《植物基因工程》PPT课件.ppt

Plant genetic engineering,Zichao Mao ,Yunnan Agricultural University,Motivation What 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 function 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 amounts of pharmaceuticals,Genetic engineering in plants,Agrobacterium tumefaciens causes crown gall disease,VIRULENCE (VIR) genes,Ti plasmid Crown gall disease, cell proliferation of plant tissue (like cancer),Crown gall disease caused by Agrobacterium,Agrobacterium usually disappears from the galls after growth of the gall begins, as bacterium itself is not necessary for tumor development.,Gall formed at the base of the stem of a rose bush.,B. A series of galls (arrowheads) along a branch of a grapevine.,Crown gall disease caused by Agrobacterium,Genetic engineering of the Ti plasmid,Some considerations: Ti plasmid is 200 kb - too big for easy cloning need to remove tumor-inducing genes from T-DNA,VIR genes,Disarmed Ti plasmid,Disarm,Agrobacterium-mediated plant transformation,Punch out leaf discs,Agrobacterium culture carrying foreign gene,-Carotene Pathway Problem in Plants,The Golden Rice Solution,Daffodil(黄水仙花 ) gene,Single bacterial gene; performs both functions,Daffodil gene,-Carotene Pathway Genes Added,Examples 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 deficiency 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 enzymes,Geranylgeranyl diphosphate (in immature rice endosperm),-carotene,Vitamin A,“Golden Rice”,Insect resistant plants,Corn borer pest of corn in North America and Europe Lepidoptera (鳞翅类 )(moths and butterflies) Bt-corn for corn borer control,What 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 )(beetles) - Bt toxin in form of powder used as insecticide spray applied to leaves where larvae feed on,2. Toxin binds to specific receptors in gut and insects stops eating.,Mode of action: Insect eats Bt crystals () and bacterial spores. Bt crystals dissolve at 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,Bt-transgenic plants are resistant to insects, no need for spraying insecticide,Bacillus thuringiensis,Bt-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 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 ecosystems,Bt 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 2000 meters away from source areas-a phenomenon that was not considered when Bt corn was licensed.,+ Glyphosate,X,Roundup Sensitive Plants,X,X,Without amino acids, plant dies,X,GM crops currently grown worldwide,Genetic 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 population,62% (herbicide resistant),Percent increase of GM crops grown worldwide,Herbicide resistance imidazolinones Mutant AHAS enzyme developed by mutagenesis Crops Canola Corn Rice Sunflower Wheat A Major Marketing Advantage but lost when stacked with a transgene,The Roundup Ready Story,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 spraying,Bacterial EPSP synthase,Shikimic acid + Phosphoenol pyruvate,3-enolpyruvyl shikimic acid-5-phosphate (EPSP),Aromatic amino acids,Roundup Resistant Plants,+ Glyphosate,With amino acids, plant lives,RoundUp has no effect; enzyme is resistant to herbicide,Introducing the Gene or Developing Transgenics,Steps,1. Create transformation cassette,2. Introduce and select for transformants,Transformation Cassettes,Contains,Transformation Steps,Prepare tissue for transformation,Introduce DNA,Culture plant tissue,Field test the 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 methods,Delivering the Gene to the Plant,Agrobacterium,Gene Gun,The Lab Steps,The Next Test Is The Field,Herbicide Resistance,Final Test of the Transgenic Consumer Acceptance,RoundUp Ready Corn,Plant Genetic Transformation,All 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-mediated Transformation,Biology 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 shrubs, 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 strain-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 element 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 interaction,Cell-cell recognition Signal transduction and transcriptional activation of vir genes Conjugal DNA metabolism Intercellular transport Nuclear import T-DNA integration,T-DNA,T-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 ); and ags, agropine).Agrobacteria are usually classified based on the type of opines specified by the bacterial T-DNA.,Ti Plasmid,Agrobacterium-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 process,1.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 between Agrobacterium and the host cell.,chvA export of beta-1,2-glucan chvB b1-2 glucan sybthesis pscA 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 in the production of cellulose fibrils.,Receptors are involved in initial binding,Plant 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 vitronectin. Animal vitronectin is an important component of the extracellular matrix and is also an receptor for several bacterial strains.,Receptors are involved in initial binding,Aside from PVN, rhicadhesin-binding protein was found in pea roots. Also, rat1(arabinogalactan protein; AGP) and rat2(potential cell-wall protein) are involved.,Plant signals,Wounded 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, acetosyringone (AS) is the most effective.,Plant signals,Sugars 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 AS.,Plant signals,These 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 signals,VirG 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 its periplasmic domain.,Structure of the T-DNA,The 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-DNA,Although 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 of the T-DNA with DNA of interest, making Agrobacterium a vector for genetic transformation of plants.,Production of T-strand,Every 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.,Production of T-strand,After 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-strand,VirC1 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.,Formation of the T-complex,The 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-complex,Whether VirE2 associates with T-strand before or after the intercellular transport is not clear.,Formation of the T-complex,If VirE2 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 intercellular transport.,Intercellular transport,Transport 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 transport,Intercellular 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.,Nuclear Import,Because 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 Import,VirD2 is 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 Import,In host plant cells VirD2 and VirE2 likely cooperate 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-specific,Flanking sequence tags (FSTs) analysis showed 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 integration,The mechanism of NHEJ makes deletions after T-DNA integration a common phenomenon.,Events of NHEJ in AgrobacteriumT-DNA integration,Integration is initiated by the 3(LB) of the T-DNA invading a poly T-rich site of the host DNA A 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 integration,A 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 the T-DNA is ligated to the bottom strand of the host DNA. This pairing frequently involves a G and another nucleotide upstream of it.,Plant genes involved in transformation,Mutant 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 Expression Vector Mono Vector System Two component Vector system,Genes = the coding system for instructions A gene = is a segment of DNA,Guanine (G),Cytosine (C),Adenine (A),Thymine (T),bases,DNA,DNA and Genes,gene,Genes and Proteins,Restriction enzymes,As biological scissors,“Gene Gun” Technique,Gene gun,Electroporation Technique,Power supply,Plant Expression Vector Mono Vector System Two component Vector system,Genes = the coding system for instructions A gene = is a segment of DNA,Guanine (G),Cytosine (C),Adenine (A),Thymine (T),bases,DNA,DNA and Genes,gene,Restriction enzymes,As biological scissors,Next Generation of Transgenic Crops,Plant-based vaccines Enhanced nutritional content Functional foods and phytoceuticals Transgenic plants for phytoremediation 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 crops,Why Should I Support the Biotech Industry?,By genetically modifying plants we can: Reduce pesticide use Conserve fuel and water Preserve non-pest insect populations Increase food production per hectare of farmland Enable crops to grow in sub-optimal conditions Increase the nutritional value of food,Current Products,Transgenic Soybean Roundup Ready Resistant to Roundup Herbicide Reduces the amount of herbicide applied to crops Altered Fatty-acid content Changes the nutritional value,Field after one round of application of Roundup herbicide,Current Products,Canola Herbicide resistant Better for the environment Altered fatty-acid composition A value-added food,/ens/pics11/ canolaprods.jpg,Current Products,Tomato Flavr Savr Tomato Delayed softening Consumers get a better tasting tomato Failed Virus resistant tomato Resistant to pests Decreases the amount of pesticide applied to crops,Transgenic tomato plants show resistance (left) while non-transformed plants are susceptible to cucumber mosaic virus under field conditions (right) /online/feature/BioTechnology/Images/2.4.jpg,Current Products,Corn Bt Corn The Monarch Butterfly debate: A microbial gene added results in the crop being resistant to insects Does it impact the Monarch Butterfly? Well planned experiments are critical to the survival of biotechnology,Wild-type corn showing infestation - Bt corn is resistant to this,Current Products,Cotton Yes clothes can be made from transgenic crops! Bollgard cotton Insect resistance Lowers pesticide usage,Insect infestation on Bt (right) and non-Bt (left) cotton bolls /programs/lifesciences/TransgenicCrops/images/cotton.jpg,Current Products,Squash Virus resistant Reduces crop loss due to infestation and decreases pesticide use,/online/feature/BioTechnology/Images/2.3.jpg Transgenic Freedom II squash showing resistance to both zucchini yellow mosaic virus and watermelon mosaic virus 2 (right) compared to non-transformed plants that are susceptible (left),Current Products,Papaya Virus resistant Restored the papaya industry in Hawaii Reduced crop loss Japan blocked