Chapter 18-Genetic Engineering of Plants: Methodology Plant transformation with the Ti plasmid of Agrobacterium tumefaciens Ti plasmid derived vector systems

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  • Chapter 18-Genetic Engineering of Plants: MethodologyPlant transformation with the Ti plasmid of Agrobacterium tumefaciensTi plasmid derived vector systemsPhysical methods of transferring genes to plantsMicroprojectile bombardmentUse of reporter genes in transformed plant cellsManipulation of gene expression in plantsProduction of marker-free transgenic plants

  • Why genetically engineer plants?To improve the agricultural, horticultural or ornamental value of a crop plantTo serve as a living bioreactor for the production of economically important proteins or metabolitesTo provide a renewable source of energyTo provide a powerful means for studying the action of genes (and gene products) during development and other biological processes

  • Plant transformation with the Ti plasmid of Agrobacterium tumefaciensA. tumefaciens is a gram-negative soil bacterium which naturally transforms plant cells, resulting in crown gall (cancer) tumorsTumor formation is the result of the transfer, integration and expression of genes on a specific segment of A. tumefaciens plasmid DNA called the T-DNA (transferred DNA)The T-DNA resides on a large plasmid called the Ti (tumor inducing) plasmid found in A. tumefaciens

  • The Ti plasmid of Agrobacterium tumafaciens and the transfer of its T-DNA to the plant nuclear genome

  • Fig. 18.3 The Ti plasmid of Agrobacterium tumafaciens and its T-DNA region containing eukaryotic genes for auxin, cytokinin, and opine production.

  • Fig. 18.1 Infection of a plant with A. tumefaciens and formation of crown galls

  • Fig. 17.3 Ti plasmid: structure & function

    The infection process:Wounded plant cell releases phenolics and nutrients.Phenolics and nutrients cause chemotaxic response of A. tumefaciensAttachment of the bacteria to the plant cell.Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone) induce vir gene transcription and allow for T-DNA transfer and integration into plant chromosomal DNA. Transcription and translation of the T-DNA in the plant cell to produce opines (food) and tumors (housing) for the bacteria.The opine permease/catabolism genes on the Ti plasmid allow A. tumefaciens to use opines as a C, H, O, and N source.

  • Fig. 18.4 The right and left borders of the T-DNA of the Ti plasmid are 25 bp direct repeats important for mobilization of the T-DNA by vir gene products Right 5-TGNCAGGATATATNNNNNNGTNANN-3Left 5-TGGCAGGATATATNNNNNTGTAAAN-3

  • Fig. 18.7 The binary Ti plasmid system involves using a small T-DNA plasmid (shown below) and a disarmed (i.e., no T-DNA) Ti plasmid in A. tumefaciens

  • Plant genetic engineering with the binary Ti plasmid systemClone YFG (your favorite gene) orthe target gene in the small T-DNAplasmid in E. coli, isolate the plasmidand use it to transform the disarmedA. tumefaciens as shown.Transgenicplant

  • Table 18.1 Plant cell DNA-delivery methods* Most commonly used methods

    MethodCommentTi plasmid-mediated gene transfer *Excellent and highly effective, but limited to dicotsMicroprojectile bombardment *Easy and effective; used with a wide range of plants Viral vectorsNot very effectiveDirect gene transfer into plant protoplastsOnly certain protoplasts can be regenerated into whole plantsMicroinjetionTedious and slowElectroporation *Limited to protoplasts that can be regenerated into whole plantsLiposome fusionLimited to protoplasts that can be regenerated into whole plants

  • Fig. 18.10 Microprojectile bombardment or biolistic-mediated DNA transfection equipment(a) lab version(b) portable version

    When the helium pressure builds to a certain point, the plastic rupture disk bursts, and the released gas accelerates the flying disk* with the DNA-coated gold particles on itslower side. The gold particles pass the stopping screen, which holds back the flying disk, and penetrate the cells of the plant.*

  • Table 18.5 Some plant cell reporter and selectable marker gene systems

    Enzyme activitySelectable markerReporter geneNeomycin phosphotransferase (kanr)YesYesHygromycin phosphotransferase (hygr)YesYesNopaline synthaseNoYesOctopine synthaseNoYesb-glucuronidase (GUS)NoYesFirefly luciferaseNoYesb-galactosidaseNoYesBromoxynil nitrilaseYesNoGreen fluorescent protein (GFP)NoYes

  • Reporter GenesFor how reporter genes work, see: http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=15000&i=15010.01&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=1322GFP Researchers Win Nobel Prize (October 8, 2008)Osamu Shimomura, Martin Chalfie, and Roger Tsien won the Nobel Prize in chemistry for their work on green flourescent protein, a tool that has become ubiquitous in modern biology as a tag and molecular highlighter, vastly improving our ability to understand what goes on inside cells. Perhaps you may even want to see a 10 minute YouTube video on GFP; if so please see http://www.youtube.com/watch?v=Sl2PRHGpYuU

  • Manipulation of gene expression in plantsStrong, constitutive promoters (35S Cauliflower mosaic virus promoter or 35S CaMV or 35S)Organ and tissue specific promoter (e.g., the leaf-specific promoter for the small subunit of the photosynthetic enzyme ribulosebisphosphate carboxylase or rbc)Promoterless reporter gene constructs to find new organ- and tissue-specific promoter (see Fig. 18.15)Inducible promotersSecretion of transgene products by inclusion of a signal peptide sequence between a root promoter and YFG and growing the transgenic plant hydroponically (YFG product will be secreted)

  • Fig. 18.26 Marker genes may be a safety issue, so it is best to remove themhere is one strategyLBRBRecombinasegeneSelectablemarkerTarget geneRecombinase recognitionsequence