chapter 18-genetic engineering of plants:...

Chapter 18-Genetic Engineering of

Plants: Methodology

• Plant transformation with the Ti plasmid of Agrobacterium tumefaciens

• Ti plasmid derived vector systems

• Physical methods of transferring genes to plants (microprojectile bombardment)

• Chloroplast engineering

• Use of reporter genes in transformed plant cells

• Manipulation of gene expression in plants

• Production of marker-free transgenic plants

Why genetically engineer plants?

• To improve the agricultural or horticultural value of

plants

• To serve as living bioreactors for the production of

economically important proteins or metabolites

• To provide a renewable source of energy (biofuels)

• To provide a powerful means for studying the

biological action of genes and gene products

Plant transformation with the Ti plasmid of

Agrobacterium tumefaciens

• A. tumefaciens is a gram-negative soil bacterium which naturally transforms plant cells, resulting in crown gall (cancer) tumors

• Tumor formation is the result of the transfer,

integration and expression of genes on a specific segment of A. tumefaciens plasmid DNA called the T-DNA (transferred DNA)

• The T-DNA resides on a large plasmid called the Ti (tumor inducing) plasmid found in A. tumefaciens

The Ti plasmid of Agrobacterium tumafaciens and the

transfer of its T-DNA to the plant nuclear genome

Fig. 18.3 The Ti plasmid of Agrobacterium tumafaciens and

its T-DNA region containing eukaryotic genes for auxin,

cytokinin, and opine production.

Copy right © 2010 ASM Press

American Society for Microbiology1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 18Genetic Engineering of Plants: Methodology

Figure 18.3

Ti plasmid structure

Copy right © 2010 ASM Press

American Society for Microbiology1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 18Genetic Engineering of Plants: Methodology

Figure 18.1

Infection of a plant with A. tumefaciensand formation of a crown gall tumor.

Fig. 18.1 Infection of a

plant with A. tumefaciens and

formation of crown galls

Fig. 28-27

Crown Gall on

Tobacco

The infection process:

1. Wounded plant cell releases phenolics and nutrients.

2. Phenolics and nutrients cause chemotaxic response of A. tumefaciens

3. Attachment of the bacteria to the plant cell.

4. Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone) induce vir gene

transcription and allow for T-DNA transfer and integration into plant chromosomal DNA.

5. Transcription and translation of the T-DNA in the plant cell to produce opines (food) and

tumors (housing) for the bacteria.

6. The opine permease/catabolism genes on the Ti plasmid allow A. tumefaciens to use

opines as a C, H, O, and N source.

Figure 18.2 and 18.3

Ti plasmid structure and

function. Note the wound-

induced plant phenolics

induce the vir genes on

the Ti plasmid.

Copy right © 2010 ASM Press

American Society for Microbiology1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 18Genetic Engineering of Plants: Methodology

Figure 18.4

Conserved nucleotides at the right and left borders of the Ti plasmid are imperfect

direct repeats.

Copy right © 2010 ASM Press

American Society for Microbiology1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 18Genetic Engineering of Plants: Methodology

Figure 18.6

Chemical structures of three

opines produced by plants.

Fig. 18.7 The binary Ti plasmid system involves using a small

T-DNA plasmid (shown below) and a disarmed (i.e., no T-

DNA) Ti plasmid in A. tumefaciens

Plant genetic engineering with

the binary Ti plasmid system

Clone YFG (your favorite gene) or

the target gene in the small T-DNAplasmid in E. coli, isolate the plasmid

and use it to transform the disarmedA. tumefaciens as shown.

Transgenicplant

(disarmed)

Disarmed

Ti plasmid

Copy right © 2010 ASM Press

American Society for Microbiology1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 18Genetic Engineering of Plants: Methodology

Table 18.1

Copy right © 2010 ASM Press

American Society for Microbiology1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 18Genetic Engineering of Plants: Methodology

Table 18.2

Fig. 18.10

Microprojectile

bombardment or

biolistic-mediated

DNA transfection

equipment

(a) lab version

(b) portable version

When the helium pressure builds to a certain point, the plastic rupture disk bursts, and

the released gas accelerates the flying disk* with the DNA-coated gold particles on itslower side. The gold particles pass the stopping screen, which

holds back the flying disk, and penetrate the cells of the plant.

*

Copy right © 2010 ASM Press

American Society for Microbiology1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 18Genetic Engineering of Plants: Methodology

Figure 18.10

Microprojectile bombardment

(biolistics) apparatus

Copy right © 2010 ASM Press

American Society for Microbiology1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 18Genetic Engineering of Plants: Methodology

Figure 18.12

Figure 18.13

Chloroplasts can be genetically engineered using microparticle bombardment.

Copy right © 2010 ASM Press

American Society for Microbiology1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 18Genetic Engineering of Plants: Methodology

Table 18.5

Table 18.5 Some plant cell reporter and

selectable marker gene systems

Enzyme activity Selectable marker

Reporter gene

Neomycin phosphotransferase (kanr) Yes Yes

Hygromycin phosphotransferase (hygr) Yes Yes

Nopaline synthase No Yes

Octopine synthase No Yes

β-glucuronidase (GUS) No Yes

Firefly luciferase No Yes

β-galactosidase No Yes

Bromoxynil nitrilase Yes No

Green fluorescent protein (GFP) No Yes

Reporter Genes

• For how reporter genes work, see:

http://bcs.whfreeman.com/lodish7e/#800911__811966__

• GFP Researchers Win Nobel Prize (October 8, 2008)

Osamu Shimomura, Martin Chalfie, and Roger Tsien won

the Nobel Prize in chemistry for their work on green

flourescent protein, a tool that has become ubiquitous in modern biology as a tag and molecular highlighter, vastly

improving our ability to understand what goes on inside

cells.

• Perhaps you may even want to see a 10 minute YouTube video on GFP; if so please see

http://www.youtube.com/watch?v=Sl2PRHGpYuU

Manipulation of gene expression in plants

• Strong, constitutive promoters (35S Cauliflower mosaic virus promoter or 35S CaMV or 35S)

• Organ and tissue specific promoter (e.g., the leaf-specific promoter for the small subunit of the photosynthetic enzyme ribulosebisphosphate carboxylase or rbc)

• Promoterless reporter gene constructs to find new organ- and tissue-specific promoter (see Fig. 18.15)

• Inducible promoters

• Secretion of transgene products by inclusion of a signal peptide sequence between a root promoter and YFG and growing the transgenic plant hydroponically (YFG product will be secreted)

Copy right © 2010 ASM Press

American Society for Microbiology1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 18Genetic Engineering of Plants: Methodology

Figure 18.21

Rhizosecretion using a

plant promoter active in

roots and a signal peptide

sequence.

Copy right © 2010 ASM Press

American Society for Microbiology1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 18Genetic Engineering of Plants: Methodology

Figure 18.26

Marker genes may be a safety issue, so it is best to remove them—here is one strategy.