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Expression of human protein C in transgenic Nicotiana tabacumPiché, Christian. January 1994 (has links)
No description available.
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Exploration of high-density oligoarrays as tools to assess substantial equivalence of genetically modified cropsBeaulieu, Julie. January 2005 (has links)
No description available.
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Genimivirus AL2 And L2 Proteins Interact With And Inactivate SNF1 KinaseHao, Linhui 14 October 2003 (has links)
No description available.
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Expression of the native cholera toxin B subunit gene as oligomers in transgenic tobacco chloroplastsHenriques, Lucinda 01 October 2000 (has links)
No description available.
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Hyper-expression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystalsDe Cosa, Brandy L. 01 October 2000 (has links)
No description available.
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Karakterisering van derivate uit 'n Thinopyrum distichum X tetraploïede rog kruisingJacobs, Johan Adolf 03 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Soil salinity is a major limiting factor of plant and crop growth, because the absorption of
water and nutrients is such a complex process while low and moderate salinity are
omnipresent. Plant growth is affected negatively if a specific ion concentration exceeds its
threshold and becomes toxic. The detrimental effect of soil affected by salt on crop
production is increasing worldwide (Tanji, 1990). The level to which plants can tolerate
high salinity levels is genetically controlled with several physiological and genetic
mechanisms contributing to salt tolerance (Epstein & Rains, 1987). The most effective way
of addressing the limitations of crop productivity in saline areas, is the development of salt
tolerant varieties. Understanding the genetics of salt tolerance is, therefore, necessary for
the development of an effective breeding strategy for salt tolerance.
The department of Genetics (US) conducts a wide crosses research programme aiming to
transfer genes for salt tolerance to wheat and triticale. The donor species, Thinopyrum
disticum, an indigenous coastal wheat grass, adapted to high concentrations of salt, was
crossed with cultivated rye (Secale cereale) in an attempt to study the genetics of salt
tolerance (Marais et al., 1998).
The primary goal of this study was to find molecular markers (RAPD and AFLP) which
associate with chromosomes promoting salt tolerance for later attempts to transfer the
genes to triticale. Seventy clones of secondary hybrids (Th disticum /4x-rye 1/2x-rye) were
tested for salt tolerance and showed different levels of salt tolerance. RAPD-marker
analyses were used to identify polymorphisms between salt tolerant and salt sensitive
plants. Twelve RAPD primers produced clear, analyzable and repetitive polymorphic
. fragments that can be used as useful markers. Different AFLP-primer combinations were
tested against the genotypes of 15 clones (Marais & Marais 2001, unpublished data) and
produced approximately 2000 clearly distinguishable AFLP fragments, of which 54 (3%)
were polymorphic fragments. Two RAPD fragments and 4 AFLP fragments that can be
used as possible markers for the presence of chromosomes that contribute to salt
tolerance were identified.
The interpretation of the markers was complicated by heterogeneity among plants with
regard to the origin of their chromosomes and the genetic diversity of the rye genome. It is also possible that chromosome re-arrangement took place during backcrossing, which
could have complicated the data. / AFRIKAANSE OPSOMMING: Versouting is een van die groot beperkende faktore op plant- en gewasgroei, omdat die
opname van water en voedingstowwe so In ingewikkelde proses is en die effek van lae of
matige versouting so alomteenwoordig is. Plantgroei word nadelig geaffekteer as 'n
spesifieke ioonkonsentrasie sy drempelwaarde oorskry en toksies word. Die nadelige effek
van soutgeaffekteerde grond op gewasproduksie, is wêreldwyd aan die toeneem (Tanji,
1990). Die vlak waartoe plante hoë konsentrasies sout kan hanteer is onder genetiese
beheer met verskeie fisiologiese en genetiese meganismes wat 'n bydrae maak tot
soutverdraagsaamheid (Epstein & Rains, 1987). Die mees effektiewe manier om die
beperkinge op gewas produktiwiteit in versoute gebiede te oorkom, is die ontwikkeling van
soutverdraagsame variëteite. Begrip van die genetika van soutverdraagsaamheid is dus
noodsaaklik vir die ontwikkeling van In effektiewe telingsstrategie.
Die departement Genetika (US) bedryf tans 'n wye-kruisings navorsingsprogram waarmee
gepoog word om gene vir soutverdraagsaamheid na korog en koring oor te dra. Die
skenkerspesie, Thinopyrum disticum, In inheemse strandkoringgras wat aangepas is by
hoë konsentrasies sout, is gekruis met verboude rog (Secale cereale) in 'n poging om die
oorerwing van soutverdraagsaamheid te bestudeer (Marais et al., 1998).
Die hoofdoel van hierdie studie was om molekulêre merkers (RAPD en AFLP) te vind,
wat assosieer met chromosome wat soutverdraagsaamheid bevorder en om nuttige
merkers daar te stel vir latere pogings om die gene na korog en koring oor te dra.
Ongeveer 70 klone van sekondêre hibriede (Th distichum I 4x-rog /I 2x-rog) is onderwerp
aan souttoetse en het verskillende grade van soutverdraagsaamheid getoon. RAPDmerker
analise is gebruik om polimorfismes te identifiseer tussen soutverdraagsame en
soutsensitiewe plante. Twaalf RAPD inleiers het duidelike, ontleedbare en herhalende
polimorfiese fragmente opgelewer en moontlike nuttige merkers uitgewys. Verskillende
AFLP-inleier kombinasies, wat getoets is teen die genotipes van 15 klone (Marais &
Marais, 2001 ongepubliseerde data) het ongeveer 2000 duidelik onderskeibare AFLP
fragmente geproduseer, waarvan 54 (3%) polimorfiese fragmente was. Twee RAPD
fragmente en 4 AFLP fragmente is geïdentifiseer wat as moontlike kandidaat merkers
gebruik kan word vir die identifisering van chromosome wat bydra tot
soutverdraagsaamheid . Die interpretasie van die merkers is bemoeilik deur heterogeniteit tussen die plante wat
betref die agtergrond van chromosome wat hulle besit en die genetiese diversiteit van die
rog genoom. Dit is ook moontlik dat chromosoom herrangskikking plaasgevind het tydens
terugkruising, wat die data verder kon kompliseer.
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Aspects of sucrose metabolism in transgenic tobaccoChampanis, Reinette 12 1900 (has links)
Dissertation (PhD) -- University of Stellenbosch, 2004. / ENGLISH ABSTRACT: In most plants the efficiency of sucrose production and the systemic distribution
thereof are the major determinants of growth, development and yield. The factors
governing sugar partitioning co-ordinate its distribution in response to intrinsic and
environmental signals. These factors include sugar transporters and invertases as
well as metabolites, including sucrose and glucose, which function as signalling
molecules to modulate gene expression.
The genetic transformation of plants and the subsequent development of
transgenic lines with disturbed sugar metabolism have made an unprecedented
impact on the study of sugar translocation and -partitioning. For instance, the
transformation of plants with a yeast-derived invertase targeted to different
subcellular compartments has led to the elucidation of several key aspects of sugar
metabolism, including phloem loading mechanisms, the regulation of photosynthesis
by sugars, the importance of sugar-metabolism compartmentation with regards to
sucrose biosynthesis, storage and distribution, as well as the role of cell-wall
invertase in phloem unloading and sink strength.
In this study, a similar strategy of transgenic plant analysis was employed to
expand our insight into the regulation of sugar partitioning. The yeast-invertase Suc2
gene, from Saccharomyces cere visiae , was overexpressed in either the cytosol,
vacuole or apoplast of transgenic tobacco plants. These transgenic lines displayed
varying increases in invertase activity, altered sugar levels and consequently
disturbed sink-source interactions and sugar partitioning. Transgenic lines
overproducing the yeast-derived invertase in either the vacuole (Vac-Inv) or apoplast
(Apo-Inv) were utilised to analyse the effect of the altered sugar levels in sink and
source organs on the expression of sugar transporters, as well as the endogenous
cell wall invertase and inhibitors in these plants.
Transcript levels of the sucrose transporter NtSUT1 and hexose transporter
NtMST1 encoding genes increased significantly in the source leaves and roots of
Vac-Inv lines, whereas increased NtMst1 transcript levels were also detected in the
roots of Apo-Inv lines. The increased mRNA levels could be correlated to the altered
invertase activities and sugar levels in these tissues. It is concluded that NtSUT1 and
NtMST1 are differentially regulated by sucrose and/or hexose content on a
transcriptional level. Furthermore, the regulatory effect of the altered sugar levels on
transporter expression depended on the subcellular compartment in which the yeast
invertase was expressed. It would seem that the subcellular compartmentation of
sugar metabolism is also fundamental to the regulation of sugar partitioning.
The transcription levels of the endogenous cell wall invertase (CWt) and cell
wall invertase inhibitor (Cwi-Inh) genes were examined in the various tissues of
Apo-Inv and Vac-Inv lines at both the vegetative and flowering growth stages. In
comparison with the control lines, the various tissues of the Apo-Inv and Vac-Inv lines displayed altered Cwi and Cwi-Inh expression levels, depending on the sink-source
status and growth stage. However, no obvious correlation between the Cwi and
Cwi-Inh expression levels and soluble sugar content of these tissues was found. It is
suggested that the post-transcriptional and post-translation control of these proteins
by sugars might play an important role in their regulation. Analysis of the Cwi:Cwi-lnh
mRNA ratio and growth observations of the various tissues of control as well as
Apo-Inv and Vac-Inv lines indicated that this transcription ratio could be an accurate
indicator of the sink strength of sink organs.
In addition, the influence of sink-source interactions on sugar partitioning was
investigated. Reciprocal grafting between Apo-Inv and control lines resulted in scions
with an altered sucrose metabolism in either the sink or source organs. These scions
were subjected to biomass distribution, soluble sugar quantification and C4C]-
radiolabelling experiments. The latter revealed an unaltered state of sugar
partitioning from the above-ground tissues of the Apo/GUS scions and a significant
shift in sugar partitioning towards the roots of the GUS/Apo scions in comparison to
the control GUS/GUS scions. Phenotypic changes, opposite to those observed in
Apo-Inv lines expressing the heterologous invertase in both sink and source organs,
could initially be observed in the GUS/Apo and Apo/GUS scions. However, no
significant differences in phenotype or biomass distribution could be observed
between the mature GUS/Apo, Apo/GUS and GUS/GUS scions seven weeks postgrafting.
This inconsistency between phenotype and sugar partitioning might be
explained by an increase in the respiration rate of the tissues as supported by the
soluble sugar content. These results highlight the complexity and adaptability of
sucrose metabolism and sugar partitioning. In addition, it confirms that sugar
partitioning can be modulated by sink-source interactions and emphasise the
importance of invertases in the regulation of sugar partitioning through its ability to
alter sink strength.
This study forms part of the rapidly expanding initiative to unravel the control
mechanisms of sugar partitioning. The results obtained in this study confirmed again
that the introduction and expression of a single heterologous gene in transgenic
plants could provide significant insight into the regulation of this process. It was
shown here that the expression of sugar transporters is closely regulated by sugar
levels and therefore fulfils a vital function in sugar sensing and consequently the
regulation of sugar partitioning. The data presented in this study also demonstrated
the intricate and flexible nature of the relationship that exists between sugar
metabolism, partitioning and growth phenomena. / AFRIKAANSE OPSOMMING: Die doeltreffendheid van sukroseproduksie, tesame met die sistemiese verspreiding
daarvan, is die vernaamste faktore wat die groei, ontwikkeling en opbrengsvermoë
van die meeste plante bepaal. Die faktore wat suikerverdeling beheer, funksioneer
om suikerverspreiding te koordineer in reaksie op beide inherente- en
omgewingsseine. Hierdie faktore sluit suikertransporters en invertases in, asook
metaboliete soos sukrose en glukose wat funksioneer as seinmolekule in die
modulering van geenuitdrukking.
Die genetiese transformasie van plante en die gevolglike daarstelling van
transgeniese lyne met veranderde suikermetabolismes het 'n beduidende inwerking
op die bestudering van suikervervoer en -verdeling gehad. Byvoorbeeld, die
transformasie van plante met 'n gis-invertase geteiken na verskillende sub-sellulêre
kompartemente, het tot die toeligting van verskeie aspekte van suikermetabolisme
gelei, insluitende dié van floëemladingsmeganismes, die regulering van fotosintese
deur suikers, die belang van kompartementalisering ten opsigte van
sukrosebiosintese, -opberging en -verspreiding, en die rol van selwand-invertases in
floëemontlaaiing en swelgpuntkrag.
In hierdie studie is van soortgelyke transgeniese plantontledings gebruik gemaak
om 'n dieper insig tot die regulering van suikerverdeling te verkry. Die gis-invertase
Suc2 geen, afkomstig van Saccharomyces cerevisiae, is ooruitgedruk in óf die
sitosol, vakuool óf apoplastiese ruimte van transgeniese tabakplante. Hierdie
transgeniese lyne het wisselende toenames in invertase-aktiwiteite en veranderde
suikervlakke getoon, asook gevolglike versteurde bron-swelgpunt interaksies en
suikerverdeling. Transgeniese lyne met ooruitdrukking van die gis-invertase in óf die
vakuool (Vac-Inv) óf die apoplast (Apo-Inv) is gebruik om die gevolg van die
veranderde suikervlakke in bron- en swelgpuntorgane op die uitdrukking van
suikertransporters, asook die endogene selwand-invertase en invertase-inhibitor in
hierdie plante te bepaal.
Transkripsievlakke van die sukrosetransporter NtSut1 en die heksosetransporter,
NtMst1, het beduidend toegeneem in die bron-blare en wortels van die
Vac-Inv lyne; 'n toename in NtMst1 transkripsievlakke is ook in die wortels van
Apo-Inv lyne bevestig. Die toenames in boodskapper RNA kon gekorreleer word met
die veranderde invertase-aktiwiteite en suikervlakke in hierdie weefsels. Die
gevolgtrekking word gemaak dat NtSUT1 en NtMST1 differensieël gereguleer word
op transkripsionele vlak deur die sukrose en/of heksose inhoud van weefsels. Meer
nog, die regulerende effek van die veranderde suikervlakke op transporteruitdrukking
het afgehang van die subsellulêre kompartement waarin die gis-invertase
uitgedruk is. Dit wil dus voorkom dat die subsellulêre kompartementalisering van
suikermetabolisme fundamenteel tot die deurgee en waarneming van suikerseine is,
met In gevolglike eweneens belangrike rol in die regulering van suikerverdeling. Die transkripsievlakke van beide die endogene selwand-invertase (CWI) en
die selwand-invertase-inhibitor (CWI-Inh) enkoderende gene is in verskeie weefsels
van die Apo-Inv en Vac-Inv lyne, tydens beide die vegetatiewe- en blomstadia,
bestudeer. Die onderskeie weefsels van die Apo-Inv en Vac-Inv lyne het, in
vergelyking met die kontrole lyne, veranderde Cwi en Cwi-inh transkripsievlakke
getoon wat bepaal is deur bron-swelgpunt status en groeistadium. Geen duidelike
korrelasie kon tussen beide Cwi en Cwi-inh uitdrukkingsvlakke en oplosbare suiker
inhoud gevind word nie. Daar word voorgestel dat post-transkripsionele en posttranslasionele
beheer deur suikers 'n belangrike rol in die regulering van hierdie
proteïne speel. Bestudering van die Cwi:Cwi-lnh mRNA verhouding, asook groei
verskynsels van die onderskeie weefsels van kontrole en Apo-Inv en Vac-Inv lyne,
dui daarop dat hierdie transkripsievlak-verhouding moontlik 'n akkurate aanwyser van
die swelgpuntkrag van 'n swelgpuntorgaan kan wees.
Voorts is die invloed van bron-swelgpuntorgaan interaksies op suikerverdeling
ondersoek. Omgekeerde enting tussen Apo-Inv en kontrole lyne het entlote met
gemodifiseerde suikermetabolisme in óf hul bron- óf hul swelgpuntorgane tot gevolg
gehad. Hierdie entlote is aan biomassaverspreidings-, oplosbare suiker kwantifisering
en C4C]-radiomerking eksperimente onderwerp. Hierdie resultate het gewys dat, in
vergelyking met die kontrole (GUS/GUS) ente, daar geen verandering in die status
van suikerverdeling vanaf die bogrondse plantdele in die Apo/GUS ente is nie, maar
wel 'n beduidende verskuiwing in suikerverdeling na die wortels van die GUS/Apo
ente. Fenotipiese veranderinge, wat teenoorgesteld van dié teenwoordig in die Apo-
Inv lyne waar die heteroloë invertase in beide bron en swelgpuntorgane uitgedruk
word, is aanvanklik in die GUS/Apo en Apo/GUS ente waargeneem. Geen verskille in
fenotipe of biomassa-verspreiding kon egter sewe weke na die entings prosedures
tussen die GUS/Apo, Apo/GUS and GUS/GUS ente gevind word nie. Dit mag
verduidelik word deur 'n moontlike toename in respirasietempo in die betrokke
weefsels; die oplosbare suikervlakke wat in die verskillende ente aangeteken is
ondersteun dié moontlikheid. Hierdie resultate as geheelonderstreep die
kompleksiteit en aanpasbaarheid van suikermetabolisme en -verdeling. Verder
bevestig dit dat suikerverdeling beïnvloed kan word deur bron-swelgpunt interaksies,
asook die belang van invertases in die regulering van suikerverdeling gegewe die
vermoë om swelgpuntkrag te verander.
Hierdie studie vorm deel van 'n vinnig groeiende inisiatief om die beheermeganismes
van suikerverdeling te ontrafel. Die resultate verkry in hierdie studie
bekragtig die belang van rekombinante DNA tegnologie in die bestudering van
fundamentele plantprosesse. Die invoeging en uitdrukking van 'n geteikende gisinvertase
in transgeniese plante het gelei tot veranderde suikervlakke en bronswelgpunt
interaksies in hierdie lyne met die gevolglike ontginning van waardevolle
inligting ten opsigte van die regulering van suikerverdeling in reaksie tot interne
seine. Daar is aangetoon dat suikertransporters onlosmaakbaar gekoppel is aan die
deurgee en waarneming van suikerseine, spesifiek op die vlak van transkripsionele regulering, en dus ook die regulering van suikerverdeling. Voorts wys die resultate op
die komplekse en aanpasbare aard van die verhouding wat bestaan tussen
suikermetabolisme, -verdeling en groeiverskynsels.
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Evaluation of transgenic grapevine lines overexpressing Vv-AMP1 antifungal peptideTredoux, Martha Maria 03 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The importance of small antimicrobial peptides in the innate immune system of plants
became increasingly apparent over the past decade. Antimicrobial peptides are unique
and diverse molecules that are found in many tissue types in a variety of invertebrate,
plant and animal species. Many of these peptides, such as plant defensins, have been
found to be ubiquitous throughout the plant kingdom and have been isolated from
flowers, leaves, roots, seeds, seedlings, pods, tubers and bark.
The growing relevance of antimicrobial peptides (including plant defensins) in
research can be largely attributed to their broad-spectrum antifungal activity. This
makes them promising potential targets, both as therapeutic agents and for their use in
crop protection and disease resistance. The continuing discovery of novel antimicrobial
peptides has advanced the development of strategies to overexpress these genes in
plants to attempt to enhance the plant’s natural ability to resist pathogenic attack.
The first grapevine antifungal peptide, Vv-AMP1, was isolated and characterized
and was shown to be tissue specific and developmentally regulated, being expressed
only in berries at the onset of berry ripening. The peptide showed strong antifungal
activity against a number of plant pathogenic fungi in vitro. In this study, the biological
role of the Vv-AMP1 peptide was further investigated, both within its native host (Vitis
vinifera) and under in vitro conditions against a panel of grapevine-specific pathogens.
As a first step, recombinant production of Vv-AMP1 using an existing bacterial
expression system was evaluated and the heterologous production of the Vv-AMP1
peptide improved. Specific optimizations targeting both production and purification of
the peptide showed to improve the yield of Vv-AMP1. Steps in the production process
targeted for improvement included induction conditions of peptide production by the
bacterial culture as well as a number of purification steps, such as lysate preparation,
binding conditions, column washing, elution conditions and thrombin protease cleavage.
The optimized purification method produced up to 3 mg of pure Vv-AMP1 peptide from
1.6 L of overnight culture. While production was markedly improved, the resultant
purified Vv-AMP1 proved biologically inactive and structurally unstable. This is
uncharacteristic of the peptide, suggesting that an important aspect necessary for
peptide activity, such as folding or the presence of specific co-factors might not be
supported in this non-host prokaryotic production system.
The study also entailed the characterization and evaluation of the Vv-AMP1
peptide against a panel of grapevine-specific pathogens that are culturable to
sporulating cultures using in vitro antifungal assays and microscopy analysis. Vv-AMP1
showed strong inhibitory activity against all pathogens tested, inhibiting the growth of
Diplodia seriata and Cylindrocarpon liriodendri by 50% at concentrations between 4.8
μg/ml and 9.6 μg/ml. Phaemoniella chlamydospora and Phomopsis viticola proved
particularly sensitive, with IC50 values of 5.5 μg/ml and 4.0 μg/ml respectively.
Microscopy analysis of the effect of the Vv-AMP1 peptide on P. viticola showed a
severe inhibition on fungal germination and growth. The peptide did not induce
morphological changes in fungal hyphae but compromises the fungal membranes,
supporting the theory that the peptide induces membrane permeabilization.
Functional analysis of a transgenic V. vinifera (cv. Sultana) population
overexpressing Vv-AMP1 was included in this study to provide the opportunity to study
the in planta role of the peptide in its native host. The genetic characterization of the
putative population included confirming gene integration and copy number through PCR
and Southern blot analysis as well as gene expression through northern blot analysis. A
confirmed transgenic population was evaluated for improved disease resistance against
Botrytis cinerea as a first test organism in an attempt to link the overexpression of the
Vv-AMP1 gene to a disease resistance phenotype. Observations of lesion type, average
lesion size and further statistical analysis concluded that the transgenic population
showed a definite, albeit slight, improved resistance when compared to the
untransformed control lines.
In conclusion, the study determined that Vv-AMP1 had a strong antifungal action
against grapevine-specific pathogenic fungi when tested in vitro. A definite link could be
established between the overexpression of Vv-AMP1 and a mild resistance phenotype
within its native host plant. The characterized transgenic population is important for
further work to evaluate the in planta activity of the peptide against more grapevine
pathogens such as the stem pathogens that were proven sensitive and specifically
those that cannot be cultured and are obligate pathogens, such as the downy and
powdery mildews. / AFRIKAANSE OPSOMMING: Die belang van klein antimikrobiese peptiede in die ingebore immuunstelsel van plante
het tydens die afgelope dekade toenemend duidelik geraak. Antimikrobiese peptide is
unieke en diverse molekules wat in verskeie weefseltipes in ‘n verskeidenheid van
invertebraat-, plant- en dierspesies gevind word. Baie van hierdie peptiede, soos bv.
“plant defensins”, word bevind om alomteenwoordig in die plantryk te wees en is reeds
geïsoleer vanuit blomme, blare, wortels, sade, saailinge, peule, knolle en bas.
Die toenemende belang van antimikrobiese peptiede (insluitend “plant defensins”)
in navorsing kan grootliks toegeskryf word aan hul breë-spektrum antifungiese aktiwiteit.
Hierdie eienskap maak hul belowende potensiële teikens, beide as terapeutiese
middels asook vir gebruik in gewasbeskerming en siekteweerstand. Die voortdurende
ontdekking van nuwe antimikrobiese peptiede bevorder tans die ontwikkeling van
strategieë om hierdie gene in plante uit te druk in ‘n poging om die plant se natuurlike
vermoeë om patogeniese aanval teen te staan te verbeter.
Die eerste wingerd antifungale peptied, Vv-AMP1, is geïsoleer en gekarakteriseer
as ‘n ontwikkelings-gereguleerde peptied wat slegs uitgedruk word in korrels, tydens die
aanvang van bessie rypwording. Die peptied het tydens in vitro toetse sterk antifungale
aktiwiteit getoon teen ‘n verskeidenheid plant-patogeniese swamme. In hierdie studie
word die biologiese rol van die Vv-AMP1 peptied verder ondersoek, beide binne sy
natuurlike gasheerplant, (Vitis vinifera) asook onder in vitro kondisies teen ‘n paneel van
wingerd-spesifieke patogene.
As ‘n beginpunt is rekombinante produksie van Vv-AMP1 met behulp van ‘n
bakteriële ekspressie sisteem evalueer en die hetereloë produksie van die Vv-AMP1
peptied stelselmatig verbeter. Spesifieke optimerings het gefokus op beide die
produksie en suiwering van die peptied en het die algehele opbrengs van Vv-AMP1
verhoog. Spesifieke stappe wat in die produksieproses vir verbetering geteiken is sluit
beide induksietoestande van peptiedproduksie deur die bakteriële kultuur in sowel as ‘n
aantal suiweringsstappe, soos lisaatvoorbereiding, bindingskondisies, kolom
wasstappe, eluasie kondisies en “thrombin” protease snyding in. Die optimale
suiweringsmetode het tot 3 mg suiwer Vv-AMP1 peptied opgelewer vanaf ‘n 1.6 L
oornag bakteriële kultuur. Hoewel die produksie van die peptide noemenswaardig
verbeter is, was die gesuiwerde Vv-AMP1 beide onaktief en struktureel onstabiel. Dit is
buitengewoon vir hierdie peptied, wat daarop dui dat belangrike aspekte benodig vir
antifungiese aktiwiteit, soos korrekte vou of die teenwoordigheid van spesifieke kofaktore,
moontlik ontbreek in hierdie nie-gasheer prokariotiese produksiesisteem.
Die studie het ook die karakterisering en evaluering van die Vv-AMP1 peptied teen
'n paneel van wingerd-spesifieke patogene wat kultureerbaar is en sporuleer, insluitend
in vitro antifungale toetse en mikroskopiese analise, behels. Vv-AMP1 toon sterk
inhiberende aktiwiteit teen alle patogene getoets. Dit inhibeer die groei van Diplodia
seriata en Cylindrocarpon liriodendri met 50% teen konsentrasies tussen 4.8 μg/ml en
9.6 μg/ml. Phaemoniella chlamydospora en Phomopsis viticola was besonders
sensitief, met IC50 waardes van 5.5 μg/ml en 4.0 μg/ml, onderskeidelik. Mikroskopiese
analise van die effek van die Vv-AMP1 peptied op P. viticola het 'n ernstige inhibisie op
swam ontkieming en groei aangedui. Die peptied het geen morfologiese veranderinge in
swam hifes veroorsaak nie maar het wel die swam membraan beskadig. Hierdie
bevinding ondersteun die teorie dat die peptied membraan permeabilisasie induseer.
Funksionele analise van ‘n transgeniese V. vinifera (cv. Sultana) populasie wat die
Vv-AMP1 geen ooruitdruk is by die studie ingesluit om ‘n geleentheid te bied om die in
planta rol van die peptide binne sy natuurlike gasheerplant te bestudeer. Die genetiese
karakterisering van die vermeende transgeniese bevolking het die bevestiging van
beide geenintegrasie en kopiegetal deur PKR en Southern-klad analise ingesluit, sowel
as geenuitdrukking d.m.v. noordelike-klad analise. ‘n Bevestigde transgeniese bevolking
is evalueer vir potensiële verbeterde weerstand (in vergelyking met die wilde tipe) deur
infeksie met Botrytis cinerea as ‘n eerste toetsorganisme in ‘n poging om ‘n
weerstandbiedende fenotipe met die ooruitdrukking van Vv-AMP1 te assosieer.
Waarnemings van letsel tipe, letsel grootte en verdere statistiese analise het tot die
gevolgtrekking gelei dat die transgeniese bevolking ‘n definitiewe (dog geringe)
verbeterde weerstand toon in vergelyking met die ongetransformeerde lyne.
Ten slotte bepaal die studie dat Vv-AMP1 ‘n sterk antifungale effek teen wingerdspesifieke
patogene toon tydens in vitro toetse. ‘n Definitiewe korrelasie is vasgestel
tussen die ooruitdrukking van Vv-AMP1 in wingerd en ‘n weerstandsfenotipe in die
transgeniese bevolking. Die gekarakteriseerde transgeniese bevolking is uiteraard
belangrik vir toekomstige werk om die in planta aktiwiteit van die peptied te evalueer
teen verdere wingerdpatogene soos bv. die stampatogene wat sensitief getoets het
teen die peptide, asook patogene wat nie kultureerbaar is nie, insluitend verpligte
patogene soos dons- en poeierskimmel.
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Estudo do papel das proteínas mitocondriais desacopladas na tolerância aos estresses abióticos empregando diferentes abordagens /Nunes, Alessandra Vasconcellos. January 2010 (has links)
Orientador: Ivan de Godoy Maia / Banca: Paulo Eduardo Martins Ribolla / Banca: Marcelo Menossi Teixeira / Resumo: As proteínas desacopladoras pertencem à família de carreadores aniônicos mitocondriais. De maneira geral, as proteínas desacopladoras dissipam o gradiente eletroquímico de prótons gerados na respiração na forma de calor, sendo dependentes de ácidos graxos e sensíveis aos nucleotídeos purínicos. O presente estudo visou investigar o comportamento de plantas transgênicas de tabaco que expressam de forma constitutiva o gene AtUCP1, frente aos estresses osmótico e salino, bem como analisar a atividade das regiões promotoras dos genes AtUCP1 e AtUCP2 de Arabidopsis thaliana, em resposta aos estresses osmótico e de baixa temperatura, e ao ácido abscísico. Numa primeira abordagem foram utilizadas sementes selvagens e de duas linhagens transgênicas, germinadas em meio MS adicionados ou não de NaCl e Manitol. O teste de germinação revelou que as linhagens transgênicas apresentam uma maior tolerância aos referidos estresses. Quando o crescimento radicular foi analisado, uma maior inibição foi constatada no controle não transgênico em relação às duas linhagens transgênicas testadas. Adicionalmente, quando submetidas aos estresses, uma maior acumulação de ânion superóxido foi verificada nas folhas de plântulas não transgênicas em relação às plântulas das linhagens transgênicas. Quanto à quantificação de GUS nas plantas transformadas com os promotores dos genes AtUCP1 e AtUCP2, nenhuma alteração significativa foi observada em nenhum dos tratamentos testados / Abstract: The uncoupling proteins belong to the mitochondrial anion carrier family. In general, the uncoupling proteins dissipate the proton electrochemical gradient generated in respiration as heat, being dependent on fatty acids and sensitive to purine nucleotides. In the present study, we investigated the behavior of transgenic tobacco plants that overexpress the AtUCP1 gene when subjected to osmotic and saline stress, as well as the activity of the promoters of the AtUCP1 and AtUCP2 genes of Arabidopsis thaliana, in response to osmotic and cold stress, and abscisic acid. In the first approach, seeds from wild type and two transgenic lines were germinated in MS medium containing (or not) NaCl and mannitol. The germination test showed that the transgenic lines have a higher stress tolerance. When root growth was analyzed, a greater inhibition was observed in non-transgenic control seedlings as compared to seedlings of the two transgenic lines tested. Additionally, when subjected to stress, a greater superoxide anion accumulation was detected in leaves of non-transgenic seedlings as compared to seedlings of transgenic lines. Quantification of GUS activity in the plants transformed with the tested promoters, revealed no treatmentspecific differences / Mestre
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RNAi para o controle de Tuta absoluta em tomateiro / RNAi for the control of Tuta absoluta in tomato plantsCamargo, Roberto de Almeida 31 January 2014 (has links)
Desde seu descobrimento, o fenômeno de silenciamento gênico por RNA (RNAi) rapidamente se tornou uma técnica amplamente estudada e utilizada nos mais diversos aspectos da biologia molecular. Uma destas possibilidades é sua aplicação no campo da entomologia agrícola, mais especificamente para o controle de insetos-praga como uma alternativa de alta eficiência, especificidade e com impacto ambiental reduzido. Por meio da geração de plantas transgênicas expressando RNAi para genes essenciais de insetos-praga específicos, a ingestão destas moléculas de RNAi pelo inseto mediante herbivoria pode resultar no silenciamento do respectivo gene, resultando em fenótipos que podem variar entre perda de apetite, infertilidade ou até a morte. Neste contexto, o presente trabalho teve como objetivo provar a viabilidade de aplicação desta técnica para a interação Tomateiro x Tuta absoluta, cultura de grande expressão econômica e social no mercado nacional e internacional e que é amplamente atacada por esta praga, com prejuizos que podem alcançar a ordem dos 100% da produção. Por meio da clonagem de genes ortólogos essenciais descritos na literatura e de genes altamente expressos nos primeiros estádios larvais, após a caracterização transcriptômica em escala do inseto, foram realizados ensaios de alimentação contendo moléculas de dsRNAs que possuíam estes genes como alvo. Também, foi realizado a transformação genética de tomateiro cultivar \"Micro-Tom\" com dois destes genes (V-ATPase A e Arginina kinase) para a realização de ensaios de herbivoria. Com os resultados obtidos nestes experimentos, foram mostradas sólidas evidências da viabilidade da técnica de RNAi para o controle de Tuta absoluta, evidenciado pelo silenciamento gênico específico observado no inseto e consequentemente os efeitos nocivos deste silenciamento. / Since their discovery, the phenomenon of gene silencing by RNA ( RNAi ) has rapidly become a widely studied and used technique in the molecular biological field. One of these possible applications is in the entomology field, more specifically for the control of insect pests, as a high efficiency, specificity and with reduced environmental impact alternative. Through the generation of transgenic plants expressing dsRNA targeting essential insect genes, their ingestion by the insect and consequently the uptake of the silencing RNA, may result in specific gene silencing, resulting in a variety of phenotypes that can range from loss of appetite, infertility to death. In this context, this study aimed to prove the feasibility of this technique to control tomato leaf miner (Tuta absoluta) in tomatoes plant, a major crop worldwide and seriously attacked by this pest, with losses that can reach 100%. For the present work, orthologous genes from successfully cases of insect gene silence described in the literature, was selected together with highly expressed genes in the early larval stages of T. absoluta, chosen after the insect molecular characterization and used in feeding assays with dsRNAs molecules to targeted these genes. Also, genetic transformation of the \"Micro-Tom\" tomato cultivar with two of these genes (V-ATPase and Arginine kinase) was conducted for testing in an herbivore assay. With these two approaches was possible to get solid evidences of the feasibility of the RNAi technique to control this insect, evidenced by specific gene silencing observed and its consequent effect on pest phenotype.
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