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1、Plant genetic engineeringZichaoMao 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 functio
2、n 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 amoun
3、ts 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 from the
4、 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 plasmidSome co
5、nsiderations: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 gene-Carotene Pa
6、thway 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 diphosphatePhytoeneL
7、ycopene -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 functionalGoldenRiceExamples of applications
8、 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 deficiency causes blindness
9、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)-caroteneVitamin A“Golden Rice”Inse
10、ct 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 in orders Lepidoptera,Coleoptera(鞘翅类)
11、(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 at high pH in insect gut.3.Toxin causes the gut
12、 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 Bt-transgenic plants are resistant to insects,no nee
13、d 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-transgenic corn on Monarch butterfly larvae(君王斑点蝶)feeding on milkwe
14、ed(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 amphibians.Parts of Bt corn,such as leaves,cobs and pollen,can travel as far as
15、 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 EPSP synthaseAromaticamino acidsWithout amino acids,plant diesX GM crops curre
16、ntly 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 use feed worlds growing population62%(herbicide resistant)Percent increase of GM cro
17、ps grown worldwideHerbicide resistanceimidazolinonesMutant AHAS enzymedeveloped by mutagenesisCrops Canola Corn Rice Sunflower WheatA Major Marketing Advantagebut lost when stacked with a transgeneThe Roundup Ready Story Glyphosate is a broad-spectrum herbicide Active ingredient in Roundup herbicide
18、 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 sprayingBacterialEPSP synthaseShikimic acid+Phosphoenol pyruvate3-enolpyruvyl shikimic acid-5
19、-phosphate(EPSP)Aromaticamino acidsRoundup Resistant Plants+GlyphosateWith amino acids,plant livesRoundUp has no effect;enzyme is resistant to herbicideIntroducing the GeneorDeveloping TransgenicsSteps1.Create transformation cassette2.Introduce and select for transformantsTransformation CassettesCon
20、tains1.Gene of interest The coding region and its controlling elements2.Selectable marker Distinguishes transformed/untransformed plants3.Insertion sequences Aids Agrobacterium insertionTransformation StepsPrepare tissue for transformationIntroduce DNACulture plant tissue Develop shoots Root the sho
21、otsField 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 are developed in the lab They are then introduced into a plant Two major delivery methodsDelivering
22、 the Geneto the Plant Agrobacterium Gene GunTissue culturerequired to generatetransgenic plantsThe Lab StepsThe Next Test Is The FieldNon-transgenicsTransgenicsHerbicide ResistanceFinal Test of the TransgenicConsumer AcceptanceRoundUp Ready CornBeforeAfterPlant Genetic TransformationAll stable trans
23、formation 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-mediated TransformationBiology of the Agrobacterium-plant interaction The only known natural example
24、 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 shrubs,herbaceous plants,dicots.Galls are spherical wart-like structures similar to tumors.The genus Agro
25、bacterium 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 strain-specific host range is unknown.Many monocot plants can be transformed(now),although they do not form c
26、rown 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 element is defined by its borders but not the sequences within.So researchers can substitute the T-DNA coding region
27、 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 transcriptional activation of vir genesConjugal DNA metabolismIntercellular transportNuclear importT-DNA integrationT-
28、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 sugar phosphate derivatives called opines(ocs,octopine(章鱼碱);mas,mannopine(甘露碱甘露碱);and ags,agropine).Agrobacteria are usual
29、ly 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,cytokinin)Opines(octopine,nopaline胭脂氨酸)Agrobacterium-host cell recognition is a two-step process1.Loosely bound step:acetylated
30、polysaccharides are synthesized.2.Strong binding step:bound bacteria synthesize cellulose filaments to stabilize the initial binding,resulting in a tight association between Agrobacterium and the host cell.chvA export of beta-1,2-glucan chvB b1-2 glucan sybthesispscA the mutants that defined the loc
31、us were initially isolated as having an altered polysaccharide composition;they were nonfluorescent on media containing Leucophor or Calcofluor,indicating a defect in the production of cellulose fibrils.Receptors are involved in initial bindingPlant vitronectin(玻连蛋白)-like protein(PVN,55kDa)was found
32、 on the surface of plant cell.This protein is probably involved in initial bacteria/plant cell binding.PVN is only immunologically related to animal vitronectin.Animal vitronectin is an important component of the extracellular matrix and is also an receptor for several bacterial strains.Receptors ar
33、e involved in initial bindingAside from PVN,rhicadhesin-binding protein was found in pea roots.Also,rat1(arabinogalactan protein;AGP)and rat2(potential cell-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(li
34、gnin,flavonoid precursors)serving as chemical attractants to agrobacteria and stimulants for virgene expression.Among these phenolic compounds,acetosyringone(AS)is the most effective.Plant signalsSugars like glucose and galactose also stimulate vir gene expression when AS is limited or absent.These
35、sugars are probably acting through the chvE gene to activate vir genes.Low opine levels further enhance vir gene expression in the presence of AS.Plant signalsThese compounds stimulate the autophosphorylation of a transmembranereceptor kinase VirA at its His-474.It in turn transfers its phosphate gr
36、oup 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,virB,virC,virD,virEand virG operons,upregulating transcription.Sugars interact with ChvE(glucose/galactose binding protein)which interacts with VirA through it
37、s periplasmic domain.Structure of the T-DNAThe existence and orientation of right border is absolutely required for Agrobacterium pathogenicity 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 delimi
38、t the transferred segments,the T-DNA content itself has no effect on the efficiency of transfer.Therefore,researchers replace most 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
39、T-strand.VirD1 and VirD2 are involved in the initial T-strand processing,acting as site-and strand-specific endonucleases.Production 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 pla
40、smid at the left border nick by its tyrosine 29.Production of T-strandVirC1 enhances T-strand production by binding to overdrive.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.Formati
41、on of the T-complexThe T-complex is composed of at least three components:one T-strand DNA molecule,one VirD2 protein,and around 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 V
42、irE2 associates with the T-strand after intercellular transport,VirE1 is probably involved in preventing VirE2-T-strand binding.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 intercell
43、ular transport.Intercellular transportTransport of the T-complex into the host cell most likely occurs through a type 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 tra
44、nsportIntercellular transport of T-DNA is probably energy dependent,requiring ATPase activities from VirB4 and VirB11.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.Nucle
45、ar ImportBecause the large size of T-complex(50,000 kD,13nm in diameter),the nuclear import of T-complex requires 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
46、 imported into the cell nucleus by a mechanism conserved between animal,yeast and plant cells(bipartite consensus 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 coo
47、perate with cellular factors to mediate T-complex nuclear import and integration into the host genome.These 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
48、no obvious site preference for integration throughout the genome.About 40%of the integrations are in 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 i
49、n AgrobacteriumT-DNA integrationIntegration is initiated by the 3(LB)of the T-DNA invading a poly 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
50、 of the duplex is degraded.Events of NHEJ in AgrobacteriumT-DNA integrationA nick in the upper host 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 th