BIO 3041: PLANT BIOLOGY AND GENETIC ENGINEERING

Barbara Kunkel 319 Monsanto Hall, Ph: 935-7284, e-mail: kunkel@biology.wustl.edu

Office hours: Weds 12:30-1:30 pm

October 16, 2003

Protein Engineering at the Codon Level:

Engineering insect resisTance using Bt toxin.

Required reading: **Research article by Kota et al, PNAS 1999 (PDF available on web).

*G+P: Ch. 15, pp. ___ Bt-toxin; Ch. 18 pp. 538- 547 on engineering insect resistance.

**Losey et al: 1999. Nature 399:214, ** Hodgson, 1999. Nature Biotech 17:627

Recommended/Supplementary reading: **Chrispeels et al, Ch. 16 on Pest Control; especially pp. 433-439; G+P, Ch. 8 pp 191-198- Site-directed mutagenesis;

**Research article by Perlak et al, 1991.

**Are Bt crops safe? Mendelsohn et al, 2003; **Pimentel and Raven, 2000

**Miscellaneous handouts on Bt resistant plants, transgenic crops, etc.

** Provided as handout; * On reserve in library

Interesting websites: http://www.colostate.edu/programs/lifesciences/TransgenicCrops/

http://www.nal.usda.gov/bic/BTTOX/bttoxin.htm

I. Methods for Monitoring Gene Expression

A. RNA:

· In situ hybridization

· RT-PCR

B. Protein

· Western blots

· ELISA

· Ezymatic or bioassay

II. Overview of pests in agriculture

· Economic impact

· Traditional methods of pest control

· Alternative approaches

III. Bt toxin (BT)

· Bacillus thuringensis: naturally produces Bt toxin (BT)

§ aka crystal protein (cry) or “insecticidal crystal protein” (ICP)

· produced as a pro-toxin, as bacteria sporulates

· protoxin is not active

· mode of action of BT is highly specific

o processed to active form after ingestion by insect

o disrupts function of cells in gut of insect larvae (caterpillar)

· Natural product used as an applied microbiological product since 1960's

· cry genes have been cloned, active regions of protein identified

IV. Expression of Bt toxin in plants

· Bt toxin genes have been introduced into plants

o In first case saw very low levels of toxin protein produced

· Review: factors affecting expression of foreign genes in plants:

o Bacterial gene is very A-T-rich

o Potential poly adenylation sites

o Codon usage

· Modifications to improve level of expression in plants

o site-directed mutagenesis (read about method in G+P text)

o synthetic gene

· Results: dramatic insect-resistance in Bt-expressing plants !!

V. Concerns regarding Bt-toxin-expressing plants

· active against non-target insects (e.g. Monarch butterfly caterpillars)

· selection for resistant pests & strategies to prevent this

· transfer of transgenes to related weed species

VI. Additional approaches for engineering resistance to pests:

· Read about these on your own in G+P, Ch. 18 pp. 543-546

· Expression of protease inhibitors