Bariloche protein symposium argentine society for biochemistry and molecular biology

143
BIOCELL, 27 (Suppl. I), 2003
PL-P39.
EFFECTS OF THE USE OF ANTIBIOTICS ON IN VITRO
CULTURE OF SUNFLOWER
Parody Betiana P.; López, Nilda, Lopez Bilbao, Marisa and
Puebla, Andrea.
Instituto de Biotecnología, CICVyA,INTA Castelar, Argentina. E-
mail: [e-mail]..gov.ar
Genetic transformation of plants mediated by Agrobacterium
tumefaciens  requires the use of antibiotics in the regeneration
media in order to control bacterial growth without interfering with
the regeneration potential of the transformed cells. Carbenicillin,
cefotaxime and more recently timentin  have been the antibiotics
most commonly used for this purpose with varying effects on
different plant species. The aim of the work presented here was to
evaluate the efficacy of these three drugs for the suppression of
strain EHA101 of A.tumefaciens and their effects on the in vitro
culture of sunflower genotype HA89 after the process of
agrotransformation.
The results demonstrated that carbenicillin was not efficient at
eliminating this strain of Agrobacterium, whereas timentin and
cefotaxime did prevent bacterial growth. To evaluate the effects
of these drugs on the explants, several parameters were analysed:
regeneration capacity, weight and height of the shoots, chlorophyll
and protein content, root development and the incidence of
undesired traits, such as hyperhydricity and premature flowering.
All the parameters studied were negatively affected when
cefotaxime was present in the culture media whereas the use of
timentin had favourable effects on all of them, especially on the
regeneration potential. As sunflower is considered a recalcitrant
specie due to its poor in vitro response, all these results indicate
that timentin could be considered as the best antibiotic to be used
in future assays with this genotype.
PL-P40.
EXPRESSION AND CHARACTERIZATION OF A POTATO
TRANSCRIPTIONAL COACTIVATOR (StMBF1) UNDER
DIFFERENT ENVIRONMENTAL CONDITIONS
Arce, Débora*; Tonón, Claudia*
1
; Zanetti, María E; Daleo,
Gustavo and Casalongué, Claudia.
*equivalent contributions, 
1
Prof.Asistente CIC.
Instituto de Investigaciones Biológicas, FCEyN, UNMDP Mar
del Plata, Buenos Aires, Argentina. E-mail:
[e-mail]
MBF is an evolutionary conserved transcriptional coactivator that
connects a regulatory factor and TATA element-binding protein
(TBP). We have reported the identification of a Solanum tuberosum
transcriptional  coactivator, named StMBF1,which is up-regulated
during  Fusarium eumartii  attack as well as wounding in potato
tubers.  StMBF1 is phosphorylated under in vivo and in vitro
experimental conditions after treatment of potato cells with hyphal
cell wall components. In addition, StMBF1 interacts in vitro with
plant transcription factors of the Hd-Zip family. Since some
members of Hd-Zip proteins are inducible by plant growth
regulators and stress conditions, we attempted to know the StMBF1
expression pattern   in potato suspension cells under different
physiological conditions. The results indicated that StMBF1
accumulates by ABA, SA and heat shock treatments. In order to
evaluate the putative interaction between StMBF1 and TBP
proteins we expressed the potato TBP protein in E. coli. Protein-
protein interaction is currently being tested by GST-pull down
assays. Our results suggest that StMBF1 may posses a versatile
regulation and it would mediate the coupling of different
environmental signals within developmental program.
Supported by IFS, CONICET, F. Antorchas, ANPCyT, UNMDP.
PL-P41.
ISOLATION AND PARTIAL CHARACTERIZATION OF AN
EXTRACELLULAR CYSTEINE PEPTIDASE FROM
JACARATIA DODECAPHILLA (Vell.) FRUITS
Argüelles, Teresa
1
 and Fernández, Graciela
2
.
1
Facultad de Ciencias Forestales, UNaM, Eldorado, Misiones.
E-mail: [e-mail]. 
2
Dpto de Ciencias Básicas.
UNLu. Luján. Buenos Aires.  E-mail: [e-mail]
Jacaratia docecaphylla (Vell.) is a  tree of the Caricaceae family,
found in the Parana Subtropical forest of the province of Misiones.
The fruit is used in popular medicine as a soft painless laxative.
The fruits that contain latex are only edible when very ripe. A
buffered, medium ionic strength phosphate extract of the fruit
mesocarp analyzed by 11% SDS-PAGE, and stained with Brilliant
blue showed 10 proteinaceous bands, plus a non resolved band
migrating with the front. Vacuum infiltration of the tissue with
water during 5 minutes followed by low-speed centrifugation for
recovery of the extracellular washings allowed the selective
extraction of a protein that showed a single band in SDS-PAGE
with an estimated molecular mass of 34.000 Da, and   isoelectric
point above 9. Proteolytic activities of the enzyme were activated
by thiol protease activators and inhibited by thiol protease
inhibitors, indicating the enzyme to be a cysteine protease. The
enzyme hydrolyzed denatured natural substrates such as casein,
azocasein and azocoll with a high specific activity. It showed a
peak of activity in alkaline conditions and at temperatures of 50
o
C.
It had not detectable carbohydrate moiety. Antibiogram assays
showed inhibitory action on the growth of hyphae of Colletotrichum
gloeosporioides and Penicillium digitatum, further characterizing
the enzyme as an extracellular plant defense cysteine peptidase.
PL-P38.
ENZYMATIC BROWNING INHIBITION IN SOLANUM
TUBEROSUM USING RNAi GENE SILENCING
TECHNIQUES
Llorente, Briardo E.; Schoijet, Alejandra C.; Alonso, Guillermo
D.; Bravo Almonacid, Fernando F.; Luppi, Juan P.; Torres, Héctor
N. and Flawiá, Mirtha M.
Instituto de Ingeniería Genética y Biología Molecular (INGEBI),
Buenos Aires, Argentina. E-mail: [e-mail]
The enzymatic browning of fruits and vegetables that takes place
when they are cut and exposed to air, is caused by the action of
the enzyme Polyphenol Oxidase (PPO) and the reaction that
catalyses is called Enzymatic Browning. PPO has also been
reported to play a role in the defense against pathogens and pests
produced by insects. There are known at least five PPO genes in
potato plants, each with a specific spatial and temporal pattern of
expression. The Pot 32 gene is strongly expressed in tubers and
roots and Pot 33 gene is mainly expressed in the outer cortex of
tubers. Due to this fact we have designed an RNAi silencing vector
against the 5' region of Pot 32 gene in order to silence it only the
tuber and avoid the later enhanced silencing of the others PPO
isoforms in the plant. The construction engineered to silence Pot
32 was cloned in the pZP200HYG vector and introduced into A.
Tumefaciens. At the moment we are transforming potato plants
and expressing recombinant Pot 32 and Pot 33 enzymes in the
heterologous system of E. coli in order to obtain antibodies to
analyze the expression pattern of the transformed plants.treated
with the glucan.

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