Technique Dna Transfer

 

 

Two main ways of getting DNA into plant chromosomes: • •

Agrobacterium- mediated gene transfer Direct gene transfer

• •

For engineering dicots, use Agro For monocots, use direct DNA transfer, but can also use Agrobacterium

 

 

Agrobacterium tumefaciens, a natural plant genetic engineer - Soil bacterium, related to Rhizobium - causes crown galls (tumors) on many dicots - Infection occurs at wound sites Brief  recitation in Weaver, pp. 85-89

 

Infected Tobacco w/teratoma

Crown galls caused by A. tumefaciens on nightshade.

 

 

Lots of  pili

complex bacterium – genome has been sequenced; 4 chromosomes with ~ 5500 genes

Agrobacterium infection and tumorigenesis • Infection occurs only at wound sites • Involves recognition and chemotaxis of the bacterium toward wounded cells • galls are “real tumors”, can be removed and grow indefinitely without hormones • genetic information must be transferred to plant cells

Tumor characteristics • hormone (auxin & cytokinin) levels altered, explains abnormal growth • synthesize a unique amino acid, called “opine” – octopine and nopaline (derived from arginine) – agropine (derived from glutamate) • specific opine depends on the strain of A. tumefaciens • opines are catabolized by the bacterium, which can use only the specific opine that it caused the plant to produce

Elucidation of the TIP (tumorinducing principle) • It was recognized early that virulent strains could be cured of virulence, and that cured strains could regain virulence when exposed to virulent strains; suggested an extrachromosomal element. • Large plasmids were found in A. tumefaciens and their presence correlated with virulence: called tumor-inducing or Ti plasmids.

Ti Plasmid • • • • •

Large (~200-kb) Conjugative ~10% of plasmid transferred to plant cell after infection transferred DNA (called T-DNA) integrates semi-randomly into nuclear DNA Ti plasmid also encodes: – enzymes involved in opine metabolism – proteins involved in mobilizing T-DNA (Vir genes)

T-DNA  LB

auxA auxB

cyt

ocs

RB

LB, RB – left and right borders (direct repeat) auxA + auxB – enzymes that produce auxin cyt – enzyme that produces cytokinin Ocs – octopine synthase, produces octopine 

 

 

Vir (virulent) genes • Found on the Ti plasmids • Transfer the T-DNA to plant cell • acetosyringone (AS) (a flavonoid) released by wounded plant cells activates vir genes • virA,B,C,D,E,F,G (A-E are operons with multiple ORFs), span about 30 kb of Ti plasmid

Vir genes functions (cont.) • virA - transports AS into bacterium, activates virG post-translationally • virG - promotes transcription of other vir genes • virD2- endonuclease that cuts T-DNA at the borders but only on one strand; attaches to the 5' end of the SS • virE2- DNA-binding protein, binds SS of TDNA – virD2 & virE2 also help T-DNA get to nucleus in plant cell, they have NLSs

• virB - 11 ORFs, helps DNA-protein complex get through cell membranes

From Covey & Grierson

Hypothetical model for virB membrane channel

From P. Zambryski

• Monocots don't produce AS in response to

wounding. • Important: Put any DNA between the LB and RB of T-DNA it will be transferred to plant cell! Engineering plants with Agrobacterium: Two problems had to be overcome: (1) Ti plasmids large, difficult to manipulate (2) couldn't regenerate plants from tumors

Binary vector system Strategy: • Move T-DNA onto a separate, small plasmid. • Remove aux and cyt genes. • Insert selectable marker (drug resistance) gene in T-DNA (usually kanamycin resistance gene). • Vir genes are retained on a separate plasmid.

Binary vector system (cont.) 5. Put foreign gene between T-DNA borders. 6. Co-transform Agrobacterium with both plasmids. 7. Infect plant with the transformed bacteria. Leaf-disc transformation common; after selection and regeneration, get plants with the introduced gene in every cell “Transgenic plant”

Binary vector system for Agrobacterium

Making a transgenic plant by leafdisc transformation with Agro.

Transgenics: Direct DNA Transfer •

Introduce naked DNA into cells (plant or animal) • Can assay expression of the gene immediately, or select cells that are permanently transformed. • DNA introduction methods: 1. Chemical 2. Microinjection 3. Electroporation 4. Particle bombardment (Biolistics)

Chemically-induced transformation • •

Usually use on cells without walls Multiple protocols: 1. put DNA inside artificial membranes (liposomes), they will fuse with plasma membrane 2. Bind DNA with polycations to neutralize charge, some cells endocytose the complex 3. Combine (2) and (1)

needle

Microinjection of DNA into the pronucleus of a newly fertilized egg. Injection is usually into the sperm’s pronucleus because its larger.

From Primrose, Molec. Biotechnology

~ 1­2  picoliter  vol is  injected. ~5­40% of  animals  will  contain  transgene.

Electroporation •

•

•

Use on cells without walls (plant protoplasts or animal cells ) High-voltage pulses cause pores to form transiently in cell membrane; DNA pulled in by electrophoresis or diffusion (?) Drawback - its more cumbersome to regenerate plants from single protoplasts than from the tissue transformations with Agrobacterium

Particle Bombardment (Biolistics) • • • •

Less limitations than electroporation Can use on cells with walls, or essentially any tissue Can transform organelles Method: 1. Precipitate DNA onto small (micron) tungsten or gold particles. 2. Accelerate particles to high speeds to penetrate cells and tissues. 3. Perform selective growth and regeneration of transgenic plants as described for Agro-mediated transformation.

Original biolistic gun, a modified .22

DNA is bound to the microprojectiles, which are accelerated by the macroprojectile and impact the tissue or immobilized cells at high speeds. J. Sanford and T. Klein, Cornell

An Air Rifle for a DNA Gun – Circa 1990

A.Thompson, Bob ?, and D. Herrin

Repairing an organellar gene: ~ 1 x 107 cells of a 

mutant of Chlamydomonas that had a deletion in the atpB  gene for photosynthesis was bombarded with the intact atpB  gene. Then, the cells were transferred to minimal medium so  that only photosynthetically competent cells could grow.

Control plate – cells were shot with tungsten  particles without DNA

The Helium Gas Gun – Circa 2000

The Hand-Held Gas Gun

Purpose: Introduce DNA into cells that are below the top surface layer of tissues (penetrate into lower layers of a tissue) One interesting use: Making DNA Vaccines in whole animals.

Transgenic Plants In Use or About to be on a Large Scale • Herbicide-resistant plants • Pest-resistant plants • Vaccine plants (just starting to be used)

Herbicide-resistant plants • Resistant to herbicide “Round-up” (Glyphosate) • Contain bacterial EPSP synthase • Advantages: better weed control, less tillage • soybeans, corn, rice, wheat

Pest-resistant plants • Resistant to certain insects • Plants carry gene(s) for Bacillus thuringiensis (Bt) toxin • Advantage: less insecticide required, better yield • corn, cotton, potatoes

Vaccine plants • cheap vaccine-delivery system • use plants producing pathogen protein to induce immunity • potatoes, bananas

Concerns that have been raised about cultivating and consuming GM crops • • • •

They may be toxic or allergenic. They may become established in the wild and outcompete other plants. They may negatively affect insects or other organisms that use crops. They may outcross to a nearby wild relative spreading the transgene into a wild population.

References on release of GM crops into the environment • Nap et al. (2003) Plant Journal 33, 1-18 – Focuses on current status and regulations • Conner et al. (2003) Plant Journal 33, 19-46 – Focuses on ecological risk assessment