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Aplicaciones
Plantas transgnicas tolerantes a aluminio mediante
la hiperexpresin de la citrato sintasa
tabaco papaya
Plantas transgnicas para fitovolatilizar el ion mercurico.
Expresin de una mercurico reductasa merA bacteriana.
50 M Hg(II)
150 M Au(II)
Fitorremediacin de mercurio
La expresin de los genes merA
(mercurio reductasa) y merB (organo
mercurial liasa) permite a las plantas
crecer en presencia
de altas concentraciones de mercurio
80 g de HgCl2
5 de acetato de fenil mercurio
La expresin en cloroplastos permite
hasta 400 de HgCL2 o acetato
de fenil mercurio
Current Opinion in Biotecnology 20 (2009) 2
Fitorremediacin
Arsnico Selenio
Plantas transgnicas que degradan el TNT.
Expresin de una PETN reductasa bacteriana
There is no applied science
without science to apply
Leloir laboratory (Argentina)
Planta tpica (resultados extrapolables)
Tamao y tiempo de generacin reducidos
Asequible al anlisis gentico
Genoma pequeo
Susceptible de modificacin gentica
Arabidopsis thaliana: una especie modelo
Susceptible al anlisis genmico
Genmica estructural: secuenciacin del genoma y
definicin de sus genes
Genmica funcional: funcin de los genes del genoma,
transcriptmica, protemica y metabolmica
Arabidopsis thaliana: una especie modelo
Discipline which aims to establish the structure and function of all
genes in a genome, using high-throughput techniques.
It is based on the important developments taking place in analytic,
robotics and informatics fields applyed in molecular biology
Provides information on the expression of each gene of the
genome in different tissues or in plants grown under
different environmental conditions
Allows to define clusters of co-regulated genes and to
identify shared cis-regulatory sequences
Is a strong phenotyping tool
Genomic chips allow the definition of transcription units
Transcriptomics
Provides information on translational
and post-translational control
Provides information on the subcellular localization
of gene products
Provides information on protein:protein interactions
at the proteome level
Allows the definition of phenotypes at the proteome level
Proteomics
Currently, there exist high resolution and sensitivity
chromatographic methods that allow the high-throughput
analysis of more than 1000 metabolites from the Arabidopsis
Provides information on relationships between
metabolites and co-regulated genes
Powerful tool for phenotyping. Metabolic profiling is
very sensitive to genetic differences or environmental
growth conditions
Metabolomics
Functional genomics relies on the integration of data
from several high-troughput platforms
Predictive plant biotechnology
200.000 independent transgenic lines that contain insertion
mutations in more than 20.000 genes from Arabidopsis.
Collections of single point mutantion
Methods making it posible the identification of allelic series
for virtually any gene. Moreover
Artificial microRNAs have proven succesful to inactivate
related, partially functionally redundant genes at once
Hightroughput mapping methods
Sequence of several Arabidopsis ecotypes.
In thisositional cloning of any gene is no longer a labour
intensive task (2 man-months)
Mutants and its cloning
Computer search of insertion mutants in Arabidopsis
Mecanismos de resistencia vegetal
frente a la infeccin de
microorganismos patgenos
Carmen Castresana
Plant defense response
Active defense
Local response
innate inmmunity
Systemic response
acquired inmmunity
Pasive defense Physical and chemical
barriers
Aniones
SAR
K
+
Plant immune system
MAMPs (microbe associated molecular patterns)
PRRs (pattern recognition receptors)
R protein
Effectors
Pathoge
n
Systemic
signal?
Systemic
signal?
Systemic defense
SA JAs ET
+
O
2
-
H
2
O
2
Ca
2+
H
Activation of defense
gene expression
Halt pathogen colonization
Antimicrobial compounds
Antimicrobial proteins Protecting enzymes
Hydrolytic proteins
Oxidase
Cell wall reinforcement
NO
NADPH
H
2
O
2 lipid peroxides
Lipid peroxidation
necrotrophs
biotrophs
Cell death
Hypersensitive reaction (HR)
Cell wall reinforcement
Systemic
signal?
Activation of gene
expression
Ca
2+
H
+
Aniones
NADPH
Oxidase
SA
SAR
JAs ET
Lipid peroxidation
lipid peroxides
Systemic defense
H
2
O
2
O
2
-
H
2
O
2
O
2
K
+
NO
Halt pathogen
colonization
Hormone pathways
Cell death control
Nutrient and water demands
Auxins, ABA, BR,
CKs, GA
Defence hormones
Plant immune system
Pathoge
n
Representative goals of the 2010 project
Identify cis regulatory sequences of all genes
Identify regulatory circuits controlled by each transcription factor
Determine biochemical function for every protein.
Describe three-dimensional structures of members of every plant-specific
protein family.
Systems analysis of the uptake, transport, and storage of ions and metabolites.
Describe globally protein-protein, protein-nucleic acid, and protein-other
interactions at organ, cellular, and subcellular levels under various environmental
conditions.
Define a predictive basis for conservation versus diversification of gene
function.
Develop bioinformatics, visualization, and modeling tools that will facilitate
access to all biological information about a representative virtual plant.
High-troughput protein resolution and identification using 2D
electrophoresis and MALDI-ToF
MALDI-ToF
SYPRO ruby
~ 1 ng/mm
2
Genome sequencing factory at Sanger Centre (UK)
Estrategias de expresin en plantas
Gen extrao
Infeccincon virus recombinantes Transformacin gentica estable
Increase of 12%, 12.3 million hectares (30 million acres), between 2006 and 2007.
Source: Clive J ames, 2007.
GLOBAL AREA OF BIOTECH CROPS
Million Hectares (1996 to 2007)
23 Biotech Crop Countries
0
20
40
60
80
100
120
140
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007
Total
Industrial
Developing
0
10
20
30
40
50
60
70
80
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007
Herbicide Tolerance
Insect Resistance
Herbicide Tolerance/Insect Resistance
Source: Clive James, 2008
Superficies de cultivos trangnicos por
rasgo introducido (millones de Ha)
Superficies de cultivos transgnicos en
diferentes pases
http://www.isaaa.org/
Agrobacterium tumefaciens,
una bacteria que produce tumores en plantas
Agrobacterium tumefaciens y el plsmido Ti
Plsmido Ti
replicacin
borde izquierdo
regin de
virulencia
DNA T
sntesis de opinas
borde derecho
catabolismo de
opinas
metabolismo de hormonas
transferencia
conjugativa
m
V
0,00
10,00
20,00
30,00
40,00
50,00
60,00
70,00
80,00
Minut es
0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00 26,00 28,00 30,00 32,00 34,00 36,00 38,00 40,
A
U
0,000
0,001
0,002
0,003
0,004
0,005
0,006
0,007
0,008
Mi nut es
2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00 26,00 28,00 30,00 32,00 34,00 36,00 38,00 40,
a)
b)
m
V
0,00
10,00
20,00
30,00
40,00
50,00
60,00
70,00
80,00
Minut es
0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00 26,00 28,00 30,00 32,00 34,00 36,00 38,00 40,
A
U
0,000
0,001
0,002
0,003
0,004
0,005
0,006
0,007
0,008
Mi nut es
2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00 26,00 28,00 30,00 32,00 34,00 36,00 38,00 40,
a)
b)
HPLC-analysis of methanol soluble phenols in roots of Myb14
a) Fluorescence-chromatograms (excitation: 300nm, emission:400nm) and
b) UV-chromatograms (280nm)
Problemas originales del sistema de plsmidos Ti
Desbalance hormonal que impeda la regeneracin de plantas
El nico marcador selectivo era la propia tumorogenicidad
Difcilmente manipulables debido a su tamao
Reducido espectro natural de huspedes
Soluciones
Eliminacin de las funciones oncognicas del T-DNA
Introduccin de marcadores selectivos en el T-DNA
Modificacin de los plsmidos Ti para hacerlos ms manejables
Espectro experimental de huspedes amplio
T-DNA transfer from Agrobacterium to plant cells
Baron and Zambryski (1995) TIBTECH 13, 356-362
Gelvin (2003) 21, 96-98
Mejora de la eficiencia de la transformacin de plantas con Agrobacterium
Mejoras en la planta
Sobreexpresin de VIP1 (ayuda en la transferencia al
ncleo del T-DNA)
VIP1
Mejoras en Agrobacterium
Dosis extra de VirG
VIRG
Sobreexpresin de H2A-1 (ayuda a la integracin del T-
DNA)
H2A-1
Cultivo de tejidos de plantas y transformacin
Walden and Wingender (1995) TIBTECH 13, 324-331
Disarmed
Ti Plasmid
Virulence
genes
Your favorite gene
Selectable marker
RB
LB
ORI
E. coli
A. tumefaciens
Binary plasmid system
I NFECCI N CON AGROBACTERI UM
Cocultivo con protoplastos
Inoculacin de discos de hojas
Infiltracin a vaco de plantas completas
Esterilizacin y
corte
Precultivo
Cultivo de
Agrobacterium
Cocultivo
Lavado
Induccin de brotes
(medio selectivo)
Enraizamiento
(medio selectivo)
Transplante a suelo
Obtencin de plantas transgnicas por transformacin de
discos de hojas con Agrobacterium
I NTRODUCCI N DI RECTA DE DNA
Permeabilizacin de protoplastos por PEG o electroporacin
Bombardeo de partculas
Electroporacin de clulas intactas
Microinyeccin
.
BOMBARDEO CON MI CROPROYECTI LES
Se producen muchas dianas por disparo
Se pueden transformar muchos tejidos
Se pueden alcanzar diferentes capas de clulas
Sistema muy eficiente para expresin transitoria,
pero poco eficiente para transformacin y regeneracin
Procedimientos altamente empricos
Placa
Disco de ruptura
Helio presurizado
Rej illade frenado
Membrana portadora
Partculas de oro
recubiertas de DNA
PDS-1000/HeParticleDeliverySystem(Bio-Rad)
Disparo
Bombardeo con microproyectiles
recubiertos con DNA
Como se hace una planta transgnica
Seleccin
Multiplicacin
Enraizamiento
Infeccin
Diferenciacin
Salud
Alergia
Resistencia a antibiticos
Toxicidad
Medio ambiente
Exterminio de insectos beneficiosos o neutrales
Aparicin de nuevos microorganismos o plagas ms dainos
(resistencia y recombinacin)
Seleccin de malas hierbas ms perjudiciales
Alteracin del equilibrio ecolgico
Disminucin de la diversidad biolgica
Posibles riesgos
Plant immune system
Halt pathogen colonization
Aniones
FLS2
K
+
Effector
triggered immunity
R protein
HR-based
defense
Effectors
TTSS
flg22
Pathogen
MAMPs (microbe associated molecular patterns)
MAMPs
PRRs (pattern recognition receptors)
Basal
defense
MAMP
triggered immunity
PRRs
ZIG/ZAG
model