectores virales
previamente desarrollados para generar los vectores clbv3¿pr y clbvINpr,
respectivamente. Ambos vectores fueron capaces de producir un nuevo sgRNA y de
expresar proteínas recombinantes.
Para determinar la estabilidad de los vectores obtenidos se clonaron en ellos
fragmentos de secuencias lineales de distinto tamaño, o en tándem invertido para la
formación de una estructura en horquilla, y se inocularon en plantas de N.
benthamiana y cítricos. Todas las construcciones derivadas del vector clbv3' se
mostraron estables a lo largo de las diferentes brotaciones analizadas durante al
menos 3 años, comprobándose la replicación viral e integridad del inserto. Sin
embargo, no se detectó multiplicación viral con ninguna de las construcciones
derivadas del vector clbvIN. La estabilidad de las construcciones derivadas de los
vectores con el promotor duplicado dependía del tamaño del inserto. Con todas
ellas se detectó replicación viral pero se observaron eventos de recombinación
cuando se clonaban fragmentos superiores a 720 nt en el vector clbvINpr o 408 nt en
el vector clbv3'pr.
Un factor importante para determinar la eficiencia y funcionalidad de los
vectores desarrollados es conocer cómo se mueve y se distribuye el virus en los
distintos tejidos de la planta. Para ello se inocularon plantas de N. benthamiana y
cítricos con la construcción clbv3¿pr-GFP, que expresa GFP en los tejidos donde se
localiza el virus. En N. benthamiana, la observación de GFP permitió detectar la
presencia de CLBV en la mayoría de tejidos, acumulándose preferentemente en
óvulos y regiones meristemáticas. En cítricos no se pudo visualizar GFP pero el virus
se detectó en regiones meristemáticas mediante RT-PCR a tiempo real e hibridación
molecular. La acumulación de CLBV en tejidos meristemáticos explicaría la dificultad
de eliminar este virus mediante microinjerto.
Para evaluar la capacidad de los vectores clbv3'pr y clbvINpr para expresar
proteínas se clonó en ellos la secuencia completa del gen gfp y se cuantificó la
cantidad de proteína GFP sintetizada en las plantas infectadas. En N. benthamiana la
cantidad de GFP estimada para el vector clbv3'pr fue de 16 µg de proteína por
gramo de peso fresco, cantidad que resultó entre 5 y 6 veces superior a la estimada para el vector clbvINpr. Sin embargo, en cítricos, debido a la inestabilidad del vector
clbv3'pr, sólo se pudo cuantificar la proteína expresada por la construcción del
vector clbvINpr, estimándose en 0.6 µg de GFP por gramo de peso fresco.
La efectividad de los vectores clbv3', clbv3'pr y clbvINpr para silenciar genes
mediante VIGS se ensayó clonando fragmentos de genes tanto endógenos de
plantas (pds, actina, sulfur) como el gen gfp introducido experimentalmente en
plantas transgénicas. En cítricos todas las construcciones de los tres vectores
indujeron fenotipo de silenciamiento del gen ensayado, aunque el vector clbv3' fue
el más efectivo para el estudio de VIGS en este huésped. Sin embargo, en N.
benthamiana sólo se desencadenó el silenciamiento en las plantas inoculadas con la
construcción clbv3¿pr-hp58PDS, que expresa una horquilla de doble cadena de un
fragmento de 58 nt del gen pds. En todas las plantas silenciadas se detectó una
disminución del correspondiente mRNA del gen ensayado y una acumulación de
siRNAs derivados tanto del mRNA del gen insertado como del RNA genómico del
virus. Por otro lado, el fenotipo de silenciamiento de los genes ensayados se observó
en sucesivas brotaciones, lo que confirma la gran estabilidad de los vectores basados
en el genoma de CLBV.
Los vectores virales desarrollados en esta tesis constituyen una herramienta
eficiente para el estudio de la función de genes mediante genética reversa utilizando
la técnica VIGS. También pueden ser útiles para estudio de genética directa
mediante expresión de proteínas o para la protección del cultivo frente a
enfermedades producidas por virus, bacterias y hongos o frente a plagas de
invertebrados. / Agüero González, J. (2013). Desarrollo de vectores virales basados en el virus del manchado foliar de los cítricos (CLBV) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34342 / TESIS
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Evaluation of RANTES analogue expression in Nicotiana benthamiana and Lycopersicon esculentum and their topical microbicidal activityMawela, Kedibone Gloria January 2013 (has links)
The HIV/AIDS pandemic has dramatically altered patterns of morbidity and mortality in
sub-Saharan Africa during the last two decades. In the absence of HIV vaccine,
microbicides may offer viable option for protection against HIV infection. Microbicides
are products that are applied topically inside the vagina or rectum that act to impede
transmission of HIV and other sexually transmitted diseases. Small human chemokines
such as RANTES (regulated upon activation, normal T cell expressed and secreted) are
currently been investigated as microbicides candidates.
A number of N-terminally modified RANTES analogues such as 5P12 and 6P4 with a
much higher antiviral potency have been developed and they have strong potential for
use as microbicides. Since plants offer an alternative option for cost effective production
of protein therapeutics, we evaluated the feasibility of expressing 5P12 and 6P4 in
Nicotiana benthamiana species. 5P12 is considered the most promising candidate for
use in the microbicide pipeline because it inhibits HIV infection through cellular receptor
antagonism. Hence its feasibility of expression was also evaluated in Lycopersicon
esculentum (tomato). The two analogues were transiently expressed in the selected
plant species via agrobacterium-mediated transfection.
For expression in N. benthamiana, two different vectors (pTRA and MagnICON) were
used to deliver the two analogues for transient expression. About 6-8 weeks-old N.
benthamiana plants were agroinfiltrated via needle injection and vacuum infiltration
methods and targeted to four subcellular compartments viz: apoplast, chloroplast,
cytosol and endoplasmic reticulum (ER). The agroinfiltrated leaves were replanted,
grown in a tissue culture laboratory and harvested after different periods. For
expression in L. esculentum, the MagnICON constructs were used to deliver the 5P12
gene into four different developmental stages of tomato fruits viz: mature green (MG),
breaker (B), pink (P) and ripe (R) via needle injection. The agroinjected tomato fruits
were incubated in a dark cupboard and harvested after different periods.
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Proteins were extracted from the harvested material and evaluated for 5P12 and 6P4
expression. ELISA results showed expression of 5P12 and 6P4 in N. benthamiana
leaves which was detectable at 3-9 days post infiltration (dpi). Similar results were
obtained for 5P12 and 6P4, consequently only results for 5P12 are reported. The
vacuum infiltrated leaves of both pTRA and MagnICON constructs led to higher yields
than the needle injected leaves. The highest yields were obtained with the MagnICON
constructs. The highest 5P12 expression level of 603 μg/kg fresh weight leaf tissues
(~0.024% TSP) was obtained in the apoplast at 9 dpi. The pTRA constructs had the
highest expression levels of 0.63μg/kg FW in the cytosol at 3 dpi.
5P12 was also detectable at 3-9 dpi in L. esculentum, based on ELISA results. The
highest 5P12 expression of 23.56 μg/kg FW and pH 4.75 tissues was obtained at the
MG stage in the apoplast at 9 dpi. Western blot analysis confirmed the size of plantmade
5P12. Moreover, the plant extracts had anti-viral activity and were not toxic to
TZM-bl cells.
Our results show that the RANTES can be made in both N. benthamiana and L.
esculentum and that the levels are not different from other systems reported previously.
Furthermore, this is the first report that a chemokine has been expressed in plants. The
quantities expressed were low making the commercial development of a microbicide
from these species impractical. However, production of bulky leaf material may enhance
the quantities. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Paraclinical Sciences / unrestricted
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