I. Why create transgenic plants?
When there is no naturally occurring genetic variation for the target trait.
- Herbicide resistance
- Vitamin A
- Flower color
- Florigene. L-3. A company dedicated to novel flower colors, through transgenics
II. What genes to transfer?
- One gene to a few genes - the CP4 ESPS example
- Multiple genes - the “Golden Rice” example
- In principle, any gene,
- CP4 ESPS L-2. Details on the gene conferring resistance to the herbicide RoundUp. The gene conferring resistance to Roundup herbicide was found in Agrobacterium. Coincidentally, this organism is also a tool for creating tansgenic plants
- "Golden Rice”. L-1. A biosynthetic pathway with genes from daffodil, bacteria, and pieces of genes from pea, rice, and virus.
III. Constructing transgenes. The minimal requirements for the transgene are a promoter, coding region, and terminator.
General structure: 5’---Promoter …..Coding region…….terminator---3’
Example (Bt gene with nptII selectable marker): 5’ --P35S…Bt…Tnos--//--P35S…nptII…Tnos---3’
A. Promoter: DNA sequence controlling spatial and/or temporal level of transgene expression. Promoters can be
- constitutive (e.g. CaMV 35S, Ubiquitin, NOS = nopaline synthase)
- tissue specific (e.g. patatin , which is tuber specific)
- inducible (e.g. heat shock, photo responsive)
B. Termination sequence: DNA sequence signaling end of the gene (e.g. T35S, T NOS).
In addition to the target gene, additional elements may be added, such as selectable markers and/or reporter genes
C. Selectable Marker: Encodes a protein (enzyme) that allows the transformed cells to grow while the growth of the non-transformed cells is inhibited. Examples include
- Antibiotic resistance (e.g. nptII, hygromycin phosphotransferase)
- Herbicide resistance (e.g. bar, phosphinothricin acetyl transferase)
- Selectable Markers. L-1. Key types of selectable markers and L-3. Details and examples of selectable markers.
D. Reporter genes: Genes that, upon expression in the transgenic plants, provide a clear indication that genetic transformation did occur, and indicate the location and the level of expression. Examples include.
- Glucuronidase (GUS) - open source molecular biology tool arising from the discovery of GUS - CAMBIA
- Luciferase, green fluorescent protein (GFP). An extreme use of GFPGUS. L-2. Blue means GUS is expressed.CAMBIA. L-3. Royalty income from GUS used to establish a biotech non-profit.GFP. L-3. So many ways to be green.An extreme use of GFP. L-3. Art meets science (?).
E. Additional components which may be included in the transgene are enhancers and introns.