Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Xiangbing meng
2009.04.02
Outline
• General transformation steps
• Model of Agrobacterium-mediated genetic transformation
• selectable marker and promoter
• Factors affecting transformation frequency
• Transgene integration and expression
• Other transformation technology
• Application
• Marker-free transgenic plants
Summary of rice transformation
• The systems to select the transformed cells, tissues or organisms from the non-
transformed ones are indispensable and selectable marker genes are vital to the
plant transformation process. Marker genes enable the transformed cells to survive
on medium containing the selective agent, while non-transformed cells and tissues
die.
• AtTPS1 (Glucose )
• Arabidopsis thaliana ATP-binding cassette (ABC) transporter gene , Atwbc19, confers antibiotic
(kanamycin) resistance to transgenic plants.
Promoters
• Transgenic rice with multitraits need different promoters.
• CaMV35S, rice ACT1 and maize UBI1 gene promoters have been used extensively to drive
high and constitutive expression of transgene in rice.
• Promoter of rice cytochrome c gene was highly active in almost every plant tissue. It was even
more active than rice ACT1 gene promoter in leaves, root, embryo and calli (2002).
• Activity of the OsAct2 promoter region is much higher than that of the OsAct1 promoter
region(2009).
Inducible or tissue or stages specific promoter
• Promoters of rice POXA and POXN were found to be root-specific and their expression was
also enhanced in the leaves on UV and wounding treatment.
• Wound inducibility has also been reported in promoter of MPI gene. Use of this promoter to
drive the expression of cry1B in transgenic rice showed better performance.
• Endosperm-specific, root/shoot-specific, seed-specific, anther-specific, and early development
stage specific promoters. Seed storage glutelin pmoters, GluA,B,C, containing endosperm
specificity-determining motifs(GCN4,AACA,and P-box).
• The specific promoter activity is controlled by introns in the case of rice α-tubulin gene
family.
Factors for transformation frequency
• The integrated DNA was stable and inherited in a Mendelian fashion in the majority of
Agrobacterium-derived lines.
• The stable inheritance and expression of foreign genes are of critical importance in the application
of genetically engineered cereals to agriculture.
• The factors contribute to variation in transgene expression: tissue culture-induced variation or
chimerism in the primary integration site (position effects), transgene copy number (dosage effects),
transgene mutation and epigenetic gene silencing.
• A pre-determined genomic locus can be achieved by the use of site-specific recombinase systems,
such as Cre/lox and FLP/frt.
• MARs (matrix attachment regions ) are DNA elements that are thought to influence gene expression
by anchoring active chromatin domains to the nuclear matrix.
• Devoid of vector backbone sequences to produce so-called ‘clean’ transgenic plants: using minimal
linear transgene constructs (promoter, coding region of the gene and terminator). Fu x.D. et
al.,2000.
• Transgene expression can be controlled temporally and spatially by the use of cell- and tissue-
specific or chemically inducible promoters.
Particle bombardment
• Rice protoplasts can be transformed with naked DNA by treatment with PEG in the
presence of divalent ions such as calcium. protoplasts are not easy to work with and
regeneration of fertile plants is problematic.
• pollen tube pathway
• LASER , imbibitions of embryo or seeds in the presence of DNA and
WHISKERSTM.
• needle dipped in Agrobacterium culture to prick the seed’s embryonic portion.
• Bioactive beads-mediated transformation (100 kb large DNA fragment).
Application
Enhancement of stress tolerances
• Antibody (ScFvT84.66)
• Industrial valuable enzyme transglutaminase
• Rice cell suspension culture systems also provide an alternative to animal/bacterial
cell lines for the production of recombinant compounds for human use as risk of
contamination is very low.
Environmental and biosafety aspects
• Tissue browning and necrosis after Agrobacterium infection are still major
obstacles in the genetic transformation of cereals.