1. Lecture 8 Genes and traits of interest Neal Stewart

2. Happy Valentine’s Day!

3. Objectives and discussion questions 1.What is genomics (and “omics” in general)? What are the tools for finding genes that might be useful in biotechnology. 2.Know the principles of two key methods: cDNA (complementary DNA) library production and PCR (polymerase chain reaction). 3.What are the differences between “input” and “output” traits? Considering the environmental and biological factors that limit production in a farmer’s field, what are some new input traits that might be good candidates for improvement using biotechnology? 4. Consider the possibility that you are employed by an agricultural biotechnology company, and they ask you to find a bacterial gene for resistance to a specific herbicide. The herbicide has been manufactured by the company for many years. Using a strategy similar to that used to find glyphosate resistance, where might you start to look for a bacterium resistant to that herbicide?

4. More objectives 5. Golden Rice producing provitamin A has the potential to help many impoverished people who might benefit from eating it. Although application of this technology is supported by many people and organizations, there are also some who oppose the technology. Considering their possible motivations and potential biases, discuss some of the reasons that groups have come out in favor or in opposition to Golden Rice. 6. What are the potential benefits of producing pharmaceutical proteins in plants? What are some of the disadvantages or potential dangers? 7. Animal genes can be inserted into plants and expressed. Would you be opposed to eating foods from plants expressing proteins encoded by animal genes? By human genes? Discuss the reasons for your answers.

5. Finding genes to modify: many strategies Biochemistry—understanding pathways Functional screens Hunting for specific genes– cDNAs Searching databases for similar genes Omics

6. http://fig.cox.miami.edu/~cmallery/150/gene/genome2.jpg How plant genome sizes compare with other organisms

7. Omics—what’s the point? Genomics Transcriptomics Proteomics Metabolomics Phenomics

8. Omics—what’s the point? Genomics Transcriptomics Proteomics Metabolomics Phenomics ID genes Transcript abundance Protein abundance ID metabolites To understand how perturbing the system results in a new phenotype—whole plant phenotyping

9. Omics—main approaches Genomics Transcriptomics Proteomics Metabolomics Phenomics Genome sequencing RNA-seq Mass spec techniques Suite of technologies that can be automated and used in the field

10. Creating cDNAs complementary DNA from mRNA

11. Introducing PCR

12. Figure 7.10 http://youtube.com/watch?v=_YgXcJ4n-kQ

13. Sample preparation Reference materialTest material Total RNA isolation mRNA purification Source: Murali Rao

14. Image Collection Sample Array Data

15. Progression of GM plants 1 st Generation: Input traits (herbicide tolerance, insect resistance, etc.) 2 nd Generation: Output traits: (enhanced nutrition, etc.)—also known as quality traits 3 rd Generation: Non-traditional– (pharmaceuticals, phytoremediation, phytosensors, next-generation biofuels)

16. First generation Input traits

17. Herbicide-resistant crops

18. Figure 8.1

19. Finding a glyphosate resistance gene Discovery of glyphosate—kills most plants Knowledge of shikimate pathway Targeting enyzme (gene) for resistance Functional screen for resistance Clone resistance gene Overexpress gene in plants

21. Discussion question Consider the possibility that you are employed by an agricultural biotechnology company, and they ask you to find a bacterial gene for resistance to a specific herbicide. The herbicide has been manufactured by the company for many years. Using a strategy similar to that used to find glyphosate resistance, where might you start to look for a bacterium resistant to that herbicide?

22. Transgenic disease resistance Viruses (yes) Bacteria (no) Fungi (no) Nematodes (no)

23. Stewart, 2004. Genetically Modified Planet 2004 Photo by Dennis Gonsalves.

24. Figure 8.5

25. Biotechnologist of the day Dennis Gonsalves From Hawaii At USDA-ARS in Hilo, Hawaii Humboldt Prize winner 2002 Virus resistant papaya credited for saving papaya industry in Hawaii

26. Figure 8.4

27. Insect resistance

28. Controlling Colorado potato beetle is not easy

29. Bt corn

30. Bt cotton

31. Stewart, 2004. Genetically Modified Planet 2004 Bacillus thuringiensis

32. Bt Cry structure I III II Stewart, 2004. Genetically Modified Planet 2004

33. Figure 8.3

34. Insect midgut cells that have bound Bt toxin. Same gut cells a few hours later– note the damage and leakage. Bt toxin Stewart, 2004. Genetically Modified Planet 2004

35. Insect midgut cells that have bound Bt toxin. Bt Mutated receptors cannot bind Bt toxin. Receptors are not present– cells cannot bind Bt Stewart, 2004. Genetically Modified Planet 2004

36. Different Bt Crys Cry 1s—kills caterpillars (lepidoptera) Cry 2s—kills caterpillars (lepidoptera) Cry 3s—kills beetles (coleoptera) Canola plant expresses a Bt cry1Ac gene

37. Discussion question Other than the products discussed in this chapter, what other sorts of genes or strategies might be useful in engineering transgenic plants resistant to insects or pathogens?

38. Second generation Output traits=quality traits

39. Improved nutrition, better foods Golden rice Modified oils from oilseeds Vitamin E enhancements Arctic apple—non-browning Innate potato—non-browning and reduced acrylamide upon browning

40. Golden rice: producing provitamin A www.goldenrice.org

41. Biotechnologist of the day: Ingo Potrykus

42. Figure 8.6

43. Third generation Non-traditional products

44. Examples Pharmaceuticals Oral vaccines Phytoremediation Phytosensors Biofuels

45. Plant-made pharmaceuticals aka Molecular pharming Duckweed Corn Genetic Engineering Protein Purification Grow in lab Or field Oral vaccine– eat the fruit, or purify the vaccine  pill or injection

46. Fraunhofer USA: one plant- based platform to produce pharmaceutical proteins: vaccines http://www.youtube.com/watch?v= oCGFW1WOFTY

47. What are the potential benefits of producing pharmaceutical proteins in plants? What are some of the disadvantages or potential dangers?

48. Phytorediation example How to remediate mercury in soil www.uga.genetics.edu/rmblab

49. So, transgenic plants could be used in a lot of applications… Are there any we should avoid?

50. PCR videos! How PCR works: http://youtube.com/watch?v=_YgXcJ4n-kQ PCR song! http://youtube.com/watch?v=x5yPkxCLads&feature=related