Презентация на тему "Молекулярные основы наследственностиГенетическая инженерия"

Презентация: Молекулярные основы наследственностиГенетическая инженерия
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  • Презентация: Молекулярные основы наследственностиГенетическая инженерия
    Слайд 1

    Молекулярные основы наследственностиГенетическая инженерия

    Лекция по курсу «Генетика» Автор – заведующий кафедрой генетики РГАУ-МСХА имени К.А.Тимирязева А.А. Соловьёв

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    Немного истории…… ‘83 ‘84 ‘85 ‘86 ‘87 ‘88 ‘89 ‘90 ‘91 ‘92 ‘93 ‘94 ‘95 ‘96 ‘97 ‘98 ‘99 ‘00 ‘01 ‘02 1-ое трансгенное растение Томаты с задержкой созревания Полевые испытания 6/92 Сорта, устойчивые к гербицидам и насекомым ГМ-кукуруза в Европе Bt –кукуруза 6/90 Томата устойчивый к бактериям

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    Трансформация Подготовка ткани для трансформации Введение ДНК Культура тканей in vitro Develop shoots Root the shoots Полевые испытания Leaf, germinating seed, immature embryos Tissue must be capable of developing into normal plants Agrobacterium or gene gun Multiple sites, multiple years

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    Лабораторный этап

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    Трансген Состоит из: 1. Целевой ген Кодирующий участок и регуляторные элементы 2. Селективный маркер Для выявления трансгенных растений 3. Последовательности для вставки Участки Т-ДНК Agrobacterium

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    Клонирование генов

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    CODING SEQUENCE INTRON poly A signal Промотор Создание трансгена Plant Transgene bacterial genes antibiotic marker replication origin Plant Selectable Marker Gene Plasmid DNA Construct Регулятор Кодирующий участок Стоп-сигнал

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    Векторные Прямые Методы трансформации Agrobacterium Генная пушка

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    The Helium Gas Gun – Circa 2000

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    Микроинъекция

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    Электропорация

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    Отбор трансгенных клеток и растений At best only 1 in 1000 cells integrate delivered DNA Transformed cells (events) are marked by co-introducing gene that provides resistance to selective agents Transformed cells are selected by killing non-transformed cells with selective agent. Three main types of selective agents: antibiotics herbicides plant growth regulators Selectable markers assist in following inheritance of transgenes. tissue culture cells under selection Herbicide Leaf Paint Assay transgenic non-transgenic resistant susceptible

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    Полевые испытания Нетрансгенные Трансгенные Устойчивость к гербицидам

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    Полевые испытания Устойчивость к RoundUp

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    GMOs = Genetically Modified Organisms Broadly defined: any microbe, plant, or animal developed through breeding and selection Narrowly defined: organisms produced by gene transfer techniques Устойчивость к насекомым хлопчатник картофель кукуруза Устойчивость к гербицидам соя кукуруза рапс другие Некоторые примеры ГМО ГМО-культуры Кукуруза, соя, рапс с улучшенными питательными качествами Культуры с запасными сахарами и маслами для промышленности Высоковитаминный “Golden Rice” Растительные вакцины Повышение урожая и устойчивости к разным стрессам

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    Повышение урожая Crops can be modified to optimize growth conditions: improve nitrogen assimilation, increase oxygen absorption, efficient photosynthetic pathway, and increase starch biosynthesis. Transgenic plant modified to have increase yield Unmodified control plant Устойчивость к насекомым Corn hybrid with a Bt gene Corn hybrid susceptible to European corn borer Various insect resistant crops have been produced. Most of these make use of the Cry gene in the bacteria Bacillus thuringiensis (Bt); this gene directs the production of a protein that causes paralysis and death to many insects.

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    Normal rice “Golden” rice «Золотой» рис Transgenic technology produced a type of rice that accumulates beta-carotene in rice grains. Once inside the body, beta-carotene is converted to vitamin A. More than 120 million children in the world suffers from vitamin A deficiency. Golden Rice has the potential to help prevent the 1 to 2 million deaths each year caused by a deficiency in this vitamin.

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    Papaya infected with the papaya ringspot virus Virus resistance gene introduced Устойчивость к вирусам The Freedom II squash has a modified coat protein that confer resistance to zucchini yellows mosaic virus and watermelon mosaic virus II. Scientists are now trying to develop crops with as many as five virus resistance genes

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    Pharmaceutical Production in Plants Genetically modified plants have been used as “bioreactors” to produce therapeutic proteins for more than a decade. A recent contribution by transgenic plants is the generation of edible vaccines. Edible vaccines are vaccines produced in plants that can be administered directly through the ingestion of plant materials containing the vaccine. Eating the plant would then confer immunity against diseases. Edible vaccines produced by transgenic plants are attractive for many reasons. The cost associated with the production of the vaccine is low, especially since the vaccine can be ingested directly, and vaccine production can be rapidly up scaled should the need arises. Edible vaccine is likely to reach more individuals in developing countries. The first human clinical trial took place in 1997. Vaccine against the toxin from the bacteria E.coli was produced in potato. Ingestion of this transgenic potato resulted in satisfactory vaccinations and no adverse effects.

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    ГМ-химозин для производства сыров Коровий соматотропин для увеличенияпродуктивности молока Source: Rent Mother Nature Source: Chr. Hansen

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    Фармацевтика

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    Применение для окружающей среды Биоремедиация Индикаторные бактерии – contamination can be detected in the environment

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    Кариес – ГМ-Streptococcus Ближайшие перспективы Вакцины – герпес, гепатит С, СПИД, малярия

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    Съедобные вакцины Transgenic Plants Serving Human Health Needs Приятные вакцины Трансгенные растения с геном патогенного белка Картофель, банан и томат Человек синтезирует антитела против патогенного белка Иммунизация против патогена

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    Создание ГМ-сортов Классическая селекция (6- 8 поколений) Транс-генная линия Сорт x x x Коммерческая трансгенная линия Генная инженерия

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    GMOs: Why the Controversy? Genetic engineering is a powerful new technology that is in general poorly understood and whose long term effects are unknown. GMOs are an innovation that have and will continue to impact all facets of the global agricultural economy. Production Consumer Products Processing Commodity Handling

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    Increased Corporate Control of Agriculture The Development of GM Crops is Expensive Intellectual property and patent protection Consolidation/vertical integration increases ability to capture profits Ag-biotech is a recent example of a century-old trend

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    Fig. 5.40 (from 5.7) Not necessarily 3:1

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    Arteries from a mouse with a KO of the low-density lipoprotein receptor-related protein (LRP). Mouse is actually a double-KO, with the LDL receptor also knocked out. This was generated by crossing the LRP-KO mouse with a LDL receptor-KO mouse. The LDL receptor-KO makes mice particularly susceptible to cholesterol feeding. Science 300, 329 (2003)

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    Has genetic engineering of foods been done?

    A FlavrSavr tomato, engineered to be more appealing. Peter Beyer demonstrating golden rice, engineered to be more nutritious.

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    9-13-2002 Purdue 38 Parts of a transgene The promoter - Usually taken from a corn gene Determines where and when the transgene will function The gene can be Tissue Specific or Constitutive The coding sequence - Determines what the gene does Must be modified to resemble a corn gene Transformation

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    “Gene Gun”

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    To knock-out a gene: Insert neo gene into the target gene. Transform KO plasmid into embryonic stem cells. Perform double-selection to get cells with the homologous integration (neo & gangcyclovir resistant). Inject cells with the knocked-out gene into a blastocyst. 1. KO KO 2,3.

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    (mouse) With DNA How to make a transgenic mouse

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    Chimeric mouse

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    Repairing an organellar gene: ~ 1 x 107 cells of a mutant of Chlamydomonas that had a deletion in the atpB gene for photosynthesis was bombarded with the intact atpB gene. Then, the cells were transferred to minimal medium so that only photosynthetically competent cells could grow. Control plate – cells were shot with tungsten particles without DNA

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    Agrobacterium tumefaciens, a natural plant genetic engineer

    Soil bacterium, related to Rhizobium causes crown galls (tumors) on many dicots Infection occurs at wound sites Infected Tobacco w/teratoma Brief recitation in Weaver, pp. 85-89

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    Methods Used in Plant Transgenesis

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    Agriculture Transgenics On the Market Source: USDA Insect resistant cotton – Bt toxin kills the cotton boll worm transgene = Bt protein Insect resistant corn – Bt toxin kills the European corn borer transgene = Bt protein Normal Transgenic

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    Virus resistance - papya resistant to papaya ringspot virus transgene = virus coat protein Source: Monsanto Herbicide resistant crops Now: soybean, corn, canola Coming: sugarbeet, lettuce, strawberry alfalfa, potato, wheat (2008?) transgene = modified EPSP synthase or phosphinothricin-N-acetyltransferase

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    Next Generation of Ag Biotech Products Source: Minnesota Microscopy Society Golden Rice – increased Vitamin A content Sunflower – white mold resistance transgene = oxalate oxidase from wheat

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    Turfgrass – herbicide resistance; slower growing (= reduced mowing) Bio Steel – spider silk expressed in goats; used to make soft-body bullet proof vests (Nexia)

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    Edible Vaccines Transgenic Plants Serving Human Health Needs Works like any vaccine A transgenic plant with a pathogen protein gene is developed Potato, banana, and tomato are targets Humans eat the plant The body produces antibodies against pathogen protein Humans are “immunized” against the pathogen Examples: Diarrhea Hepatitis B Measles

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    Crown galls caused by A. tumefaciens on nightshade.

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    complex bacterium – genome has been sequenced; 4 chromosomes with ~ 5500 genes Lots of pili

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    auxA auxB cyt ocs LB RB LB, RB – left and right borders (direct repeat) auxA + auxB – enzymes that produce auxin cyt – enzyme that produces cytokinin Ocs – octopine synthase, produces octopine T-DNA

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    Vir genes functions (cont.)

    virA - transports AS into bacterium, activates virG post-translationally virG - promotes transcription of other vir genes virD2- endonuclease that cuts T-DNA at the borders but only on one strand; attaches to the 5' end of the SS virE2- DNA-binding protein, binds SS of T- DNA virD2 & virE2 also help T-DNA get to nucleus in plant cell, they have NLSs virB - 11 ORFs, helps DNA-protein complex get through cell membranes

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    From Covey & Grierson

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    Hypothetical model for virB membrane channel From P. Zambryski

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    Binary vector system

    Strategy: Move T-DNA onto a separate, small plasmid Remove aux and cyt genes Insert selectable marker (drug resistance) gene in T-DNA Vir genes are retained on a separate plasmid

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    Risk management of transgenic plant

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    Binary vector system (cont.)

    5. Put foreign gene between T-DNA borders 6. Co-transform Agrobacterium with both plasmids 7. Infect plant with the transformed bacteria Leaf-disc transformationcommon; after selection and regeneration, get plants with the introduced gene in every cell - “Transgenic plant”

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    Binary vector system for Agrobac-terium

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    Making a transgenic plant by leaf-disc transformation with Agro.

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    Floral dip method

    Female part gets transformed & seeds are heterozygous. You can select homozygous in the later generations.

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    GFP marker for field trials

    RB LAT52 pro Recombinase NOS ter RS Bar NOS ter LB RS NOS pro GFP LAT59 pro 35S ter RB LAT52 pro NOS ter RS Bar NOS ter LB RS NOS pro GFP LAT59 pro 35S ter Cre recombinase with loxP recognition sites ParA recombinase with MRS recognition sites CinH recombinase with RS2 recognition sites Cre recombinase with fused loxP-FRT recognition sites No recombinase with loxP recognition sites

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    GM-gene-deletor system(Luo et al. 2007 Plant Biotechnol J 5:263)

    Cre-loxP/FRT vector No recombinase vector

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    Important Plant Improvement Methods Breeding Crossing two individuals from the same species; produces a new, improved variety; not a biotechnology procedure Transformation Adding a gene from another species; the essential biotechnology procedure to produce transgenics Source: USDA Source: USDA

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    Wheat Rye Triticale X Interspecific Cross New species, but NOT biotechnology products

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    ATTCGA ATTGGA Susceptible Normal Gene Resistant Mutant Gene Mutagenesis Treatment Mutagenesis Changes the DNA Sequence

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    The Golden Rice Solution IPP Geranylgeranyl diphosphate Phytoene Lycopene  -carotene (vitamin A precursor) Phytoene synthase Phytoene desaturase Lycopene-beta-cyclase ξ-carotene desaturase Daffodil gene Single bacterial gene; performs both functions Daffodil gene -Carotene Pathway Genes Added Vitamin A Pathway is complete and functional Golden Rice

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    Transgenic Example: Bt

    ( 71 Bt genes originate from the soil bacteria, Bacillus thuringiensis Researchers can identify and isolate the genes. “Blasted” into corn DNA using gold particle gene guns Bt genes that successfully incorporate into corn DNA express the insecticidal Bt protein in corn plant grown by farmer.

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    Bacillus thuringiensis (Bt)

    Bacillus thuringiensis is a gm+ soil bacterium that produces an insecticidal crystal protein from the cry genes (over 100 genes in different strains) Bt crystal proteins are toxic to insects such as the ECB (European Corn Borer) and other related pests. When ingested, the Bt crystal toxin is activated by enzymes in the insect's gut. The activated toxin attaches to specific gut receptors, destroys cells in the gut wall, puncturing the gut and poisoning the insect.

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    Solution: "In Plant Protection"-

    Isolate the cry genes from bacteria, and introduce into corn plants = transgenic Bt-corn, followed by Bt-cotton, etc. These GM crops make the cryproteins in each and every cell of the plant. Reduces or eliminates the traditional spraying of Bt in the field.

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    Introducing the Gene or Developing Transgenics Steps 1. Create transformation cassette 2. Introduce and select for transformants

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    Transformation Cassettes Contains 1. Gene of interest The coding region and its controlling elements 2. Selectable marker Distinguishes transformed/untransformed plants 3. Insertion sequences Aids Agrobacterium insertion

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    Transformation Steps Prepare tissue for transformation Introduce DNA Culture plant tissue Develop shoots Root the shoots Field 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

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    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 Tissue culture required to generate transgenic plants

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    The Lab Steps

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    The Next Test Is The Field Non-transgenics Transgenics Herbicide Resistance

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    Final Test of the Transgenic Consumer Acceptance RoundUp Ready Corn Before After

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    6.4 Health and Environmental Concerns

    Human Health Opponents fear the effects of foreign genes, bits of DNA not naturally found in plants Allergic reactions Antibiotic-resistance marker genes could spread to disease-causing bacteria in humans Cause cancer To date, science has not supported any of these concerns

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    Environmental Concerns Genes for pest or herbicide resistance could spread to weeds Few experts predict this will happen; further studies are needed Regulation FDA regulates foods on the market USDA oversees growing practices EPA controls use of Bt proteins and other pesticides

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    BT corn Non- BT corn R S S S S S Resistant (R) and susceptible (S) European corn borer adults

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    Bioenergy and plant genomics:Expanding the nation’s renewable energy resources

    Whole Genome Microarrays Yesterday Today Tomorrow Accelerated Domestication Metabolic Profiling Carbon allocation Conventional Forestry Short rotation hardwoods High yield wood crops Brian Davison ORNL

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