California State University Northridge

Biology 470 - Biotechnology


Plant genetic engineering

Resource: Glick and Pasternack slides from Chapter 17


http://www.monsanto.com/ag/articles/FieldsOfPromise/framemonmag.html

Agrobacterium-mediated transfer

The crown gall, and the wounded plant - acetosyringone and hydroxyacetosyringone


The distribution of the vector (requires shockwave)




Agrobacterium tumefaciens, chromosome and plasmid



http://www.ppws.vt.edu/~sforza/prokaryote/agrobacterium1.jpg


Rough draft of
Ti plasmid (requires shockwave)

The military analogy: Is Agrobacterium like a "mercenary commando"?

Resource pictures: The Pathogenesis of Crown Gall, by Sforza et al.



Octopine-type Ti plasmid

T-region - the segment transferred to plants, between Right and Left border

Genes for production of:

  • Auxin
  • Cytokinin
  • Opine

Outside the T-region - the segment not transferred

  • Opine catabolism genes
  • origin of replication
  • vir genes
    • example: virD encodes a site-specific endonuclease for the Right border sequence, makes a nick.

What is the difference between an element required in cis, and one required in trans?

...a trans element can be moved to a different location, for example from the Ti plasmid to the genome, and it will still function effectively. A gene that makes a diffusible protein product can be moved generally, and that is an example of a "trans" element. An example of a "cis" element is an origin of replication. That cannot be moved, and still serve its purpose. It must be "on" the DNA that needs replicating.

A T-border sequence is a "cis" active element, whereas the vir genes are "trans" active. Vir genes do not need to be on the plasmid carrying the T-DNA region.


From CAMBIA Intellectual Property Resource, a 262 page "white paper" with detailed information on Agrobacterium transformation, intellectual property, and patents: (pdf format)


Binary vector systems - widely used





http://www.cambiaip.org/cambiaIP/books/whole.html#agri_page25

Elements:

1. A wide host-range plasmid with

2. A "helper" Ti that lacks the T-DNA, but contains the vir genes



Co-integrated or hybrid Ti vector system - oldies but goodies

Elements:

1. A disarmed Ti plasmid

2. An ori-less plasmid that can integrate into the Ti plasmid, containing (typically) cis elements necessary for T-DNA mobilization. (Fig 17.6)

Depends on a single crossover event




Modified Co-integrated vectors -
using cre/lox to prepare for directed recombination



Engineering issues:

1. In vitro methodology

2. Transformation of E. coli

3. Transformation or conjugation (with cis/trans function) into A. tumifaciens

4. Transformation of plants

http://www.cambiaip.org/cambiaIP/diags/transfer_1.jpg



General Plant Applications:

Monocots

Dicots





Microprojectile bombardment - biolistics


Biorad's
Helios Gene Gun




The gene is in the plant, so now what?


Detection of foreign DNA expression - Table 17.4

Selectable markers vs. Reporters


e.g.

GUS - beta-D-glucuronidase (from E. coli) plus 5-bromo-4-chloro-3-inolyl B-D-glucuronic acid

Luciferase -

GFP






Reading assignment: Chapter 18

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Dr. Stan Metzenberg
Department of Biology
California State University Northridge
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Stan Metzenberg, Department of Biology, California State University Northridge, 18111 Nordhoff St., Northridge CA 91330-8303.

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