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Sathiyaraj Srinivasan 사티야라지 스리니바산 Dept. of Oriental Medicinal materials and Processing Kyung Hee University, Republic of Korea – 449 701
Gene Transfer Technique
To transfer a gene from one DNA molecule to another DNA molecule. Gene Transfer includes isolation of gene, manipulation (target gene insertion ) and reintroduction of DNA into the cells or model organisms. It used to make a crop to resistant to a particular herbicide, pest, weeds or introducing a novel trait, or producing a new protein or enzyme. The gene transfer methods normally include three categories: 1. transduction by biochemical methods, 2. transduction by physical methods, 3. virus-mediated transduction. The gene transfer results can be transient and stable transduction.
Transient transfection In transient transfection, the transfected DNA is not integrated into host chromosome. DNA is transferred into a recipient cell in order to obtain a temporary but high level of expression of the target gene. Fast, flexible, not influenced by position effects, can provide large amounts of protein for detailed characterization, does not require regeneration system, gene expression limited to specific plant tissues. Stable transfection Stable transfection is also called permanent transfection. By the stable transfection, the transferred DNA is integrated (inserted) into chromosomal DNA and the genetics of recipient cells is permanent changed. Time consuming, labor intensive, influenced by position effects, large production volumes, requires regeneration system, large amounts of glasshouse space, gene expression in all plant tissues
Generally, there are 9 ways for gene transfer: (as published by Sambrook, 2001).
(1) Lipid-mediated method, (2) Calcium-phosphate mediated, (3) DEAE-dextran-mediated, (4) Electroporation, (5) Biolistics, (6) Viral vectors, (7) Polybrene, (8) Laser transfection, (9) Gene transfection enhanced by elevated temperature.
Introduction to Agrobacterium tumefaciens
Agrobacterium tumefaciens is a ubiquitous soil borne pathogen responsible for Crown Gall disease, affecting many higher species of plant. It’s a Gram negative, motile, rod shaped bacterium which is non sporing, and is closely related to the N-fixing rhizobium bacteria which form root nodules on leguminous plants. The bacterium is surrounded by a small number of peritricious flagella.
Virulent bacteria contain one or more large plasmids, one of which carries the genes for tumour induction and is known as the Ti (tumour inducing) plasmid.
Crown Gall Disease
Mechanism of infection of Bacterium in plants
Ti Plasmid :
The tumor inducing plasmid (pTi) contains a portion that is transferred to the plant cell is Transfer DNA (T-DNA)
Ti plasmids can be classified according to the opines produced
1. Nopaline plasmids: carry gene for synthesizing nopaline in the plant and for utilization (catabolism) in the bacteria. Tumors can differentiate into shooty masses (teratomas). 2. Octopine plasmids: carry genes (3 required) to synthesize octopine in the plant and catabolism in the bacteria. Tumors do not differentiate, but remain as callus tissue. 3. Agropine plasmids: carry genes for agropine synthesis and catabolism. Tumors do not differentiate and die out. 4. Ri plasmids: induce hairy root disease on some plants and crown gall on others; have agropine-type genes and may have segments from both nopaline and octopine plasmids
Vir Genes and their Functions
Vir A, Vir G
Sense phenolic compounds from wounded plant cells and induce expression of other virulence genes Endonuclease; cuts T-DNA at right border to initiate T-strand synthesis Topiosomerase; Helps Vir D2 to recognise and cleave within the 25bp border sequence Covalently attaches to the 5I end of the T-strand, thus forming the T-DNA Complex. Also guides the T-DNA complex through the nuclear pores Binds to the 'overdrive' region to promote high efficiency T-strand Synthesis Binds to T-strand protecting it from nuclease attack, and intercalates with lipids to form channels in the plant membranes through which the T-complex passes Acts as a chaperone which stabilises Vir E2 in the Agrobacterium Assemble into a secretion system which spans the inner and outer bacterial membranes. Required for Export of the T-complex and Vir E2 into the plant cell
VirD2 Vir D1 Vir D2 Vir C Vir E2 Vir E1 Vir B & Vir D4
Important genes encoded by Ti plasmid
1. Cytokinins (plant hormone for cell plant division and tumorous growth) 2. Enzymes for indoleacetic acid (auxin) synthesis
Another plant hormone (inducing stem and leaf elongation, inducing parthenocarpy and preventing aging)
3. Enzymes for synthesis and release of novel plant metabolites: the opines (uniques amino acid derivatives) the agrocinopines (phosphorylated sugar derivatives) .
Nopaline Opines and agrocinopines are NUTRIENTS for A.tumefacies. They can not be used by other bacterial species It provides unique niche for A.tumefaciens
Ti plasmids and the bacterial chromosome act in concert to transform the plant 1.
Agrobacterium tumefaciens chromosomal genes: chvA, chvB, pscA required for initial binding of the bacterium to the plant cell and code for polysaccharide on bacterial cell surface. Virulence region (vir) carried on pTi, but not in the transferred region (T-DNA). Genes code for proteins that prepare the T-DNA and the bacterium for transfer.
3. T-DNA encodes genes for opine synthesis and for tumor production. 4. oc (opine catabolism) genes carried on the pTi and allows the bacterium to utilize opines as nutrient.
Agrobacterium chromosomal DNA
pscA chvB T-DNA
vir genes transfer to the plant oriV
tra bacterial conjugation
opine catabolism inc pTi’s are in the same inc group.
DNA between L and R borders is transferred to plant as ssDNA; T-DNA encoded genes can be substituted by target genes
Opine catabolism Virulence region ORI
The basis of Agrobacterium-mediated genetic engineering
T-DNA of A. tumefaciens is excised and integrates into the plant genome as part of the natural infection process. Any foreign DNA inserted into the T-DNA will also be integrated.
Ti-plasmid based vectors
Binary systems Needs 2 vectors:
Disarmed Ti plasmid with gene of interest Form co-integrated plasmid (no vir genes) Helper vector for infection (with vir genes)
Needs 3 vectors
Disarmed Ti plasmid capable for infection
Intermediate vector with T-region after homologous and gene of interest recombination on T-DNA (transferred by conjugation) Helper vector for transfer of intermediate plasmid into A.tum
Co-integrated vectors (hybrid ti-plasmids)
In a modern labs rarely used
DISADVANTAGES: 1) Long homologies required between the Ti plasmid and the E. coli plasmids (pBR322 based Intermediate vectors) making them difficult to engineer and use 2) Relatively inefficient gene transfer compared to the binary vector
Ti plasmid vector systems are often working as binary vectors
DISADVANTAGE: Depending on the orientation, plasmids with two different origins of replication may be unstable in E. coli. ADVANTAGE: small vectors are used, which increases transfer efficiency from E. coli to Agrobacterium. No intermolecular recombination is needed
Promoters useful for expression in transgenic plants
35S, cauliflower mosaic virus 35S promoter
CaMoV is a circular dsDNA genome virus
CaMoV 35S is a strong promoter that is active in essentially all dicot plant tissues.
Vir Gene expression induced
Vir D1 & D2 cut T-DNA at right and left borders.
Formation of Tcomplex
Phenolics detected by the VirA/VirG two component sensor system.
T-DNA VirD2 attaches to exposed 5I end
Formation of T-Pilus
Phenolics Produced by Wounded Plant cell
VIP1 associates with the complex to target it to the nucleus VIP2 associates the complex to transcriptionally T-DNA integrates into plant DNA active DNA and gall production is initiated.
Procedure for creation a transgenic plant
1. Both plasmids are transfected into A.tumefaciens 2. Plant cell culture is infected with A.tumefaciens 3. Products of Vir genes excised gene of interest within T-DNA and transfer it to plant chromosome
T-DNA Repeat Polylinker Kan-resistance gene T-DNA Repeat
Gene of interest
4. Plant cells are selected on kanamycine 5. Presence of transgene confirmed by PCR 6. Whole plant could be grown from transformed cells !!!
Currently 16 countries permit the cultivation of transgenic crops 99% of the total acreage is in 6 countries USA, China, Argentina, Canada, Brazil* & Australia Five crops are currently in release soybean, cotton, corn, canola, squash
tomato, potato and flax in past
Since the first widespread release in 1996, land devoted to transgenic crops has increased by 10 - 50% yearly 90% of Canadian canola is GMO - mostly transgenic 20+ countries suspected of growing transgenic crops without official approval
(ISAA Brief. Global Review of Commercialised Transgenic crops: 1998 & 2001)
Global area of transgenic crops
Acreage of transgenic crops has gone from nothing in 1995 to around 135 million acres in 2001.
Millions of hectares
50 40 30 20 10 0 1995 1997 1999 2001
Approved Transgenic plants
Soybean Corn Cotton Oil Carnations Potato Flax Papaya Chicory Rice Melon
Seed rape Sugarbeet Squash Tomato Tobacco
Types of GM crops (1998)
40 35 30 25 20 15 10 5 0 Soybean Cotton Corn Oil Seed
* * *
Soybean and corn are the major GM crops Large acreage Grown in the USA Can be regenerated Acreage of potatoes is small (<0.1 million hectares)
Types of genetic modification
Millions of hectares 25 20 15 10 5 0
>99% of all transgenic crops are either herbicide or insect resistant <1% have other traits
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