1 Factors Affecting Rates of Respiration • Temperature- For every 10 degree C rise in temperature between 0-35 C the rate of respiration increases 2X – 4X. • Storage temperature for harvested plant parts is often critical because these parts continue to respire after harvest ( a catabolic process) which causes a build up of heat, and the breakdown of the product.
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1 Factors Affecting Rates of Respiration Temperature- For every 10 degree C rise in temperature between 0-35 C the rate of respiration increases 2X – 4X.
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1
Factors Affecting Rates of Respiration
• Temperature- For every 10 degree C rise in temperature between 0-35 C the rate of respiration increases 2X – 4X.
• Storage temperature for harvested plant parts is often critical because these parts continue to respire after harvest ( a catabolic process) which causes a build up of heat, and the breakdown of the product.
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Factors Affecting Rates of Respiration
• Most plants grow better when night time temperatures are 5 degrees C lower than day time temperatures.
• This is because lower night time respiration reduces the use of carbohydrates and allows more carbohydrates to be stored or used for growth.
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Factors Affecting Rates of Respiration
• Oxygen concentration- Generally speaking, lower oxygen level results in the reduction of respiration.
• Controlled atmosphere (CA) storage in which oxygen is decreased is useful in storage of fruits and vegetables because of lower respiration rates.
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Factors Affecting Rates of Respiration
• Soil conditions- Compacted and/or wet soil conditions result in low oxygen in the root zone and reduced root respiration.
• Consequently, roots don’t function well in supplying mineral nutrients essential for the activity of respiratory enzymes which decreases overall respiration.
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Factors Affecting Rates of Respiration
• Light- Lower light intensities result in lower respiration rates. – Lower photosynthesis rates in low light
supply fewer carbohydrates essential for respiration.
• Plant growth- As a plant grows it depends on energy to be supplied by respiration. – The more growth that is occurring, the higher
the respiration rate must be.
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Summary of Respiration
• Aerobic Respiration– Glycolysis– Transition Rx.– Kreb’s Cycle– Electron Transport Chain
• Anaerobic Respiration– Pyruvate
• Lactic Acid• Mixed Acids• Alcohol + CO2
– Recycle NADH– 2 ATP / Glucose
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Amino Acid Catabolism
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Amino AcidsAmino Acids
• Building blocks for polymers called proteins
• Contain an amino group, –NH2, and a carboxylic acid, –COOH
• Can form zwitterions: have both positively charged and negatively charged groups on same molecule
• 20 required for humans
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Peptide Bond
• Connect amino acids from carboxylic acid to amino group
• Produce amide linkage: -CONH-• Holds all proteins together• Indicate proteins by 3-letter abbreviation
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Sequence of Amino AcidsSequence of Amino Acids
• Amino acids need to be in correct order for protein to function correctly
• Similar to forming sentences out of words
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Catalyze the reversible transfer of an amino group between two -keto acids.
Aspartate donates its amino group, becoming the -keto acid oxaloacetate.
-Ketoglutarate accepts the amino group, becoming the amino acid glutamate.
Example of a Transaminase reaction:
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In another example, alanine becomes pyruvate as the amino group is transferred to -ketoglutarate.
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Essential amino acids must be consumed in the diet.
Mammalian cells lack enzymes to synthesize their carbon skeletons (-keto acids). These include:
Isoleucine, leucine, & valine
Lysine
Threonine
Tryptophan
Phenylalanine (Tyr can be made from Phe.)
Methionine (Cys can be made from Met.)
Histidine (Essential for infants.)
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Amino Acid MetabolismAmino Acid Metabolism
•Metabolism of the 20 common amino acids is considered from the origins and fates of their:
(1) Nitrogen atoms (2) Carbon skeletons
•For mammals: Essential amino acids must be obtained from dietNonessential amino acids - can be synthesized
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The Nitrogen Cycle and Nitrogen Fixation
• Nitrogen is needed for amino acids, nucleotides
• Atmospheric N2 is the ultimate source of biological nitrogen
• Nitrogen fixation: a few bacteria possess nitrogenase which can reduce N2 to ammonia
• Nitrogen is recycled in nature through the nitrogen cycle
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Fig 17.1 The Nitrogen cycleFig 17.1 The Nitrogen cycle
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NitrogenaseNitrogenase
• An enzyme present in Rhizobium bacteria that live in root nodules of leguminous plants
• Some free-living soil and aquatic bacteria also possess nitrogenase
• Nitrogenase reaction:
N2 + 8 H+ + 8 e- + 16 ATP
2 NH3 + H2 + 16 ADP + 16 Pi
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Assimilation of AmmoniaAssimilation of Ammonia
• Ammonia generated from N2 is assimilated into low molecular weight metabolites such as glutamate or glutamine
• At pH 7 ammonium ion predominates (NH4+)
• At enzyme reactive centers unprotonated NH3 is the nucleophilic reactive species
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A. Ammonia Is Incorporated into GlutamateA. Ammonia Is Incorporated into Glutamate
• Reductive amination of -ketoglutarate by glutamate dehydrogenase occurs in plants, animals and microorganisms
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Glutamine Is a Nitrogen Carrier in Many Glutamine Is a Nitrogen Carrier in Many Biosynthetic ReactionsBiosynthetic Reactions
• A second important route in assimilation of ammonia is via glutamine synthetase
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Glutamate synthase transfers a Glutamate synthase transfers a nitrogen to nitrogen to -ketoglutarate-ketoglutarate
Prokaryotes & plants
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Alternate amino acid production in prokaryotes
Especially used if [NH3] is low. Km of Gln synthetase lower than Km of Glu dehydrogenase.
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The First Step in Amino Acid Degradation is the Removal of Nitrogen
•Amino acids released from protein turnover can be resynthesized into proteins.•Excess amino acids are degraded into specific compounds that can be used in other metabolic pathways. •This process begins with the removal of the amino group, which can be converted to urea and excreted.•The -ketoids that remain are metabolized so that their carbon skeletons can enter glycolysis, gluconeogenesis, or the TCA cycle.
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The Biosynthesis of Amino Acids
•Amino acids are the building blocks of proteins and the nitrogen source of many other important molecules including nucleotides, neurotransmitters, and prosthetic groups such as porphyrins.
•Ammonia is the source of all nitrogen for all of the amino acids.
•The carbon backbones come from the glycolytic pathway, the pentose phosphate pathway, and/or the TCA cycle.
•Amino acid biosynthesis is feedback regulated to ensure that all amino acids are maintained in sufficient amounts for protein synthesis and other processes.
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Summary of Protein and Amino Acid Degradation
•Proteins are degraded to amino acids.
•Protein turnover is tightly regulated.
•The first step in amino acid degradation is the removal of nitrogen.
•Ammonium ion is converted into urea in most terrestrial vertebrates.
•Carbon atoms of degraded amino acids emerge as major metabolic intermediates.
•Inborn errors of metabolism can disrupt amino acid degradation.
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Summary of Amino Acid Biosynthesis
•Microorganisms use ATP and a powerful reductant to reduce atmospheric nitrogen to ammonia.
•Amino acids are made from intermediates of the TCA cycle and other major pathways.
•Amino acid metabolism is regulated by feedback inhibition.
•Amino acids are precursors of many molecules.
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Overview of Nucleotide Biosynthesis
•Nucleotides serve as active precursors of nucleic acids.
•ATP is the universal currency of energy.
•Nucleotide derivatives such as UDP-glucose participate in bioynthetic processes.
•Nucleotides are essential components of signal transduction pathways.
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Two Classes of Pathways for the Synthesis of Nucleotides.
•In the salvage pathway, a base is attached to a ribose, activated in the form of 5- phosphoribosyl-1-pyrophosphate (PRPP).
•In de novo synthesis, the base itself is synthesized from simpler starting materials, including amino acids.
•ATP hydrolysis is necessary for de novo synthesis.
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Summary of Nucleotide Biosynthesis
•In de novo synthesis, the pyrimidine ring is assembled from bicarbonate, aspartate, and glutamine.
•Purine bases can be synthesized de novo or recycled by salvage pathways.
•Deoxyribonucleotides are synthesized by the reduction of ribonucleotides.
•Key steps in nucleotide biosynthesis are feeback regulated.
•NAD+, FAD, and Coenzyme A are formed from ATP.
•Disruptions in nucleotide metabolism can cause pathological conditions.
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Proteins Proteins Proteins Proteins
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Structure of ProteinsStructure of Proteins
• Four organizational levels• Primary structure: amino acid sequence• Secondary structure: arrangement of chains
around an axis– Pleated sheet– Alpha helix: right-handed helix
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Pleated SheetsPleated Sheets
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Alpha HelixAlpha Helix
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Tertiary StructureTertiary Structure
• Spatial relationships of amino acids relatively far apart in protein chain
• Globular proteins: compact spherical shape
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Quaternary Structure
• Structure when two or more amino acid sequences are brought together
• Hemoglobin has four units arranged in a specific pattern
TransaminationTransamination: use the essential AA to : use the essential AA to synthesize the others!synthesize the others!
TransaminationTransamination: use the essential AA to : use the essential AA to synthesize the others!synthesize the others!
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Protein metabolism
Another route:Another route:
Intestinal bacteria -> ammonia (toxic) -> Intestinal bacteria -> ammonia (toxic) -> liver uses it to make amino acidsliver uses it to make amino acids
Another route:Another route:
Intestinal bacteria -> ammonia (toxic) -> Intestinal bacteria -> ammonia (toxic) -> liver uses it to make amino acidsliver uses it to make amino acids
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Protein metabolism
Amino acids: C, H, O plus amine group Amino acids: C, H, O plus amine group with Nwith N
Amino acids: C, H, O plus amine group Amino acids: C, H, O plus amine group with Nwith N
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Protein metabolismProtein metabolism
Amino acids are broken down into:Amino acids are broken down into:
a) ammonia -> ureaa) ammonia -> urea
b) pyruvate or molecules that are part of b) pyruvate or molecules that are part of the krebs cycle -> respired for energy, or the krebs cycle -> respired for energy, or converted to fats or glucoseconverted to fats or glucose
Amino acids are broken down into:Amino acids are broken down into:
a) ammonia -> ureaa) ammonia -> urea
b) pyruvate or molecules that are part of b) pyruvate or molecules that are part of the krebs cycle -> respired for energy, or the krebs cycle -> respired for energy, or converted to fats or glucoseconverted to fats or glucose
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Proteins are degraded into amino acids.
Protein turnover is tightly regulated.
First step in protein degradation is the removal of the nitrogen
Ammonium ion is converted to urea in most mammals.
Carbon atoms are converted to other major metabolic intermediates.
Inborn errors in metabolism
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• Amino acids used for synthesizing proteins are obtained by degrading other proteins
– Proteins destined for degradation are labeled with ubiquitin.
– Polyubiquinated proteins are degraded by proteosomes.
• Amino acids are also a source of nitrogen for other biomolecules.
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Excess amino acids cannot be stored.
Surplus amino acids are used for fuel.
Carbon skeleton is converted to
Acetyl–CoA
Acetoacetyl–CoA
Pyruvate
Citric acid cycle intermediate
The amino group nitrogen is converted to urea and excreted.
Glucose, fatty acids and ketone bodies can be formed from amino acids.
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proteins are a vital source of amino acids.
Discarded cellular proteins are another source of amino acids.
1. Protein Degradation1. Protein Degradation
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Biotechnology
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What Is Biotechnology?
• Using scientific methods with organisms to produce new products or new forms of organisms
• Any technique that uses living organisms or substances from those organisms to make or modify a product, to improve plants or animals, or to develop microorganisms for specific uses
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What Is Biotechnology?What Is Biotechnology?
• GMO- genetically modified organisms.• GEO- genetically enhanced organisms.• With both, the natural genetic material of the
organism has been altered.• Roots in bread making, wine brewing,
cheese and yogurt fermentation, and classical plant and animal breeding
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What Is Biotechnology?What Is Biotechnology?
• Manipulation of genes is called genetic engineering or recombinant DNA technology
• Genetic engineering involves taking one or more genes from a location in one organism and either– Transferring them to another organism– Putting them back into the original organism in
different combinations
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What is the career outlook in biotechnology?What is the career outlook in biotechnology?
• Biotech in 1998– 1,300 companies in the US– 2/3 have less than 135 employees– 140,000 jobs
• Jobs will continue to increase exponentially• Jobs are available to high school graduates
through PhD’s
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What Subjects Are Involved With Biotechnology?
• Multidisciplinary- involving a number of disciplines that are coordinated for a desired outcome
• Science– Life sciences– Physical sciences– Social sciences
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What Subjects Are Involved With Biotechnology?What Subjects Are Involved With Biotechnology?
• Mathematics• Applied sciences
– Computer applications– Engineering– Agriculture
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What Are the Stages of Biotechnology Development
• Ancient biotechnology- early history as related to food and shelter; Includes domestication
• Classical biotechnology- built on ancient biotechnology; Fermentation promoted food production, and medicine
• Modern biotechnology- manipulates genetic information in organism; Genetic engineering
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What Are the Areas of Biotechnology?What Are the Areas of Biotechnology?
• Organismic biotechnology- uses intact organisms; Does not alter genetic material
• Molecular biotechnology- alters genetic makeup to achieve specific goals– Transgenic organism- an organism with
artificially altered genetic material
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What Are the Benefits of Biotechnology?What Are the Benefits of Biotechnology?
• Medicine– Human– Veterinary– Biopharming
• Environment• Agriculture• Food products• Industry and manufacturing
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What Is Molecular Biology?
• Molecular biology- study of molecules in cells
• Metabolism- processes by which organisms use nutrients
• Anabolism- building tissues from smaller materials
• Catabolism- breaking down materials into smaller components
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What Is a Cell?What Is a Cell?
• Cell- a discrete unit of life
• Unicellular organism- organism of one cell
• Multicellular organism- organism of many cells
• Prokaryote- cells that lack specific nucleus
• Eukaryote- cells with well-defined nucleus
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What Is a Cell?What Is a Cell?
• Cells are building blocks:– Tissue- collection of cells with specific functions– Organs- collections of tissues with specific
functions– Organ systems- collections of organs with
specific functions
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What Are the Structures in Molecular Genetics?
• Molecular genetics- study of genes and how they are expressed
• Chromosome- part of cell nucleus that contains heredity information and promotes protein synthesis
• Gene- basic unit of heredity on a chromosome
• DNA- molecule in a chromosome that codes genetic information
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Deoxyribonucleic Acid (DNA)
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What Is Ribonucleic Acid (RNA)?
• Transcription- process of RNA production by DNA
• DNA-thread-like molecule which decodes DNA information
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What Is Ribonucleic Acid (RNA)?
• Kinds of RNA:– mRNA- RNA molecules that carry information that
specifies amino acid sequence of a protein molecule during translation
– rRNA- RNA molecules that form the ribosomal subunits; Mediate the translation of mRNA into proteins
– tRNA- molecules that decode sequence information in and mRNA
– snRNA- very short RNA that interconnects with to promote formation of mRNA
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What Are Genetic Engineering Organisms?
• Genetic engineering- artificially changing the genetic information in the cells of organisms
• Transgenic- an organism that has been genetically modified
• GMO- a genetically modified organism• GEO- a genetically enhanced organism
• Donor cell- cell that provides DNA• Recipient cell- cell that receives DNA• Protocol- procedure for a scientific process• Three methods used in gene transfer
1. Use genetic techniques to find marker near gene
Gene Marker
2. Find cosegregating markerGene/Marker
3. Discover overlapping clones (or contig) that contains the marker Gene/Marker
4. Find ORFs on contigGene/Marker
5. Prove one ORF is the gene by transformation or mutant analysis
Mutant + ORF = Wild type?Yes? ORF = Gene
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Gene Manipulation
• It is now routine to isolate genes
• But the target gene must be carefully chosen
• Target gene is chosen based on desired phenotype
Function:Glyphosate (RoundUp) resistance EPSP synthase enzymeIncreased Vitamin A content Vitamin A biosynthetic pathway enzymes
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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
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+ Glyphosate
X
RoundUp Sensitive Plants
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X
Shikimic acid + Phosphoenol pyruvate
3-Enolpyruvyl shikimic acid-5-phosphate(EPSP)
Plant EPSP synthase
Aromaticamino acids
Without amino acids, plant dies
X
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BacterialEPSP synthase
Shikimic acid + Phosphoenol pyruvate
3-enolpyruvyl shikimic acid-5-phosphate(EPSP)
Aromaticamino acids
RoundUp Resistant PlantsRoundUp Resistant Plants
+ Glyphosate
With amino acids, plant lives
RoundUp has no effect;enzyme is resistant to herbicide
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The Golden Rice Story
• Vitamin A deficiency is a major health problem
• Causes blindness• Influences severity of diarrhea, measles
• >100 million children suffer from the problem
• For many countries, the infrastructure doesn’t existto deliver vitamin pills
• Improved vitamin A content in widely consumed cropsan attractive alternative
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-Carotene Pathway in Plants
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
-carotene(vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-beta-cyclase
ξ-carotene desaturase
Problem:Rice lacks
these enzymes
NormalVitamin A
“Deficient”Rice
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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 APathway
is completeand functional
GoldenRice
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Metabolic Pathways are ComplexMetabolic Pathways are Complexand Interrelatedand Interrelated
Understanding pathways is critical to developing
new products
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Modifying Pathway ComponentsCan Produce New Products
Modified Lipids =New Industrial Oils
Turn On Vitamin Genes = Relieve Deficiency
Increase amino acids = Improved Nutrition
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Trait/Gene Examples
RoundUp Ready Bacterial EPSP
Golden Rice Complete Pathway
Plant Virus Resistance Viral Coat Protein
Male Sterility Barnase
Plant Bacterial Resistance p35
Salt tolerance AtNHX1
Trait Gene
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Introducing the Gene orDeveloping 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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Gene of InterestGene of Interest
Coding Region• Encodes protein product
ex.: EPSP -carotene genes
Promoter Region• Controls when, where and how much the gene is expressed
ex.: CaMV35S (constitutive; on always) Glutelin 1 (only in rice endosperm during seed development)
Promoter Coding RegionTP
Transit Peptide• Targets protein to correct organelle
ex.: RbCS (RUBISCO small subunit; choloroplast target
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Selectable Marker
Coding Region• Gene that breaks down a toxic compound;non-transgenic plants die