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Estudos iniciais de ineraçãos da HSP90 através da caracterização funcioanl de um transgênico e biofísica de uma co-chaperona / Insights on Hsp90 chaperone interactions using transgenic and biophysical approachesGonçalves, Danieli Cristina, 1986- 20 August 2018 (has links)
Orientadoesr: Carlos Henrique Inácio Ramos, Gonçalo Amarante Guimarães Pereira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T06:21:14Z (GMT). No. of bitstreams: 1
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Previous issue date: 2012 / Resumo: Chaperonas moleculares (Heat Shock proteins - HSPs) são componentes chave do sistema de controle de qualidade de proteínas (PQC - Protein Quality Control), que é essencial para a vida, sendo responsável por manter a homeostase proteica e a adequada função de diversas vias. Problemas no processo de enovelamento estão relacionados a doenças degenerativas, amilóides e câncer. Em plantas, as chaperonas moleculares desempenham um papel crucial na proteção contra estresses bióticos e abióticos, pois como organismos sésseis, as plantas devem ser capazes de responder rapidamente a mudanças na temperatura, salinidade, déficit hídrico, entre outros. A chaperona molecular Hsp90 (Heat Shock protein 90 kDa) compreende uma família ubíqua, considerada um 'hub' por interagir com chaperonas, co-chaperonas e ter como clientes proteínas regulatórias essenciais como fatores de transcrição, quinases, receptores de hormônios, entre outros. A Hsp90 age em conjunto com co-chaperonas, as quais modulam e direcionam sua função. Uma destas co-chaperonas é a Hop (Hsp70-Hsp90 organizing protein), capaz de interagir simultaneamente com a Hsp90 e Hsp70, mediando a transferência de substratos. A Hop é composta por três domínios com repetições de tetratricopeptídeos (TPR) (TPR1, TPR2A e TPR2B), responsáveis pela interação com as chaperonas, porém a dinâmica desta interação não está bem entendida, uma vez que ainda não há estrutura da Hop inteira e o estado oligomérico desta co-chaperona ainda é controverso na literatura. Neste trabalho apresentamos a classificação de um gene de Hsp90 de cana-de-açúcar, e o início de sua caracterização funcional através de transgenia em Arabidopsis thaliana. Apresentamos também a caracterização biofísica de uma importante co-chaperona da Hsp90, a Hop (Hsp70-Hsp90 organizing protein) humana. Através da análise de sequências a Hsp90 de cana-de-açúcar foi classificada como Hsp90-3, uma isoforma citosólica. Plantas transgênicas de A. thaliana, produzidas a partir da inserção do gene da Hsp90-3 de cana-de-açúcar, apresentaram níveis reduzidos de Hsp90. Tal perturbação nos níveis de Hsp90 parece ter afetado a expressão de outras proteínas da rede de interações, relacionadas com processos diversos como resposta imune e fotossíntese. As plantas transgênicas também exibiram germinação mais rápida e raízes mais longas em relação ao controle. Sob estresse térmico, linhagens transgênicas apresentaram maior suscetibilidade à alta temperatura em relação ao controle. Tais resultados sugerem que a Hsp90 tem um importante papel na fisiologia celular e no desenvolvimento, e que os níveis de Hsp90 são críticos para a resposta frente a estresses. A caracterização biofísica do mutante Hop D456G, uma mutação no domínio TPR2B, mostrou que esta proteína é uma mistura de monômeros, dímeros e oligômeros maiores, porém com prevalência do estado monomérico. O resíduo D456 pode ter uma participação na dinâmica de dimerização e é possível que o estado oligomérico da Hop seja regulado entre os estados monomérico e dimérico, com a finalidade de facilitar sua atividade adaptadora / Abstract: Molecular chaperones (heat shock proteins - HSPs) are key components of protein quality-control system (PQC - Protein Quality Control), which maintains protein homeostasis and the proper function of several pathways, being essential for life. Defects in folding processes are related to degenerative diseases, amyloidosis and cancer. In plants, which as sessile organisms must be able to respond rapidly to changes in temperature, salinity, water deficit, and others, molecular chaperones play a crucial role in protecting against such biotic and abiotic stresses. Molecular chaperone Hsp90 (Heat Shock Protein 90 kDa) comprise an ubiquitous family, considered a hub as it interacts with chaperones, co-chaperones, and have as clients key regulatory proteins such as transcription factors, kinases, hormone receptors, and others. The chaperone acts together with co-chaperones, which modulate and guide Hsp90 function. The co-chaperone Hop (Hsp70-Hsp90 organizing protein), interacts simultaneously with Hsp90 and Hsp70, mediating substrate transfer. Hop has three TPR domains (TPR1, and TPR2A TPR2B) responsible for interaction with the chaperones, but this interaction dynamics remains unclear, since there is no structure of full length Hop and its oligomeric state is controversial in literature reports. This work presents the classification of an Hsp90 gene from sugarcane, and primary functional characterization studies in Arabidopsis thaliana transgenic lines. We also present the biophysical characterization of the human Hsp90 co-chaperone Hop (Hsp70-Hsp90 organizing protein). Through sequence analysis the Hsp90 from sugarcane has been classified as Hsp90-3, a cytosolic isoform. Transgenic A. thaliana, produced by Hsp90-3 insertion, exhibited reduced transcript levels of Hsp90. This disruption in Hsp90 levels seems to affect the expression of other proteins from the interaction network, which are related to various processes such as immune response and photosynthesis. Transgenics also exhibited faster germination and longer roots than the control. Under heat stress, transgenic lines showed increased susceptibility to high temperature. These results suggest that Hsp90 has an important role in cellular physiology and development; in addition the levels of Hsp90 are critical for responses to stresses. The biophysical characterization of the mutant D456G Hop, a mutation in domain TPR2B showed that this protein is a mixture of monomers, dimers and higher oligomers, but the monomeric state is majoritary. The residue D456 may be involved in dimerization dynamics, and it is possible that Hop is regulated between monomeric and dimeric species, to enable its adaptor functions / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular
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Molecular analysis of genetic diversity in dometicated pigeonpea (Cajanus cajan (L.) Millsp.) and wild relatives / Molecular analysis of genetic diversity in domesticated pidgeonpea (Cajanus cajan Milsp.) and wild relatives : insights into the domestication of pidgeonpeaKassa, Mulualem Tamiru January 2011 (has links)
Cajanus cajan (L.) Millsp. (Pigeonpea) belongs to the Leguminosae genus Cajanus which is composed of 34 species. Pigeonpea is the only cultivated member of the genus, while the remaining species are wild relatives belonging mainly to the secondary gene pool. DNA sequence data from the nuclear ITS region and the chloroplast trnL-F spacer were utilized to investigate the phylogenetic relationships between Cajanus and five other allied genera in the subtribe Cajaninae. This study revealed the non-monophyly of Cajanus and Rhynchosia and supported the monophyly of Eriosema and Flemingia, but more sampling ,especially from the large genera of Rhynchosia and Eriosema, is recommend to adequately test the hypothesis of generic monophyly. The phylogenetic relationships within the genus Cajanus resolved Cajanus scarabaeoides (L.) Thouars as the most basal species in the Cajanus clade. The study also utilized Single Nucleotide Polymorphism (SNP) markers derived from low copy orthologous genes and genotyped using the high throughput SNP-OPA Illumina golden gate assay. The aim was to understand phylogenetic and domestication history, genetic structure, patterns of genetic diversity, gene flow and historical hybridization between Cajanus cajan (pigeonpea) and wild relatives. The neighbor-joining tree resolved well-supported clusters, which reflect the distinctiveness of species and congruence with their geographical origin. It supported the ITS based phylogeny and resolved C. scarabaeoides as basal to the Cajanus clade. The phylogenetic signal and genetic signatures revealed insights into the domestication history of pigeonpea. Our results supported Cajanus cajanifolius as the presumed progenitor of pigeonpea and we speculate that for pigeonpea there was a single major domestication event in India. Genetic admixture and historical hybridization were evident between pigeonpea and wild relatives. Abundant allelic variation and genetic diversity was found in the wild relatives, with the exception of wild species from Australia, as compared to the domesticated pigeonpea. There was a reduction of about 75% in genetic polymorphism in domesticated pigeonpea as compared to the wild relatives, indicating a severe “domestication bottleneck” during pigeonpea domestication. We discovered SNP markers associated with disease resistance (NBS-LRR) loci. The SNPs were mined in a comparison of BAC-end sequences (BES) of C. cajan and amplicons of the wild species, C. scarabaeoides. A total of ~3000 SNPs were identified from 304 BES. These SNPs could potentially be used in constructing a genetic map and for marker assisted breeding.
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PCR-based Synthesis of Codon Optimized cry2Aa Gene for Production of Shoot and Fruit Borer (Leucinodes orbonalis) Resistant Eggplant (Solanum melongena L.) CultivarsGupta, Rahul 20 January 2006 (has links)
Brinjal shoot and fruit borer (Leucinodes orbonalis Guenee) is a major limiting factor in commercial cultivation of eggplant in southeast Asia. Extensive use of pesticides as well as the conventional breeding methods have been ineffective in controlling the borer so there is a need for Integrated Pest Management (IPM) strategies for its control. Bacillus thuringiensis (Bt) is known to produce a variety of insecticidal crystal proteins toxic to lepidopteran, dipteran and coleopteran pests. The Cry2Aa protein has been found to be more toxic to brinjal shoot and fruit borer than Cry1Ab. My objective was to develop eggplant cultivars that express a codon-optimized cry2Aa gene, the sequence of which is based on that of an Indian isolate of Bt, with the eventual goal of producing fully resistant cultivars. The cry2Aa gene was modified for optimal expression in eggplant using the codon usage frequencies based on solanaceous sequences (eggplant, tomato and pepper). The GC content was increased from 34.3% in the native gene to 41.3% in the optimized gene, thus removing the AT-rich regions that are typical for Bt cry genes. Also, other mRNA destabilizing and hairpin forming structure sequences were removed. The gene was synthesized in four different parts with complementary restriction sites. A total of 152 oligonucleotides (oligos) was used to assemble the 1.9 kb gene using dual asymmetric (DA) and overlap extension (OE) PCR techniques. The individual parts were subsequently ligated using the complementary restriction sites and inserted into vector pCAMBIA 1302. Also, the transformation efficiency of 12 different eggplant cultivars was tested using plasmid pHB2892 to predict utility for transformation with the synthetic cry2Aa. / Master of Science
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Plant defence genes expressed in tobacco and yeastBecker, John van Wyk 03 1900 (has links)
Thesis (MSc (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2002. / Pathogen devastation of food products has been the topic of extensive research efforts
worldwide. Fungal infections are foremost amongst these pests, contributing not only to
losses in product yield, but also significantly affecting the quality thereof. It is not surprising
then that producers of these foodstuffs and their derived products continually strive
towards the highest possible product quality. Therefore, it remains imperative that
satisfactory methods are implemented to control these fungal pathogens. The current
strategies are all hampered by drawbacks, and severe crop losses are still experienced.
New technologies are being explored; one such technology is the genetic
transformation of plant species. This method has enabled scientists to introduce foreign
genes, with known functions and predictable outcomes, into plants. Genes identified to be
involved in disease resistance have become the focus of numerous research efforts
concerned with the improvement of the plant's innate defence response. This study aimed
to enhance disease resistance to fungal pathogens by means of the genetic transformation
of two genes previously shown to be involved in disease resistance. These genes encode
polygalacturonase-inhibiting proteins (PGIPs) from Phaseolus vulgaris and resveratrol
synthase from Vitis vinifera. PGIPs specifically inhibit the action of fungal
polygalacturonases (PGs), which are enzymes responsible for the hydrolytic breakdown of
plant cell walls. These enzymes were also found to be the first enzymes that are secreted
by fungal pathogens during infection of the host plant. Additionally, PGIP-PG interaction
results in the existence of molecules involved in the activation of plant defence responses.
Resveratrol, the product of resveratrol synthase, exerts its antifungal action by destruction
of the microbial cellular membranes. These mentioned genes were transformed alone, and
in combination, into Nicotiana tabacum and the resultant transgenic lines were evaluated
for enhanced disease resistance and for possible synergistic effects between the
transgenes.
Several independent transgenic lines were regenerated with genes integrated into the
tobacco genome. Almost all the plants harbouring only pgip or vst1 genes also expressed
these genes at a high frequency. Some non-expressing lines were identified from the
transgenic plants that had integrated both genes, but several lines were obtained
expressing both transgenes. Good correlations were observed between transgene product
activity and enhanced resistance to the fungus Botrytis cinerea in an antifungal in planta
assay. Lines showing the highest PGIP activity and resveratrollevels were more resistant
to the pathogen, leading to disease resistance of up to 80% seven days after inoculation in
comparison to an untransformed control. These lines maintained their strong inhibition,
even three weeks post-inoculation, showing a complete halt in disease development and
fungal growth. These results provide good indications of the efficacy of these transgenes
in the upregulation of plant defence. However, the study will have to be expanded to include even more transgenic lines to elucidate the possible synergistic effects of these
genes.
In an additional pilot study, genes encoding for precursors and for the formation of
resveratrol were introduced into the yeast Saccharomyces cerevisiae. The resultant
recombinant yeast strains were evaluated for their ability to produce the phenolic
substance, resveratrol. This compound has been implicated in beneficial aspects relating
to human health, including positive effects on atherosclerosis and platelet aggregation as a
direct result of its antioxidant and anti-inflammatory activities.
Recombinant yeast strains were constructed that expressed genes coding for
coenzyme A ligase and resveratrol synthase. These strains were shown to be able to
produce the phenolic compound resveratrol from the precursors present in the yeast as
well as from the products introduced with the transformation. The resveratrol was
complexed with an added glucose moiety. These results are extremely positive,
considering the possibility of manipulating wine yeasts to produce resveratrol during the
wine fermentation, thereby adding to the health aspects of both red and white wine. This is
the first report of the production of this compound by the introduction of genes necessary
for its biosynthesis in a foreign host.
This study has confirmed the importance of PGIPs and resveratrol in the effort to
enhance disease resistance in plants through genetic transformation technology. It has
also shown that the health benefits of resveratrol could be exploited more optimally in the
wine industry, by producing wine yeasts with the ability to synthesise this important
antioxidant.
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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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Cultura de tecidos e transformação genética com o gene Ddm1 no estudo do silenciamento de elementos de transposição em cana-de-açúcar / Tissue culture and genetic transformation with the Ddm1 gene to study silencing of the transposable elements in sugarcanePicelli, Eduardo da Cruz Maduro 27 August 2010 (has links)
A cana-de-açúcar é uma das principais culturas agroindustriais do Brasil, sendo amplamente cultivada para a produção de açúcar e etanol. Esta cultura se torna a cada dia mais importante no cenário mundial, devido à busca constante por fontes de energia alternativas e mais sustentáveis. Para atender a crescente demanda, é necessária a liberação frequente de novas variedades, mais adaptadas às regiões de cultivo e tolerantes às alterações ambientais. Assim, o estabelecimento da metodologia de transformação genética além de contribuir para o estudo funcional de genes de interesse é uma metodologia alternativa para obtenção de novas variedades. O processo de obtenção de transgênicos é dependente de um eficiente protocolo de regeneração de plantas in vitro, que geralmente envolve uma fase de formação de células indiferenciadas (calos). A indução e a manutenção dos calos são favoráveis ao aumento da atividade de elementos de transposição (ETs) os quais são muito freqüentes no genoma de cana e podem acarretar variabilidade no genoma vegetal pela alteração dos padrões e funções gênicas devido a essa mobilização, afrontando a fidelidade genética dos cultivares transgênicos obtidos. Baseando-se na importância de reduzir o período de cultura de tecidos e controlar a atividade dos ETs durante o desenvolvimento in vitro, o objetivo desse trabalho foi buscar alternativas no controle e na redução do tempo para regeneração de plantas, inclusive com a aplicação de peptídeos hormonais, assim como de transformar geneticamente as variedades RB835089 e RB835486 com o gene Ddm1 de Arabidopsis, visando o silenciamento dos elementos de transposição em cana-de-açúcar. Para isso, foram analisados os meios de cultura MS3c e ML1G1 e o efeito da água de coco na indução e formação de calos como também na regeneração de plantas. Foram testados os meios de regeneração de plantas MSAc, SRM, ML1R3 e ML1R4, obtendo-se em média 5,2 plantas por explante no meio MSAc, que foi superior aos demais meios. Este meio foi utilizado para testar o efeito individual dos peptídeos hormonais CLV3 e PSK- em calos embriogênicos, os quais apresentaram acréscimo na regeneração de plantas para 9,3 plantas por explantes com doses de 30 µM de PSK-a. A transformação genética por biolística através da cotransformação dos genes neo e AtDdm1 resultou em 34 plantas transgênicas. O estudo da mobilização dos ETs durante o desenvolvimento in vitro foi realizado para quatro retrotransposons. A expressão heteróloga do gene AtDdm1 em cana-de-açúcar mostrou atuar no controle da expressão do retroelemento TE010. O estudo da mobilização dos retrotransposons e do gene Ddm1 endógeno de cana (SsDdm1) durante o desenvolvimento in vitro confirmou que o gene SsDdm1 foi chave no controle da expressão dos retroelementos. A transformação genética com o gene AtDdm1 aliada a rápida regeneração de plantas a partir de discos foliares possibilitam condições que minimizam a expressão dos ETs em cana-de-açúcar. / Sugarcane is one of the major agro-industrial crops of Brazil being widely cultivated for the production of sugar and ethanol. This culture has become increasingly more important on the world stage each day due to the constant search for alternative and sustainable energy sources. In order to meet growing demand, it is necessary to often release new varieties, better adapted to cultivated expansion area and tolerant to environmental changes. Thus, the establishing of genetic transformation methodology beyond of contributing to the functional study of genes of interest and it is an alternative method for obtaining new varieties. The process of obtaining transgenic plants is dependent of an efficient protocol for in vitro plant regeneration, which generally involves a phase of undifferentiated cells (callus). The induction and maintenance of callus are favorable to increase the activity of transposable elements (TEs) which are very frequent in the genome of sugarcane and may cause variability in the plant genome by altering patterns and gene functions due to this mobilization, confronting the genetic fidelity of the transgenic cultivars obtained. Based on the importance of reducing the period of tissue culture and control the activity of TEs during in vitro development, the objective of this work was to seek alternatives to control and reduce the time for plant regeneration, including the use of peptides hormone, as well as to genetically transform sugarcane varieties RB835089 and RB835486 with the Ddm1 Arabidopsis gene to silence the transposable elements in cane sugar. For this, we tested the culture media MS3c and ML1G1and the effect of coconut water in callus induction and growth as well as on plant regeneration. We tested the plant regeneration media MSAc, SRM, ML1R3 and ML1R4, obtaining an average of 5.2 plants per explants using MSAC, superior to other medium tested. It was used to test the individual effect of peptides hormones such as CLV3 and PSK- in embryogenic callus, which showed an increase in plant regeneration to 9.3 plants per explant with doses of 30µM PSK-a. Genetic co-transformation with the neo and AtDdm1 genes by biolistic resulted in 34 transgenic plants. A study of TEs during in vitro development was performed for four retrotransposons. Heterologous expression of the AtDdm1 gene in sugarcane showed to control the expression of the retroelement TE010. The study of mobilization of retrotransposons and the endogenous Ddm1 gene (SsDdm1) during in vitro development confirmed that SsDdm1 was key gene in controlling the expression of retrotransposons. Genetic transformation with the AtDdm1 gene and the fast regeneration of plants provide positive conditions to minimize the expression of ETs in sugarcane.
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Plant as bioreactor: transgenic expression of malaria surface antigen in plants.January 2001 (has links)
by Ng Wang Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 131-139). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / List of Tables --- p.ix / List of Figures --- p.x / List of Abbreviations --- p.xiii / Table of Contents --- p.xv / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter Chapter 2: --- Literature Review --- p.3 / Chapter 2.1 --- Malaria --- p.3 / Chapter 2.1.1 --- Global picture --- p.3 / Chapter 2.1.2 --- Malaria mechanics --- p.4 / Chapter 2.1.3 --- Life cycle of malaria parasite --- p.4 / Chapter 2.2 --- Treatment of malaria ´ؤ malaria drugs --- p.5 / Chapter 2.2.1 --- Antimalarial drugs --- p.5 / Chapter 2.2.2 --- Drug resistance --- p.6 / Chapter 2.3 --- Treatment of malaria - malarial vaccines --- p.7 / Chapter 2.3.1 --- Malarial vaccine developments --- p.7 / Chapter 2.3.2 --- Transmission blocking vaccines --- p.7 / Chapter 2.3.3 --- Pre-erythrocytic vaccines --- p.9 / Chapter 2.3.4 --- Blood stage vaccines --- p.10 / Chapter 2.4 --- The major merozoite protein - gpl95 --- p.11 / Chapter 2.5 --- Plants as bioreactors --- p.12 / Chapter 2.5.1 --- Products of transgenic plants --- p.13 / Chapter 2.6 --- Transgenic plants for production of subunit vaccines --- p.14 / Chapter 2.6.1 --- Norwalk virus capsid protein production --- p.15 / Chapter 2.6.2 --- Hepatitis B surface antigen production --- p.15 / Chapter 2.7 --- Tobacco and Arabidopsis as model plants --- p.16 / Chapter 2.7.1 --- Arabidopsis --- p.16 / Chapter 2.7.2 --- Tobacco --- p.17 / Chapter 2.8 --- Transformation methods --- p.17 / Chapter 2.8.1 --- Direct DNA uptake --- p.17 / Chapter 2.8.1.1 --- Plant protoplast transformation --- p.17 / Chapter 2.8.1.2 --- Biolistic transformation --- p.18 / Chapter 2.8.2 --- Agrobacterium-mediated transformation --- p.18 / Chapter 2.8.2.1 --- Leaf-disc technique --- p.18 / Chapter 2.8.2.2 --- In planta transformation --- p.19 / Chapter 2.9 --- Phaseolin --- p.20 / Chapter 2.10 --- Detection and purification of recombinant products - Histidine tag --- p.21 / Chapter 2.11 --- Aims of study and hypotheses --- p.22 / Chapter Chapter 3: --- Materials and Methods --- p.24 / Chapter 3.1 --- Introduction --- p.24 / Chapter 3.2 --- Chemicals --- p.24 / Chapter 3.3 --- Expression in tobacco system --- p.24 / Chapter 3.3.1 --- Plant materials --- p.24 / Chapter 3.3.2 --- Bacterial strains --- p.25 / Chapter 3.3.3 --- Chimeric gene construction for tobacco transformation --- p.25 / Chapter 3.3.3.1 --- The cloning of pTZPhasp/flgp42-His/Phast (F1) --- p.26 / Chapter 3.3.3.2 --- The cloning of pBKPhasp-sp/flgp42-His/Phast (P9) --- p.30 / Chapter 3.3.3.3 --- The cloning of pHM2Ubip/flgp42-His/Nost (C2) --- p.30 / Chapter 3.3.4 --- Confirmation of sequence fidelity of chimeric gene by DNA sequencing --- p.33 / Chapter 3.3.5 --- Cloning of chimeric gene into binary vector --- p.34 / Chapter 3.3.6 --- Triparental mating of Agrobacterium tumefaciens LBA4404/pAL4404 --- p.35 / Chapter 3.3.7 --- Tobacco transformation and regeneration --- p.36 / Chapter 3.3.8 --- GUS assay --- p.37 / Chapter 3.3.9 --- Genomic DNA isolation --- p.37 / Chapter 3.3.10 --- PCR amplification and detection of transgene --- p.38 / Chapter 3.3.11 --- Southern blot analysis --- p.38 / Chapter 3.3.12 --- Total seeds RNA isolation --- p.39 / Chapter 3.3.13 --- RT-PCR --- p.39 / Chapter 3.3.14 --- Northern blot analysis --- p.40 / Chapter 3.3.15 --- Protein extraction and SDS-PAGE --- p.40 / Chapter 3.3.16 --- Western blot analysis --- p.41 / Chapter 3.4 --- Expression in Arabidopsis system --- p.42 / Chapter 3.4.1 --- Plant materials --- p.42 / Chapter 3.4.2 --- Bacterial strains --- p.42 / Chapter 3.4.3 --- Chimeric gene construction --- p.42 / Chapter 3.4.3.1 --- The cloning of pBKPhasp-sp/His/EK/p42/Phast (DH) --- p.43 / Chapter 3.4.3.2 --- The cloning of pTZPhaSp/His/EK/p42/Phast (EH) --- p.45 / Chapter 3.4.3.3 --- The cloning of pBKPhasp-sp/His/EK/flgp42/Phast (DHF) and pTZPhasp/His/EK/flgp42/Phast (EHF) --- p.45 / Chapter 3.4.4 --- Confirmation of sequence fidelity of chimeric genes --- p.45 / Chapter 3.4.5 --- Cloning of chimeric gene into Agrobacterium binary vector --- p.49 / Chapter 3.4.6 --- Transformation of Agrobacterium tumefaciens GV3101/pMP90 with chimeric gene constructs --- p.49 / Chapter 3.4.7 --- Arabidopsis Transformation --- p.49 / Chapter 3.4.8 --- Vacuum infiltration transformation --- p.50 / Chapter 3.4.9 --- Selection of successful transformants --- p.51 / Chapter 3.4.10 --- Selection for homozygous plants with single gene insertion --- p.51 / Chapter 3.4.11 --- GUS assay --- p.52 / Chapter 3.4.12 --- Genomic DNA isolation --- p.52 / Chapter 3.4.13 --- PCR amplification and detection of transgenes --- p.52 / Chapter 3.4.14 --- Southern Blot analysis --- p.52 / Chapter 3.4.15 --- Total siliques RNA isolation --- p.53 / Chapter 3.4.16 --- RT-PCR --- p.53 / Chapter 3.4.17 --- Northern blot analysis --- p.53 / Chapter 3.4.17 --- Protein extraction and SDS-PAGE --- p.54 / Chapter 3.4.18 --- Western blot analysis --- p.54 / Chapter 3.5 --- In vitro transcription and translation --- p.54 / Chapter 3.5.1 --- In vitro transcription --- p.54 / Chapter 3.5.2 --- In vitro translation --- p.55 / Chapter 3.6 --- Particle bombardment of GUS fusion gene --- p.56 / Chapter 3.6.1 --- Chimeric gene constructs --- p.56 / Chapter 3.6.2 --- Particle bombardment using snow bean cotyledon --- p.61 / Chapter Chapter 4: --- Results --- p.63 / Chapter 4.1 --- Tobacco system --- p.63 / Chapter 4.1.1 --- Chimeric gene constructs --- p.63 / Chapter 4.1.2 --- Tobacco transformation and regeneration --- p.65 / Chapter 4.1.3 --- GUS activity assay --- p.67 / Chapter 4.1.4 --- Molecular analysis of transgene integration --- p.68 / Chapter 4.1.4.1 --- Genomic DNA extraction and PCR --- p.68 / Chapter 4.1.4.2 --- Southern blot analysis --- p.70 / Chapter 4.1.5 --- Molecular analysis of transgene expression --- p.72 / Chapter 4.1.5.1 --- Total RNA isolation and RT-PCR --- p.72 / Chapter 4.1.5.2 --- Northern blot analysis --- p.75 / Chapter 4.1.6 --- Genomic PCR to confirm whole gene transfer --- p.76 / Chapter 4.1.7 --- Biochemical analysis of transgene expression --- p.78 / Chapter 4.1.7.1 --- Protein extraction and SDS-PAGE --- p.78 / Chapter 4.1.7.2 --- Western blot analysis --- p.78 / Chapter 4.2 --- Arabidopsis system --- p.83 / Chapter 4.2.1 --- Chimeric gene constructs --- p.83 / Chapter 4.2.2 --- Arabidopsis transformation and selection --- p.85 / Chapter 4.2.3 --- Selection of transgenic plants --- p.87 / Chapter 4.2.4 --- Assay of GUS activity --- p.91 / Chapter 4.2.5 --- Molecular analysis of transgene integration --- p.92 / Chapter 4.2.5.1 --- Genomic DNA extraction and PCR --- p.92 / Chapter 4.2.5.2 --- Southern blot analysis --- p.96 / Chapter 4.2.6 --- Molecular analysis of transgene expression --- p.99 / Chapter 4.2.6.1 --- Total RNA isolation and RT-PCR --- p.99 / Chapter 4.2.6.2 --- Northern blot analysis --- p.106 / Chapter 4.2.7 --- Genomic PCR for confirmation of whole gene transfer --- p.107 / Chapter 4.2.8 --- Biochemical analysis of transgene expression --- p.108 / Chapter 4.2.8.1 --- Protein extraction and SDS-PAGE --- p.108 / Chapter 4.2.8.2 --- Western blot analysis --- p.108 / Chapter 4.3 --- In vitro transcription and translation --- p.112 / Chapter 4.4 --- Particle bombardment of p42/ GUS fusion gene --- p.115 / Chapter Chapter 5: --- Discussion and Future perspectives --- p.117 / Chapter 5.1 --- Failure in detecting transgene expression --- p.117 / Chapter 5.2 --- Poor transgene expression --- p.120 / Chapter 5.2.1 --- Bacillus thuringiensis toxin and green fluorescent protein --- p.120 / Chapter 5.2.2 --- AT-richness --- p.121 / Chapter 5.2.3 --- Deleterious sequence - AUUUA --- p.123 / Chapter 5.2.4 --- Presence of AAUAAA or AAUAAA-like motifs --- p.125 / Chapter 5.2.5 --- Codon usage --- p.126 / Chapter 5.3 --- Future perspectives --- p.127 / Chapter Chapter 6: --- Conclusion --- p.129 / References --- p.131
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Isolation and molecular characterisation of tomato spotted wilt virus (TSWV) isolates occuring in South Africa.Sivparsad, Benice. January 2006 (has links)
Tomato spotted wilt virus (TSWV), a Tospovirus, is one of the ten most economically
destructive plant viruses worldwide, causing losses exceeding one billion U.S. dollars
annually on several crops. In South Africa (SA), TSWV has become an important
virus in many economically important crops. The main objective of this research
project was to isolate, identify and characterise TSWV isolates occurring in SA.
A review of current literature assembled background information on TSWV molecular
biology, epidemiology, transmission, detection and control.
A TSWV isolate infecting pepper (Capsicum sp.) occurring in KZN was isolated and
partially characterised. The virus was positively identified as TSWV using the
enzyme-linked immunosorbent assay (ELISA) and the presence of typical necrotic
TSWV symptoms on Nicotinia rustica L. Symptomatic leaves were harvested and the
virus was partially purified using standard procedures. Under the transmission
electron microscope (TEM), typical quasi-spherical and dumbbell-shaped particles of
80-100nm in diameter were observed in negatively stained preparations of both crude
and purified virus samples. In negatively stained ultra-thin virus infected leaf
sections, an abundance of mature viral particles (100nm) housed in the cisternae of
the endoplasmic reticulum (ER) were observed among typical viroplasm inclusions
(30nm) and hollow tubules (200-300nm). A viral protein migrating as a 29kDa band,
which corresponds to the TSWV nucleocapsid (N) protein, was observed after sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Total
plant RNA, isolated from N. rustica displaying typical symptoms was subjected to reverse-transcription polymerase chain reaction (RT-PCR)
using .primers specific to
the nucleocapsid (N) gene. An expected 760bp product was amplified. The results
obtained in this study confirm the presence of TSWV in infected pepper plants from
KZN.
The genetic diversity of TSWV isolates occurring in SA was examined. The
nucleocapsid (N) gene sequences of six SA TSWV isolates originating from Gauteng, KwaZulu-Natal, North West, Limpopo and Mpumulanga provinces were determined
and used in a phylogenetic tree comparison with TSWV isolates occurring in different
geographical locations in the world. Nucleotide sequence comparisons of the N gene
revealed high levels of similarity between the SA isolates and TSWV isolates from
Asia and Europe. SA isolates showed a high degree of sequence similarity (99-100%)
which was reflected in their distinct clustering pattern.
The resistance of tomato (Lycopersicon escuJentum Mill.) plants with natural and
transgenic resistance against mechanical inoculation with TSWV isolates occurring in
SA was evaluated. The Stevens cultivar which has natural resistance conferred by
the Sw-5 gene and the transgenic 13-1 line, which expresses the nucleocapsid (N)
protein gene of the TSWV-BL isolate, was used as test cultivars. Plants were
assessed for TSWV resistance using a disease severity rating scale and
measurements of virion accumulation levels (A405nm). There were no significant
differences among the reactions produced by the six TSWV isolates on the test
plants. Although both plants were susceptible to the SA TSWV isolates by exhibiting
similarly high viral accumulation levels, the transgenic tomato line showed milder
disease severity compared to the natural resistant cultivar. Results suggest that
transgenic resistance is a more effective approach in the control of TSWV in SA.
The information generated in this study will be useful in formulating effective control
measures using genetic engineering approaches for this economically important virus. Such approaches will be used as a tool to make strategic decisions in an
integrated control programme for ISWV. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.
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Dispersal propensity of adult Colorado potato beetles (Coleoptera:Chrysomelidae) on potato and its implications on the insect resistance management planMbungu, Nsitu T. January 2006 (has links)
A three-year (1998-2000) field and laboratory study conducted in Fredericton, New Brunswick, Canada analyzed and quantified the dispersal of adult Colorado potato beetles within and between conventional and transgenic potato plots established according to the high-dose/refuge strategy. More specifically, the study addressed the following four predictions: (1) Adult Colorado potato beetle abundance or flight activity in transgenic potato fields is positively correlated to the abundance or flight activity in the immediately adjacent refuge field. (2) Colorado potato beetle intraspecific competition on potato plants will increase the flight take-off frequency of adult CPB; tolerating relatively high numbers of CPB egg masses or larvae or a high level of CPB defoliation on potato plants in the refuge could therefore be considered to increase the movement of beetles from the refuge to the transgenic field. (3) CPB flight take-off frequency will be higher on potato plants at the bloom than at the vegetative stage; planting of the non transgenic potato crop in the refuge earlier than the Bt transgenic potato crop in the main field could therefore be considered to increase movement of the Colorado potato beetles from the refuge to the transgenic field. (4) The aggregated distribution of CPB populations in the potato crop is caused by the presence of mating pairs; strategies changing the distribution of males and females in the refuge field could therefore be considered to increase dispersal from the refuge to the main crop field. / Population monitoring using plant counts, flight interception traps, flight landing traps and pitfall traps established that a transient population of adult CPB is present in the transgenic potato fields throughout the crop season and that the abundance of the beetle is higher than that required by the high dose/refuge strategy models. Furthermore results showed that the beetles invading the transgenic field population originate as much from the surrounding fields of conventional cultivars as from the adjacent refuges. It would therefore be possible to relax existing requirements for the refuge to be located immediately adjacent to the transgenic crop. / Like most insects, the adult CPB populations are aggregated and can be fitted to a negative binomial distribution over the crop season. This study revealed that the distribution results from the presence of mating pairs for the overwintered population and from the clumped pupation for the non breeding summer population. The activity of the males in search of females is at least partly responsible for the higher dispersal activity observed with the overwintered than with the summer populations. The comparatively low level of dispersal activity with the summer population could affect the efficacy of the high/dose refuge strategy during the later part of the crop season. / Results of flight chamber tests demonstrated that plant phenology and intraspecific competition have a positive effect on flight take-off frequency. These findings suggest that summer adult dispersal between the refuges and the transgenic crops could be stimulated by manipulating planting dates and the abundance of the different CPB life stages on the plants. / Together, the results of the thesis provide support for some of the premises of the high dose/refuge strategy and offer new information on the CPB dispersal that could be used to further improve its efficacy. Although the transgenic potato (NewLeaf) is not commercially available at this time, the threat of CPB resistance to new products or resistant cultivars under development makes it important to continue the research required by CPB resistance management plans.
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The development of transgenic sweet potato (Ipomoea batatas L.) with broad virus resistance in South Africa.Sivparsad, Benice. 20 November 2013 (has links)
Sweet potato (Ipomoea batatas Lam.) is ranked as the seventh most important food crop in the world and its large biomass and nutrient production give it a unique role in famine relief. However, multiple virus infection is the main disease limiting factor in sweet potato production worldwide. The main objective of this research project was to develop a transgenic sweet potato cultivar with broad virus resistance in South Africa (SA).
A review of current literature assembled background information pertaining to the origin, distribution and importance of the sweet potato crop; viruses and complexes infecting sweet potato; and the strategies used in sweet potato virus detection and control.
A survey to determine the occurrence and distribution of viruses infecting sweet potato (Ipomoea batatas Lam.) was conducted in major sweet potato-growing areas in KwaZulu-Natal (KZN). A total of 84 symptomatic vine samples were collected and graft inoculated onto universal indicator plants, Ipomoea setosa Ker. and Ipomoea nil Lam. Six weeks post inoculation, typical sweet potato virus-like symptoms of chlorotic flecking, severe leaf deformation, stunting, chlorotic mosaic, and distinct interveinal chlorotic patterns were observed on indicator plants. Under the transmission electron microscope (TEM), negatively stained preparations of crude leaf sap and ultra-thin sections from symptomatic grafted I.setosa plants revealed the presence of elongated flexuous particles and pinwheel type inclusions bodies‟ that are characteristic to the cytopathology of Potyviruses. Symptomatic leaf samples from graft-inoculated I. setosa and I. nil were assayed for Sweet potato feathery mottle virus (SPFMV), Sweet potato mild mottle virus (SPMMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato chlorotic fleck virus (SPCFV), Sweet potato virus G (SPVG), Sweet potato mild speckling virus (SPMSV), Sweet potato caulimo-like virus (SPCaLV), Sweet potato latent virus (SPLV), Cucumber mosaic virus (CMV), and Sweet potato C-6 virus (C-6) using the nitrocellulose membrane enzyme-linked immunosorbent assay (NCM-ELISA). The majority of leaf samples (52%) tested positive for virus disease and showed the
occurrence of SPFMV, SPMMV, SPCSV, SPCFV, SPVG, SPMSV, and SPCaLV. Of these 7 viruses, the most frequently detected were SPFMV (39%), SPVG (30%), followed by SPCSV (13%) and SPMMV (12%). SPCaLV and SPCFV at 10% and SPMSV at 7% were found exclusively in samples collected from one area. SPFMV, SPVG, SPCSV, and SPMMV were identified as the most prevalent viruses infecting sweet potato in KZN.
The genetic variability of the three major viruses infecting sweet potato (Ipomoea batatas Lam.) in KZN was determined in this study. A total of 16 virus isolates originating from three different locations (Umbumbulu, Umfume and Umphambanyomi River) in KZN were analyzed. These comprised of 10 isolates of Sweet potato feathery mottle virus (SPFMV), five isolates of Sweet potato virus G (SPVG) and one isolate of Sweet potato chlorotic stunt virus (SPCSV). The phylogenetic relationships of the SPFMV, SPVG and SPCSV isolates from KZN relative to isolates occurring in SA and different parts of the world were assessed. The division of SPFMV into four genetic groups (strains) according to the phylogenetic analysis of coat protein encoding sequences revealed mixed infections of the O (ordinary) and C (common) strains in sweet potato crops from KZN. All SPFMV isolates showed close lineage with isolates from South America, East Asia and Africa. The SPVG isolates showed high relatedness to each other and close lineage with other isolates, especially those from China and Egypt. Analysis of the partial sequence of the Heat shock protein 70 homologue (Hsp70h) gene indicated that the SPCSV isolate from KZN belongs to the West African (WA) strain group of SPCSV and showed close relatedness to an isolate from Argentina. The knowledge of specific viral diversity is essential in developing effective control measures against sweet potato viruses in KZN.
Multiple virus infections of Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG) and Sweet potato mild mottle virus (SPMMV) cause a devastating synergistic disease complex of sweet potato (Ipomoea batatas Lam.) in KZN. In order to address the problem of the multiplicity and synergism of sweet potato viruses in KZN, this study aimed to develop transgenic sweet
potato cv. Blesbok with broad virus resistance. An efficient and reproducible plant regeneration protocol for sweet potato (Ipomoea batatas Lam.) cultivar Blesbok was also developed in this study. The effect of different hormone combinations and type of explants on shoot regeneration was evaluated in order to optimize the regeneration protocol. Coat protein (CP) gene segments of SPFMV, SPCSV, SPVG and SPMMV were fused to a silencer DNA, the middle half of the nucleocapsid (N) gene of Tomato spotted wilt virus (TSWV) and used as a chimeric transgene in a sense orientation to induce gene silencing in the transgenic sweet potato. Transformation of apical tips of sweet potato cv. Blesbok was achieved by using Agrobacterium tumefaciens strain LBA4404 harboring a modified binary vector pGA482G carrying the plant expressible neomycin phosphotransferase ll gene (nptll), the bacterial gentamycin-(3)-N-acetyl-transferase gene and the expression cassette. A total of 24 putative transgenic plants were produced from the transformed apical tips via de novo organogenesis and regeneration into plants under 50mg/L kanamycin and 200 mg/L carbenicillin selection. Polymerase chain reaction (PCR) and Southern blot analyses showed that six of the 24 putative transgenic plants were transgenic with two insertion loci and that all plants were derived from the same transgenic event. The six transgenic sweet potato plants were challenged by graft inoculation with SPFMV, SPCSV, SPVG and SPMMV- infected Ipomoea setosa Ker. Although virus presence was detected using NCM-ELISA, all transgenic plants displayed delayed and milder symptoms, of chlorosis and mottle of lower leaves when compared to the untransformed control plants. These results warrant further investigation under field conditions. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.
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