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1、Plant genetic engineeringZichaoM Yunnan Agricultural University MotivationWhat is genetic engineering?Why do scientists genetically engineer organisms? Introduction of genes from one species of organism into organism of a different species (transgenic organism) Cloning a gene to determine its functi
2、on Change the phenotype of an organism to improve a desired trait Traditional genetic manipulation Selective breeding to improve nutritional value of plants too slow Genetic engineering by molecular techniques Allows to create larger genetic changes at a much more rapid pace Production of large amou
3、nts of pharmaceuticalsGenetic engineering in plants Agrobacterium tumefaciens causes crown gall diseaseTi plasmidT-DNARBLBVIRULENCE (VIR) genesTi plasmid Crown gall disease, cell proliferation of plant tissue (like cancer) Crown gall disease caused by AgrobacteriumAgrobacterium usually disappears fr
4、om the galls after growth of the gall begins, as bacterium itself is not necessary for tumor development.A.Gall formed at the base B.of the stem of a rose bush. B. A series of galls (arrowheads) along a branch of a grapevine. Crown gall disease caused by AgrobacteriumGenetic engineering of the Ti pl
5、asmidSome considerations: Ti plasmid is 200 kb - too big for easy cloningneed to remove tumor-inducing genes from T-DNAVIR genesDisarmedTi plasmid DisarmTi plasmidAgrobacteriumCloning vectorYFGT-DNAAgrobacterium-mediated plant transformationPunch outleaf discsAgrobacterium culture carrying foreign g
6、ene -Carotene Pathway Problem in PlantsIPPGeranylgeranyl diphosphatePhytoeneLycopene -carotene(vitamin A precursor)Phytoene synthasePhytoene desaturaseLycopene-beta-cyclase-carotene desaturase Problem:Rice lacksthese enzymesNormalVitamin A“Deficient”RiceThe Golden Rice SolutionIPPGeranylgeranyl diph
7、osphatePhytoeneLycopene -carotene(vitamin A precursor)Phytoene synthasePhytoene desaturaseLycopene-beta-cyclase-carotene desaturase Daffodil(黄水仙花黄水仙花 ) geneSingle bacterial gene;performs both functionsDaffodil gene -Carotene Pathway Genes AddedVitamin APathwayis completeand functionalGoldenRiceExamp
8、les of applications of genetic engineering in agriculture“Golden Rice” containing genes for production of vitamin A Rice is a major staple food for half of the worlds population Rice grains (endosperm) lack several essential nutrients, including vitamin A and its precursors (-carotene) Vitamin A def
9、iciency causes blindness and weakness of the immune system (effects 400 million people worldwide)Wild-type Rice“Golden Rice”“Golden Rice” Generate transgenic rice containing entire new metabolic pathway -carotene pathway includes 4 enzymesGeranylgeranyl diphosphate (in immature rice endosperm) -caro
10、teneVitamin A“Golden Rice” Insect resistant plantsCorn borer pest of corn in North America and Europe Lepidoptera (鳞翅类 )(moths and butterflies) Bt-corn for corn borer controlWhat is Bt and how does it work?Bacterium Bacillus thuringiensis produces protein, delta-endotoxin, that is toxic to insects i
11、n orders Lepidoptera, Coleoptera (鞘翅类 )(beetles)- Bt toxin in form of powder used as insecticide spray applied to leaves where larvae feed on2. Toxin binds to specific receptors in gut and insects stops eating.Mode of action: 1.Insect eats Bt crystals ( ) and bacterial spores. Bt crystals dissolve a
12、t high pH in insect gut.3. Toxin causes the gut wall to break down, allowing spores and normal gut bacteria to enter the body.4. Insect dies as spores and gut bacteria proliferate in the body. Genetic engineering of Bt-resistant corn (Bt-corn) Clone Bt toxin gene from bacteria and express in plants
13、Bt-transgenic plants are resistant to insects, no need for spraying insecticideBacillus thuringiensisBt-crops for corn borer control Currently in crops of corn (control of corn borer 螟 ) cotton (control of bollworm蛾的幼虫 ) Controversial study (Scientists at Cornell University 1999) effect of Bt-transg
14、enic corn on Monarch butterfly larvae(君王斑点蝶 ) feeding on milkweed (乳草属 ) ? Bt corn may harm stream ecosystems (Scientists at Indiana University; Rosi-Marshall et al. 2007 PNAS 104 p16204)Bt corn may harm stream ecosystemsBt corn may harm the caddis fly(石蛾) , which serves as food for fish and amphibi
15、ans. Parts of Bt corn, such as leaves, cobs and pollen, can travel as far as 2000 meters away from source areas-a phenomenon that was not considered when Bt corn was licensed.+ GlyphosateXRoundup Sensitive PlantsXXShikimic acid + Phosphoenol pyruvate3-Enolpyruvyl shikimic acid-5-phosphate(EPSP)Plant
16、 EPSP synthaseAromaticamino acidsWithout amino acids, plant diesX GM crops currently grown worldwideGenetic engineering in plants Ethical questions Do we need GM crops? Contamination of non-GM crops (cross pollination) damage to wildlife human health risks? improved health benefits reduced agrichem
17、use feed worlds growing population62%(herbicide resistant)Percent increase of GM crops grown worldwideHerbicide resistanceimidazolinonesMutant AHAS enzymedeveloped by mutagenesisCrops Canola Corn Rice Sunflower WheatA Major Marketing Advantagebut lost when stacked with a transgeneThe Roundup Ready S
18、tory Glyphosate is a broad-spectrum herbicide Active ingredient in Roundup herbicide Kills all plants it come in contact with Inhibits a key enzyme (EPSP synthase) in an amino acid pathway Plants die because they lack the key amino acids A resistant EPSP synthase gene allows crops to survive sprayin
19、gBacterialEPSP synthaseShikimic acid + Phosphoenol pyruvate3-enolpyruvyl shikimic acid-5-phosphate(EPSP)Aromaticamino acidsRoundup Resistant Plants+ GlyphosateWith amino acids, plant livesRoundUp has no effect;enzyme is resistant to herbicideIntroducing the GeneorDeveloping TransgenicsSteps1. Create
20、 transformation cassette2. Introduce and select for transformantsTransformation CassettesContains1. Gene of interest The coding region and its controlling elements2. Selectable marker Distinguishes transformed/untransformed plants3. Insertion sequences Aids Agrobacterium insertionTransformation Step
21、sPrepare tissue for transformationIntroduce DNACulture plant tissue Develop shoots Root the shootsField test the plants Leaf, germinating seed, immature embryos Tissue must be capable of developing into normal plants Agrobacterium or gene gun Multiple sites, multiple years Transformation cassettes a
22、re developed in the lab They are then introduced into a plant Two major delivery methodsDelivering the Geneto the Plant Agrobacterium Gene GunTissue culturerequired to generatetransgenic plantsThe Lab StepsThe Next Test Is The FieldNon-transgenicsTransgenicsHerbicide ResistanceFinal Test of the Tran
23、sgenicConsumer AcceptanceRoundUp Ready CornBeforeAfterPlant Genetic TransformationAll stable transformation methods consist of three steps: Delivery of DNA into a single plant cell.Integration of the DNA into the plant cell genome.Conversion of the transformed cell into a whole plant. Agrobacterium-
24、mediated TransformationBiology of the Agrobacterium-plant interaction The only known natural example of inter-kingdom DNA transfer Infects at root crown or just below the soil line. Can survive independent of plant host in the soil. Infects plants through breaks or wounds.Common disease of woody shr
25、ubs, herbaceous plants, dicots. Galls are spherical wart-like structures similar to tumors.The genus Agrobacterium has a wide host range:Overall, Agrobacterium can transfer T-DNA to a broad group of plants.Yet, individual Agrobacterium strains have a limited host range.The molecular basis for the st
26、rain-specific host range is unknown.Many monocot plants can be transformed (now), although they do not form crown gall tumors.Under lab conditions, T-DNA can be transferred to yeast, other fungi, and even animal and human cells.Why is Agrobacterium used for producing transgenic plants?The T-DNA elem
27、ent is defined by its borders but not the sequences within. So researchers can substitute the T-DNA coding region with any DNA sequence without any effect on its transfer from Agrobacterium into the plant.Steps of Agrobacterium-plant cell interactionCell-cell recognitionSignal transduction and trans
28、criptional activation of vir genesConjugal DNA metabolismIntercellular transportNuclear importT-DNA integrationT-DNAT-DNA carries genes involved in the synthesis of plant growth hormones (auxin, auxin synthesis; cyt, cytokinin synthesis) and the production of low molecular weight amino acid and suga
29、r phosphate derivatives called opines (ocs, octopine(章鱼碱); mas, mannopine(甘露碱甘露碱 ); and ags, agropine).Agrobacteria are usually classified based on the type of opines specified by the bacterial T-DNA.Ti PlasmidAgrobacterium-induced plant tumors contain high concentrations of :Plant hormones (auxin,
30、cytokinin)Opines (octopine, nopaline胭脂氨酸 )Agrobacterium-host cell recognition is a two-step process1.Loosely bound step: acetylated polysaccharides are synthesized.2.Strong binding step: bound bacteria synthesize cellulose filaments to stabilize the initial binding, resulting in a tight association
31、between Agrobacterium and the host cell.chvA export of beta-1,2-glucan chvB b1-2 glucan sybthesispscA the mutants that defined the locus were initially isolated as having an altered polysaccharide composition; they were nonfluorescent on media containing Leucophor or Calcofluor, indicating a defect
32、in the production of cellulose fibrils. Receptors are involved in initial bindingPlant vitronectin(玻连蛋白 )-like protein (PVN, 55kDa) was found on the surface of plant cell. This protein is probably involved in initial bacteria/plant cell binding.PVN is only immunologically related to animal vitronect
33、in.Animal vitronectin is an important component of the extracellular matrix and is also an receptor for several bacterial strains. Receptors are involved in initial bindingAside from PVN, rhicadhesin-binding protein was found in pea roots.Also, rat1(arabinogalactan protein; AGP) and rat2(potential c
34、ell-wall protein) are involved.Plant signalsWounded plants secrete sap with acidic pH (5.0 to 5.8) and a high content of various phenolic compounds (lignin, flavonoid precursors) serving as chemical attractants to agrobacteria and stimulants for virgene expression.Among these phenolic compounds, ace
35、tosyringone (AS) is the most effective. Plant signalsSugars like glucose and galactose also stimulate vir gene expression when AS is limited or absent. These sugars are probably acting through the chvE gene to activate vir genes.Low opine levels further enhance vir gene expression in the presence of
36、 AS.Plant signalsThese compounds stimulate the autophosphorylation of a transmembranereceptor kinase VirA at its His-474. It in turn transfers its phosphate group to the Asp-52 of the cytoplasmic VirG protein.Plant signalsVirG then binds to the vir box enhancer elements in the promoters of the virA,
37、 virB, virC, virD, virEand virG operons, upregulating transcription.Sugars interact with ChvE (glucose/galactose binding protein) which interacts with VirA through its periplasmic domain.Structure of the T-DNAThe existence and orientation of right border is absolutely required for Agrobacterium path
38、ogenicity but not the left border. Transfer of the T-DNA is polar from right to left.Structure of the T-DNAAlthough right border and left border are required to delimit the transferred segments, the T-DNA content itself has no effect on the efficiency of transfer.Therefore, researchers replace most
39、of the T-DNA with DNA of interest, making Agrobacterium a vector for genetic transformation of plants.Production of T-strandEvery induced Agrobacterium cell produces one T-strand.VirD1 and VirD2 are involved in the initial T-strand processing, acting as site-and strand-specific endonucleases.Product
40、ion of T-strandAfter cleavage, VirD2 covalently attaches to the 5 end of the T-strand at the right border nick and to the 5-end of the remaining bottom strand of the Ti plasmid at the left border nick by its tyrosine 29.Production of T-strandVirC1 enhances T-strand production by binding to overdrive
41、.Overdrive is a cis-active 24-base pair sequence adjacent to the right border of the T-DNA. It stimulates tumor formation by increasing the level of T-DNA processing.Formation of the T-complexThe T-complex is composed of at least three components: one T-strand DNA molecule, one VirD2 protein, and ar
42、ound 600 VirE2 proteins.Formation of the T-complexWhether VirE2 associates with T-strand before or after the intercellular transport is not clear.Formation of the T-complexIf VirE2 associates with the T-strand after intercellular transport, VirE1 is probably involved in preventing VirE2-T-strand bin
43、ding.Judging from the size of the mature T-complex (13nm in diameter) and the inner dimension of T-pilus (10nm width), the T-strand is probably associated with VirE2 after intercellular transport. Intercellular transportTransport of the T-complex into the host cell most likely occurs through a type
44、IV secretion system.In Agrobacterium, the type IV transporter (called T-pilus) comprises proteins encoded by virD4 and by the 11 open reading frames of the virB operon.Intercellular transportIntercellular transport of T-DNA is probably energy dependent, requiring ATPase activities from VirB4 and Vir
45、B11.Physical contact between Agrobacterium and the plant cell is required to initiate T-complex export. Without recipient plant cells, T-strands accumulate when vir genes are induced.Nuclear ImportBecause the large size of T-complex (50,000 kD, 13nm in diameter), the nuclear import of T-complex requ
46、ires active nuclear import.The T-complex nuclear import is presumably mediated by the T-complex proteins, VirD2 and VirE2. Both of them have nuclear-localizing activities.Nuclear ImportVirD2 is imported into the cell nucleus by a mechanism conserved between animal, yeast and plant cells (bipartite c
47、onsensus motif).VirE2 has a plant-specific nuclear localization mechanism. It does not localize to the nucleus of yeast or animal cells.Nuclear ImportIn host plant cells VirD2 and VirE2 likely cooperate with cellular factors to mediate T-complex nuclear import and integration into the host genome.Th
48、ese host factors have been identified through two-hybrid screens, however their functions are not clear.T-DNA integration is not highly sequence-specificFlanking sequence tags (FSTs) analysis showed no obvious site preference for integration throughout the genome.About 40% of the integrations are in
49、 genes and more of them are in introns.Non-homologous end-joining (NHEJ) occurs during T-DNA integrationThe mechanism of NHEJ makes deletions after T-DNA integration a common phenomenon.Events of NHEJ in AgrobacteriumT-DNA integrationIntegration is initiated by the 3(LB) of the T-DNA invading a poly
50、 T-rich site of the host DNAA duplex is formed between the upstream region of the 3-end of T-DNA and the top strand of the host DNA.The 3-end of T-DNA is ligated to the host DNA after a region downstream of the duplex is degraded.Events of NHEJ in AgrobacteriumT-DNA integrationA nick in the upper ho
51、st DNA strand is created downstream of the duplex and used to initiate the synthesis of the complementary strand of the invading T-DNA.Events of NHEJ in AgrobacteriumT-DNA integration The right end of the T-DNA is ligated to the bottom strand of the host DNA. This pairing frequently involves a G and
52、 another nucleotide upstream of it.Plant genes involved in transformationMutant screen, 2-hybrid screens and other methods have revealed a number of plant genes that are involved in transformation.Rat mutants (resistant to Agrobacterium transformation), VIP genes and other genes were isolated.Plant
53、Expression VectorMono Vector SystemTwo component Vector system GenesGenes =the coding system for instructionsA geneA gene =is a segment of DNAGuanine(G)Cytosine(C)Adenine(A)Thymine(T)basesDNADNA and GenesgeneGenes and ProteinsGene(a piece of DNA)traitproteintranslationtranslationmRNAtranscriptiontra
54、nscriptionRestriction enzymesAs biological scissors“Gene Gun” TechniqueDNA coated golden particlesGene gunCell divisionA plant cell withthe new geneTransgenic plantPlant cellCells DNADuracellDNA containingthe gene of interestPlant cellProtoplastElectroporation TechniquePower supplyDNA inside the pla
55、nt cellThe plant cell withthe new genePlant Expression VectorMono Vector SystemTwo component Vector system GenesGenes =the coding system for instructionsA geneA gene =is a segment of DNAGuanine(G)Cytosine(C)Adenine(A)Thymine(T)basesDNADNA and GenesgeneRestriction enzymesAs biological scissorsNext Ge
56、neration of Transgenic CropsPlant-based vaccines Plant-based vaccines Enhanced nutritional contentEnhanced nutritional contentFunctional foods and Functional foods and phytoceuticalsphytoceuticalsTransgenic plants for Transgenic plants for phytoremediationphytoremediation Plant-derived plastics and
57、polymers Plant-derived plastics and polymers Where is the technology being used?Valued-added food?In 1999 approximately 70% of foodstuffs in North America contain components derived from GM cropsWhy Should I Support the Biotech Industry?By genetically modifying plants we can:Reduce pesticide useCons
58、erve fuel and waterPreserve non-pest insect populationsIncrease food production per hectare of farmlandEnable crops to grow in sub-optimal conditionsIncrease the nutritional value of foodCurrent ProductsTransgenic SoybeanRoundup Ready Resistant to Roundup HerbicideReduces the amount of herbicide app
59、lied to cropsAltered Fatty-acid contentChanges the nutritional valueField after one round of application of Roundup herbicideCurrent ProductsCanolaHerbicide resistantBetter for the environmentAltered fatty-acid compositionA value-added foodhttp:/ canolaprods.jpg Current ProductsTomatoFlavr Savr Toma
60、toDelayed softening Consumers get a better tasting tomatoFailedVirus resistant tomatoResistant to pestsDecreases the amount of pesticide applied to cropsTransgenic tomato plants show resistance (left) while non-transformed plants are susceptible to cucumber mosaic virus under field conditions (right
61、)http:/www.apsnet.org/online/feature/BioTechnology/Images/2.4.jpgCurrent ProductsCornBt CornThe Monarch Butterfly debate:A microbial gene added results in the crop being resistant to insectsDoes it impact the Monarch Butterfly?Well planned experiments are critical to the survival of biotechnologyWil
62、d-type corn showing infestation - Bt corn is resistant to thisCurrent ProductsCottonYes clothes can be made from transgenic crops!Bollgard cotton Insect resistanceLowers pesticide usageInsect infestation on Bt (right) and non-Bt (left) cotton bollshttp:/www.colostate.edu/programs/lifesciences/Transg
63、enicCrops/images/cotton.jpgCurrent ProductsSquashVirus resistantReduces crop loss due to infestation and decreases pesticide usehttp:/www.apsnet.org/online/feature/BioTechnology/Images/2.3.jpgTransgenic Freedom II squash showing resistance to both zucchini yellow mosaic virus and watermelon mosaic v
64、irus 2 (right) compared to non-transformed plants that are susceptible (left)Current ProductsPapayaVirus resistantRestored the papaya industry in HawaiiReduced crop lossJapan blocked imports of transgenic papayaCurrent ProductsGolden RiceBiotechnologys poster child?A true value added foodVitamin A e
65、nriched rice prevents disease and blindnessGolden in colourGolden rice and normal (white) wsjbiotech.htmlCan GMOs have health benefits?Some plants have been altered to increase the nutritional valuePharmaceuticals, Plantibodies, and Edible VaccinesResearch has looked at transgenic plants as producti
66、on vehicles for anti-cancer antibodiesEdible vaccines are closer than we thinkEdible VaccinesPlants producing vaccines could eliminate or simplify vaccine distribution problems in developing nationsHow a transgenic plant containing a vaccine is madewww.niaid.nih.gov/final/ infds/la_infds.htmEdible V
67、accinesMay have advantages over injected vaccinesPlants being studied include potato, banana, papaya, tomato, lettuce, carrot, rice, wheat, corn and soybean Quite a salad!Potatoes are one of many plants being used to produce vaccineshttp:/ VaccinesTomato and potato plant can make antigens from Hepat
68、itis B, E. Coli and V. choleraeFeeding to test animals induces an immune responsePotatoes fed to human volunteers induced and immune response to an inactive form of the E. coli toxinWhats next?How will all of these products be regulated?Who decides if they are safe?How do I know if I am eating food
69、derived from a GMO?Other Cool Plant Biotech ProductsBlue Carnations and RosesNature can not make theseNon-allergenic peanutsKids can take peanut butter sandwiches to school again!Decaffeinated coffeeLess processingWhats next?How will all of these products be regulated?Who decides if they are safe?Ho
70、w do I know if I am eating food derived from a GMO?Whoa! Who is Keeping us Safe?CanadaHealth Canada Human SafetyStringent evaluation of safety of GM foodsHow was the product made?Is the GM product different from the non-modified product (Composition and Nutritional value)?Is there potential for toxi
71、city of allergic reactions?SafetyCanadian Food Inspection Agency (CFIA)Regulation of the non-human effects Stringent Safety testingEnvironmental impact field trialsImpact on Livestock feeding trialsRegistration of all crop products under the Seeds ActFood SafetyGM products must meet ALL standardsAdd
72、itional testing can be requested at any time before or after approvalWhat about allergic reactions?All new food products (GM and non-GM) must undergo strict testingTesting includes:Potential for an allergic responseComparison of the food versus all known allergensWhat about allergic reactions?Many p
73、eople have food allergiesThe brazil nut example:A transgenic soybean with an inserted gene from Brazil nuts was createdIt was later found that this gene encoded a major allergenAs a result of this assessment, commercial interest in this transgenic soybean variety was abandonedSteve L. Taylor Departm
74、ent of Food Science & Technology, University of Nebraska, Lincoln, NE 68583-0919, USAhttp:/ Are GM Foods Labeled?Policy is controlled by Health Canada and the CFIACurrently approximately 70% of foodstuffs in North America contain components derived from GM cropsVoluntary labellingLabeled can of toma
75、toes in BritainSource: Nature, April 22, 1999Public AcceptanceWhat is it? What do I get out of it? Is it safe? Do I have choice? Is it natural? Public AcceptanceWhat is it?Producers and consumers must come together to educate each other on what the technology is and also what consumers wantProducers
76、 have not done a good jobConsumers must decide!Public AcceptanceWhat do I get?Much of the current products directly benefit farmersWhat would the reaction be if a non-allergenic peanut or edible vaccine was produced?Perhaps resources should be devoted to “useful” productsPublic AcceptanceIs it safe?
77、Safety testing is stringent, but short term onlyResults made public?LabelingDo I have a choice?Labeling products would give consumers the choice to use or not to usePublic AcceptanceIs it natural?Genetic modification of plants is a natural process Recall the Agrobacterium exampleSummaryTransgenic pl
78、ants have potential to impact many areas, including our food supply and our healthcare systemThere is no doubt that the technology worksIt is essential that proper testing of all products be carried out prior to commercializationLabeling products would allow consumers to choose what they want to consumeEducate yourself!Cre/loxPFlp/FrtCrefrtflpfrtloxPloxPM1M2Target 1Target 2Hybrid Target 1
