Download Binary Ti vector plasmids

Transgénesis Vegetal. El diseño
transgénico.
Disarmed binary vectors
Disarmed binary vectors
• The combined action of the vir genes achieves the
delivery of the T-DNA to the nucleus of the host plant
• Removal of all the genes within the T-DNA does not
impede the ability of A.t. to transfer this DNA but does
prevent the formation of tumours
• Ti plasmids and their host Agrobacterium strains that are no
longer oncogenic are termed ‘disarmed’
Disarmed binary vectors
• Two key advances have made Agrobacterium transformation
the method of choice:
– the development of binary Ti vectors
– the development of a range of disarmed Agrobacterium strains
Binary Ti vector plasmids
• T-DNA and the vir region can reside on separate plsmids.
These form the basis of modern Ti plasmid vectors, termed
binary Ti vectors
• The vir gene functions are provided by the disarmed Ti
plasmids resident in the A.t. Strain
• The T-DNA , within which are the gene(s) to be transferred,
is provided on the vector
• Most binary Ti vectors replicate in both E. Coli and A.t.
Komori et al. 2007. Current status of binary and superbinary vectors.
Plant Physiology 145:1155-1160.
Binary Ti vector plasmids
Antibiotic resistance genes for plasmid selection and maintenance in culture
• A.t. strains are marked with antibiotic resistances that are
either in the chromosome or Ti-plasmid localized => the
binary Ti vector and bacterial antibiotic resistance markers
cannot be duplicated
• Penicillin-based antibiotics (e.g. carbenicillin and
amoxycillin) are used to inhibit the further growth of A.t.
several hours or days after co-cultivation with target
Binary Ti vector plasmids
Plasmid replication, size matters, and host species
• A broad host range ori for a wide range of Gram(-) bacteria
in most binary Ti vectors. Alternatively, two separate origins
for E. coli and A. t.
• Progressive reduction in size through elimination of
unrequired DNA, smaller oris, and moving genes to the
resident plasmid or bacterial chromosome
• Bacterial conjugation between E. coli and A.t. or direct DNA
transfer (electroporation or freeze-thaw techniques)
Binary Ti vector plasmids
The binary Ti vector T-DNA
• Older designs: RB and LB from several different Ti plasmids.
Not anymore, because of little variation in borders between
Ti plasmids and good functioning in heterologous A.t. strains
• ‘Overdrive’ sequence from the octopine strains.
Incorporated in the more recent binary Ti vectors
• Selectable marker gene in the T-DNA. Early vectors at the
RB. More recent vectors, nearest to the LB
Binary Ti vector plasmids
T-DNA integration
• Accuracy: LB is prone to incomplete nicking and,
consequently, vector DNA adjacent to LB might be
transferred.
• Transferred vector DNA has been associated with aberrant
gene expression
• Designed variations for specific purposes
Binary Ti vector plasmids
Specific purposes
Binary vectors have been adapted for:
Promoter and enhacer trapping
Gene activation tagging
T-DNA insertional inactivation of genes
Transposon mutagenesis
Promoter transcrip. or translat. fusions to reporters
Direct cloning of a coding sequence (35S, poly(A) site)
Inducible promoters
Double selection (LB and RB), for improved expression
BiBACs
Binary Ti vector plasmids
Future developments
• User-friendly vectors for a wide range of species
• Plant genomics. Increasing use of map-based cloning for gene-isolation.
Complementation of mutants in a given species. Need for more flexible
BiBACs
• Environmental issues. Removal of extraneous DNA sequences not
needed for expression of the introduced trait
• Concept and development of superbinary vectors. vir genes from A.t.
strain pTiBo542, a strain with a supervirulence phenotype.
Superbinary vectors
Tzfira et al. 2007. Advanced Expression Vector Systems: New Weapons for
Plant Research and Biotechnology. Plant Physiology 145:1087-1089.
Constitutive, inducible, and tissuespecific promoters
• A guiding bibliography:
– Biochemistry and Molecular Biology of Plants. 2000. B. Buchanan, W.
Gruissem, R. Jones. Chapter 7. I.S.B.N. 0- 943088-39-9
– Datla et al. 1997. Biotechnology Annual Review 3:269-296
– Zuo and Chua. 2001. Current Opinion in Biotechnology 11:146-151
– Caddick et al. 1998. Nature Biotechnology 16:177-180
- Tomsett et al. 2004. Trends in Plant Science 9:159-161
- Venter, M. 2007. Trends in Plant Science 12:118-124
Promoters for transgenic expression
• In addition to coding sequences, regulatory sequences
or promoters to target expression to appropriate cells,
tissues or developmental stages are essential for
engineering traits in transgenic plants
• Characterization of isolated plant promoters has
identified DNA elements responsible for conferring
respective specifities
Stomatal guard cells express a drought stress gene
in response to dehydration and ABA.
Transgenic Arabidopsis plants containing the promoter of a drought
responsive gene fused to GUS, show expression in the stomata when
exposed to drought or induced by ABA
Structure and organization of a eukaryotic gene
Key promoter elements
• The most basic cis-element is the TATA box, located around
position -30
• The TATA box is often juxtaposed to another basic cis-element,
the CAAT box
• Inducible genes almost always contain a TATA box and at least two
other cis-elements that play a role in the final stages of
environmental signal transduction
• Housekeeping genes have less diversity in their cis-elements and
may not even contain a recognizable TATA box
Enhancers
• Regulatory elements found upstream from the TATA box, downstream in the 3’
flanking regions, in the 5’ UTR, and even within introns
• They may span hundreds of basepairs and can contain cassettes of repeated
sequences, each of which may function independently as cis-elements
• They can function in either orientation in the chromosome and can be located
at a considerable distance from the coding region of the gene
• They can also dictate whether a gene is expressed in a particular organ or
tissue
A gene enhancer element can function in gene regulation at a distance
Comparison of the promoters of several stress-response genes
containing the G-box
Three major categories of transcription factor proteins
Transcription factors
• Transcription factors are often encoded by families of genes that
collectively code for a population of related proteins.
Heterodimers allow for variation in the DNA binding specificity
• Example of the G-box
– It is recognized by G-box binding factors, a family of leucine zipper proteins
that freely form heterodimers
– Heterodimers with slightly different binding affinities may confer specificity
among the wide variety of promoters that contain the G-box
Plant promoters for transgenic
expression
• Plant promoter analysis has allowed the
identification and characterization of over 200
promoters that can be classified in six major
classes
• Together with some promoters of non-plant
origin, they form one of the most important
elements for transgenic designs
Plant promoters for transgenic
expression
1.- Constitutive. Function in all cells, tissues, and organs
through different stages of development, e.g.,
promoters of house-keeping genes
2.- Tissue-specific. Direct expression in particular tissues
only, e.g., phloem, xylem, tapetum and meristem
3.- Cell-specific. Direct expression in specific cells, e.g.,
pollen and guard cells
(...)
Plant promoters for transgenic
expression
4.- Developmental stage-specific. Regulate expression
of genes at specific developmental stages and particular
organs, e.g., root, stem, leaf, flower, fruit and seed
5.- Hormone-regulated. Induce gene expression in
response to phytohormones at different stages of plant
growth, e.g., auxin inducible
6.- Environmental condition-controlled. Respond to
external factors, e.g., heat, cold, light, UV, stress, salt
and pathogen attack
Chemical-inducible systems for regulated
expression of plant genes
• They offer a general and flexible solution for the control
of gene expression
• They allow to regulate gene expression in transgenic
plants at a particular developmental-stage and for a
specific duration
• Useful for basic plant biology research and for
biotechnological applications
PROMOTORES SINTÉTICOS
Diseño de
promotores
sintéticos