Молекулярные основы наследственности Генетическая инженерия Лекция по курсу

Молекулярные основы наследственности Генетическая инженерия Лекция по курсу «Генетика» Автор – заведующий кафедрой генетики РГАУМСХА имени К. А. Тимирязева А. А. Соловьёв

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

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

Лабораторный этап

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

Клонирование генов

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

Методы трансформации • Векторные • Прямые • Agrobacterium • Генная пушка

The Helium Gas Gun – Circa 2000

Микроинъекция

Электропорация

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

Полевые испытания Устойчивость к гербицидам Нетрансгенные Трансгенные

Полевые испытания Устойчивость к Round. Up

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

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 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. Corn hybrid with a Bt gene Corn hybrid susceptible to European corn borer

«Золотой» рис Transgenic technology produced a type of rice that accumulates betacarotene in rice grains. Once inside the body, beta-carotene is converted to vitamin A. Normal rice 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. “Golden” rice

Устойчивость к вирусам Papaya infected with the papaya ringspot virus Virus resistance gene introduced 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

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 vaccines producedof edible Edible vaccines are is the generation in plants vaccines. that can be administered directly through the ingestion of plant materials containing the vaccine. Eating the plant would then Edible vaccines produced by transgenic plants are confer immunity attractive for many reasons. The cost associated against diseases. 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.

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

Фармацевтика Продукт Использование Инсулин Диабет Интерферон Онкология Интерлейкин Онкология Гормон роста Карликовость Нейроактивные ферменты Болевые эффекты

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

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

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

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

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 Processing Commodity Handling Consumer Products

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

Not necessarily 3: 1 Fig. 5. 40 (from 5. 7)

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)

Has genetic engineering of foods been done? A Flavr. Savr tomato, engineered to be more appealing. Peter Beyer demonstrating golden rice, engineered to be more nutritious.

Transformation 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 9 -13 -2002 Purdue 38

“Gene Gun”

To knock-out a gene: 1. Insert neo gene into the target gene. 2. Transform KO plasmid into embryonic stem cells. 3. Perform doubleselection to get cells with the homologous integration (neo & gangcyclovir resistant). 4. Inject cells with the knocked-out gene into a blastocyst. 1. KO KO 2, 3.

How to make a transgenic mouse With DNA (mouse)

Chimeric mouse

Repairing an organellar gene: ~ 1 x 107 cells of a mutant of Chlamydomonas that had a deletion in the atp. B gene for photosynthesis was bombarded with the intact atp. B 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

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

Methods Used in Plant Transgenesis

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

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

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

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

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: üDiarrhea üHepatitis B üMeasles

Crown galls caused by A. tumefaciens on nightshade.

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

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

Vir genes functions (cont. ) n n vir. A - transports AS into bacterium, activates vir. G post-translationally vir. G - promotes transcription of other vir genes vir. D 2 - endonuclease that cuts T-DNA at the borders but only on one strand; attaches to the 5' end of the SS vir. E 2 - DNA-binding protein, binds SS of TDNA n n vir. D 2 & vir. E 2 also help T-DNA get to nucleus in plant cell, they have NLSs vir. B - 11 ORFs, helps DNA-protein complex get through cell membranes

From Covey & Grierson

Hypothetical model for vir. B membrane channel From P. Zambryski

Binary vector system Strategy: 1. Move T-DNA onto a separate, small plasmid 2. Remove aux and cyt genes 3. Insert selectable marker (drug resistance) gene in T-DNA 4. Vir genes are retained on a separate plasmid

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 transformation common; after selection and regeneration, get plants with the introduced gene in every cell - “Transgenic plant”

Binary vector system for Agrobacterium

Making a transgenic plant by leafdisc transformation with Agro.

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

GFP marker for field trials LB RB RS LAT 52 pro Recombinase NOS ter LAT 59 pro GFP 35 S ter NOS pro Bar NOS ter RS LB RB • Cre recombinase with lox. P recognition sites • Par. A recombinase with MRS recognition sites • Cin. H recombinase with RS 2 recognition sites • Cre recombinase with fused lox. P-FRT recognition sites • No recombinase with lox. P recognition sites

GM-gene-deletor system (Luo et al. 2007 Plant Biotechnol J 5: 263) No recombinase vector Cre-lox. P/FRT vector

Important Plant Improvement Methods Source: USDA • 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

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

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

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

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

Bacillus thuringiensis (Bt) n Bacillus thuringiensis a gm+ soil bacterium is that produces an insecticidal crystal protein from the cry genes (over 100 genes in different strains) n 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,

Solution: "In Plant Protection"n Isolate the crygenes from bacteria, and introduce into corn plants = transgenic Btcorn, followed by Bt-cotton, etc. n 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.

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

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

Transformation Steps Prepare tissue for transformation • Tissue must be capable of developing into normal plants • Leaf, germinating seed, immature embryos Introduce DNA • Agrobacterium or gene gun Culture plant tissue • Develop shoots • Root the shoots Field test the plants • Multiple sites, multiple years

Delivering the Gene to the Plant • Transformation cassettes are developed in the lab • They are then introduced into a plant • Two major delivery methods • Agrobacterium • Gene Gun Tissue culture required to generate transgenic plants

The Lab Steps

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

Final Test of the Transgenic Consumer Acceptance Round. Up Ready Corn Before After

6. 4 Health and Environmental Concerns n Human Health n Opponents fear the effects of foreign genes, bits of DNA not naturally found in plants n Allergic reactions n Antibiotic-resistance marker genes could spread to disease -causing bacteria in humans n Cause cancer n To date, science has not supported any of these concerns

6. 4 Health and Environmental Concerns n n n Genes for pest or herbicide resistance could spread to weeds Few experts predict this will happen; further studies are needed Regulation n FDA regulates foods on the market USDA oversees growing practices EPA controls use of Bt proteins and other pesticides

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

Bioenergy and plant genomics: Expanding the nation’s renewable energy resources Tomorrow Short rotation hardwoods Carbon allocation Today d n ate atio r ele stic c Ac ome D Conventional Forestry Yesterday Metabolic Profiling Whole Genome Microarrays Brian Davison ORNL High yield wood crops