Genetic Engineering Methods

Outline

Why do it?Research examples: poplar trees

Plant gene transfer concepts and methodsGetting genes ready for transfer (recombinant DNA/plasmids)Analysis of transgenic plants

Why do it?To create new, desired trait not in accessible gene poolTo breed faster, more directly

Dominant, rare allelesTo amplify genetic diversity for particular genes/traits for breeding

To breed with more direction, science basisTo use plants as bioproduct factories for industry/medicineTo promote biosafety for exotic/unsafe crop (domestication, sterility)In all cases, new traits/diversity feeds into breeding to check yield/stability of whole organism in the field: integrated health test

Native gene alteration example:Glutamine synthetase (GS) over-expression stimulates growth of

poplarsHyper-expression of GS to stimulate ammonium nitrogen incorporation into proteins3-year field trial (Spain)Increased nitrogen storage in stemTree height up 41%Many examples, abiotic stress (F. Cánovas, U. Málaga, Spain)

Gene suppression example:Lignin modification improves

pulping

Energy and chemical costs of pulping great

70 million tons pulp (USA)~$25 billion lignin removal (USA)

Success in changing lignin amount and chemistry

CAD- example354045505560

15 17 19 21 23 25 27

Pulp

yiel

d(%

) GE

Control

Pilate et al., INRA-France and UK

Active alkali (%)

Exotic gene function example:Gene from bacterium makes trees more effective at bioremediation

Thousands of square miles in U.S. mercury contaminated

Neurotoxin, biomagnified

merA transgenic cottonwood tolerates ~400 ppm mercury Volatilizing gene

Wood-sequestering genes under development

S. Merkle & R. Meagher, U Georgia

Malonyl-CoA+coumaroyl-CoA

Biosynthetic pathway example:Modifying color/antioxidants

AnthocyaninsPhobaphenes

Chalcone

Flavan-4-ol

Flavanone

Dihydroflavonol

A1

Bz1

A1

C2C2

P

Flavan-3,4-diol

3-Hydroxy-anthocyanidine

Vp1

C1 (ABA)

R/B

PI

R/B

Biosafety exampleSterile trees can dramatically

reduce gene dispersal

Variety of genetic mechanismsA tool to reduce risk of invasion by new exotic nursery/forestry species

Invasive Douglas-firs in Argentina (B Bond)

Plant gene transfer concepts and methods

Summary of steps in plant genetic engineering

Antibiotic selection

Transformation

Callus formation Shoot generation Root generation

Antibiotic selection of transgenic cells

TRANSCRIPTION

TRANSLATION

Regulatory Elements

When HowMuch

Promoter Gene Terminator

STOPGO

Cell Receptor

DNA encodes both genes and signals for their control

External Signal

Where

mRNA

Protein

Examples of Promoter:Gene Combos

Promoter Gene

35S-CAMV (virus) Bt (bacteria)

Corn Stem Bt (bacteria)

Round-up Ready© (bacteria)35S-CAMV

Tomato Fruit 5X Lycopene (tomato)

Reporter genes help visualize transgenic cells, promoter activity

Any promoter Any easily visualized geneFused to

Ubiqutious Fluorescence(JELLY FISH)

GLO-FISH

FLORAL(POPLAR)

GUS(BACTERIA)

Insertion of DNA into cells via biolistics (“gene gun”)

Transgenic cassava via biolistics

Agrobacterium tumefaciensagent of crown gall disease

A common soil pathogen that infects an large and taxonomically diverse range of plantsA natural genetic engineer--gene transfer is essential to its pathogenic life styleIt transfers DNA into plant cells to cause gall formation, which provides a home and nutrition for it

Agrobacterium is the method of choice for plant transformation

Agrobacterium has a sophisticated gene transfer machinery

Pathogenesis depends on presence of a very large plasmid, called the Ti (tumor inducing) plasmid, the source of its transferred genes (T-DNA)For biotechnology, the pathogenic genes are removed, replaced by useful genes

Agrobacterium life cycle

Agrobacterium transfer machinery

Agrobacterium Ti plasmid genes

LB RBauxin cytokinin opine

vir genes ori opine catabolism

T - DNA

Ti plasmid

Right and left border (RB,LB) sequences are the only parts of T-DNA needed to enable transfer into plants—removal of other T-DNA genes creates a disarmed plasmid

Disarmed and binary vector systemBinary vectors live in E. coli too, and are used to modify & shuttle genes

Agrobacterium

DisarmedTi plasmid

Binary vectorRB

LBvir

genes T- DNA

Plant Cell

A sterilized paper punch is used to cut “disks” from plant leaves as the first step in genetic engineering.

The cells on the edges of the disk are wounded in the process of cutting so they can receive a new gene from Agrobacteriumtumefaciens.

Step-by-step view of poplar transformation

Summary of steps in regenerating transgenic plants using Agrobacterium

Getting genes ready for transfer

Restriction Enzymes cut DNA at specific DNA sequences

(Alcamo. 1999. DNA Technology,2nd Ed. Harcourt Press.)

Electrophoresis separates DNA fragments based on their length

(Alcamo. 1999. DNA Technology, 2nd Ed. Harcourt Press.)

Construction of recombinant DNA molecules

(Alcamo. 1999. DNA Technology, 2nd Ed. Harcourt Press.)

Boyer-Cohen Experiment, 1973 showed how genes could be cloned

Plasmids are small, Circular DNA molecules that can replicate independently in a host cell.Foreign DNA inserted into plasmids can generate millions of copies of the inserted gene.

(Alcamo. 1999. DNA Technology, 2nd Ed. Harcourt Press.)

Example of a map of binary plasmid used in plant transformation

Map of binary plasmid used in pre-commercial plant transformation

Analysis of transgenic plants

Example of repeated transgenes in a plant genome caused by

transformation process

Southern (DNA) and northern (RNA) blots of transgenic cassava

Produced via biolistics

DNA –gene presence RNA-gene expression

Expression analyses of Agrobacteriumtransformed tobacco

Level of expression varies widely among independent gene transfer events

Many transgenic events need to be tested to find ones that are

agronomically suitable

Dozens to hundreds tested prior to commercial useStable gene and trait expression (look for Mendelian inheritance like native gene)Single gene insertion for stability and simple breedingDesired level and pattern of expression (position effects)

No deleterious effects on plant health/nearby genes (i.e., lack of somaclonal variation = unintended mutations)Introgression or insertion into other varieties for commercial useRegulation considers: Plant biochemistry, novel protein safety, allergenic potential, environmental impacts

Many transgenic events need to be tested to find ones that are

agronomically suitable

The genome is a complex, messy, mutagenized, recombinant place!

Natural transposable elements in the maize genome