Chapter 19 Flashcards
A local crop is being ravaged by a non-enveloped virus with a single-stranded RNA genome (8000bo long). The virus and the RNA can readily be isolated. You have antibodies against all four proteins encoded within the viral genome. Describe a strategy that you could use to protect the crop against this viral infection and prevent subsequent damage.
Assume that one of the proteins are the encoded by the virus should be a coat proteins. Take the viral RNA, purify for coat protein RNA, create the cDNAwith linkers. Insert to a Ti plasmid under the control of 35s Promoter in the antisense direction and transform into plants
Suggest several different strategies for developing insect resistance plants
Engineer plants to express a truncated Bt toxin in chloroplast
Use a Ti plasmid vector to introduce protease and a-amylase inhibitors
Engineer plants that produces streptomyces cholesterol oxidase gene
Engineer plants that produce VIP proteins which can be used alongside Cry proteins
Engineer plants to express RNAi that inhibits insect proliferation
How can protease inhibitors, alpha amylase inhibitor, cholesterol oxidase, Vip proteins and ricin each protect a plant against damage from insect predation
Protease inhibitors – when ingested, protease inhibitors prevent insect from hydrolyzing plant proteins and starves the insect
Alpha amylase inhibitor- when larvae eat alpha-amylase inhibitor, the insect is no longer able to digest starch causing the insect to eventually die
Cholesterol oxidase – disrupts the insect’s midgut epithelial membrane killing the insect
Vegetative Insecticidal Toxins (Vip) – secreted insecticidal protein which can be used in tandem with cry genes because they use different receptors to bind to the gut of the insect
Ricin – ricin binds to N-acetylgalactosamine residues which binds to different receptors on the gut membrane, can be used in conjunction with Bt toxin to prevent resistance insects
How can RNAi be used to protect plants against damage from insect predation
Method 1 – ingestion or microinjection of dsRNA into insects to silence genes in organisms, then create larval cDNA library, test the dsRNAs for ability to elicit RNAi and inhibit larval proliferation, clone into gene and transform into plants. RNAi targets the transcript for vacuolar ATPase
Method 2 – identify method that insect uses to protect itself against gossypol, create RNAi against gene for cytochrome P452 monooxygenase . Insect damages plant, plant synthesizes pssypol, RNAi destroys gossypol resistance and kills the insect
Suggest a couple of strategies for simultaneously protecting a plant against damage from several different viruses?
Create Ti plasmid binary vectors expressing more than one virus protein coat in the antisense direction
Transform plant with gene for RNAse III which only cleaves dsRNA which is what most plant viruses are
Transform plants with PAP proteins which are ribosome inactivating proteins causing the ribosomes to be inactivated when ingested by the plant
Engineer plants to produce antibodies directed against invading viruses through expression of single chain Fv antibodies against tobacco mosaic viruses.
Engineering plants to produce miRNA’s that interfere with viral replication by targeting the viral RNA for degredation.
How can RNAi be used to protect plants against damage from plant viruses?
RNAi would act to target specific mRNAs (e.g., mRNAs
encoding viral coat proteins) for nuclease digestion.
What general strategies can be employed in genetically engineering plants to be resistant to herbicides
Inhibit uptake of herbicide
Overproduce herbicide-sensitive target protein so that enough remains available for cellular functions despite presence of herbicide
Introduce a bacterial or fungal gene that produces a protein that is not sensitive to herbicide but performs the same function as the plant protein
Reduce the ability of a herbicide-sensitive target protein to bind to a herbicide
Endow plants with the capability to metabolically inactivate the herbicide
Suggest two different strategies for engineering plants that are resistant to the herbicide glyphosate. Why is this important?
Method 1 – place the EPSPS gene from glycophosate resistant strain of E. coli under control of plant promorter with TT-polyA sequence and clone into plant
Method 2 – find an enzyme that deactivates glycophosate. ID 3 strains producing N-acetylglyphosate from N-acetyltransferase gene, isolate the genes, shuffle the gene fragments, introduce altered N-acetyltransferase gene in plants
How can crop plants be engineered to be resistant to the herbicide dicamba
Gene: promoter- enhancer-transit peptide- dicamba monooxygenase gene-terminator
Transit peptide to target chloroplast which is a ready source of reduced ferredoxin which supplies electrons for monooxygenase.
Monooxygenase gene inactivates dicamba herbicide
How can plants be engineered to resist damage from pathogenic soil fungi
Place chitinase under control of 35S promoter which will resist damage from fungi in general
How can plants be genetically engineered to be resistant to pathogenic bacteria?
Overexpress NPR1 gene which controls Pathogen related protein in the plant.
How can single-chain Fv antibodies be engineered to protect plants against fungal pathogens?
Fuse cDNA’s for different antimicrobial peptides and chitinase to Fv antibody that binds to cell wall protein.
How can a plant’s systematic acquired resistance response be engineered to confer resistance to a broad spectrum of both fungal and bacterial pathogens?
Overproduce salicylic acid and add genes encoding enzymes that convert chorismate into salicyclic acid which will induce PR proteins for fungal and bacterial pathogen resistence
What is the effect of increasing the level of oxidized glutathione within a plant? How would you genetically manipulate a plant to do this
Increased levels of oxidized glutathione can reduce oxidative stress. Create cDNA with glutathione S-transferase and glutathione peroxidase under 35S promoter and introduced via Ti plasmid.
Suggest several strategies that could be used to engineer plants that are resistant to growth inhibition by salt and by drought
-Engineer plants to increase cellular accumulation of osmoprotectants such as
Betaine – osmolyte that accumulates during water stress (gene: 35S promoter – choline dehydrogenase gene )
Trehalose – protects plants against inhibition when high levels of salt in the soil (gene: RB-ABA-protein-3’UTR-selectable marker-LB or RB-RBC-S-transit peptide-protein-3’UTR-selectable marker-LB)
- engineer salt tolerance by sequestering Na ion in large intracellular vacuole, overproduce Na/H antiport protein
- overproduction of cytokinin
