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เทคนิคทางชีวโมเลกลุ กับ การศึกษาฮอร์ โมน Why at Molecular Level? 1 hormone many responses different responses Different hormones same responses Interaction / Induction / Inhibition Molecular strategies for better understanding Hormone Studies physiology biochemistry molecular biology Molecular Studies gene gene product gene expression control of gene expression molecular biology & hormone study synthesis signal transduction action/response Approach for molecular study genetics reverse genetics Advantages/Disadvantages Genetic Approach Mutagenesis:making mutants Mutant: organisms with altered arrangement or altered amount of genetic materials Phenotypic changes: yes / no Genetic Approach Mutation: inversion translocation deletion duplication point Point mutation base substitution Ethylmethane sulfonate (EMS) GC to AT transition Sodium azide (NaN3) AT to GC transition Point mutation base substitution Results: no change (GGG/GGA = glycine) amino acid substitution (effects on function: yes/no) stop codon (TGG tryp TGA stop) Deletion mutation (ionizing radiation) Reading frame of Genetic codes: Codon NNN NNN NNN NNN NN- N NNN NNN NN Frame shift mutation resulting in nonsense peptide or premature stop codon Deletion mutation (ionizing radiation) Large scale deletion resulting in loss of entire coding sequence or chromosome rearrangement Insertion mutation Duplication / Translocation Transposon (Ac, Ds, etc.) T-DNA Results: +/- Functional recombinant protein (in-frame insertion) No/nonsense protein (off-frame or promoter insertion) Effect of Mutation Mostly recessive to wild type allele Heterozygote with normal phenotype Homozygous mutant effect of mutation Effect of Mutation Loss of function mutation: inadequate gene product of mutant allele usually Recessive Effect of Mutation Gain of function mutation: overproduction of normal gene product production of novel/toxic gene product usually Dominant Mutant Screen Visible screen: morphology anatomy development Biochemical screen: hormone precursor intermediate Mutant check Heritability (selfing): mutation of germ cells Pattern of inheritance (crossing): dominant/recessive trait single/multiple gene Allelic test: complementation group phenotypic epistasis Allelic test Crossing of homozygous mutants Phenotypes of F1 compared to parents Mutation at the same locus same complementation group phenotype of F1 = ? xx x xx xx Allelic test Mutation at different loci Different complementation group dominant x dominant F1? recessive x recessive F1? dominant x recessive F1? Allelic test dominant x dominant F1? xx x xx x x Check for phenotypic epistasis Hormone mutant With hormone no response Without hormone responsive phenotype Hormone mutant changes in Synthesis: synthetic pathway Sensitivity: perception / signaling Regulation: responsive phenotype Synthesis mutant Analysis of hormone level Use of hormone hormone inhibitor Rescue WT phenotypes Synthesis mutant Reproducible Clear Complete penetrance Ethylene mutants Triple response Ethylene overproduction: ctr1 and eto2 chromosome 5 GA mutants seed germination stem elongation flowering GA-deficient mutants Seeds unable to germinate on basal medium Germinated after being transferred to medium with GA 5 complementation groups: all recessive ga1, ga2, ga3, ga4, and ga5 GA-deficient mutants Mutant phenotypes complemented by chemical compounds ga1, ga2, and ga3 dwarf plants tall with exogenous/supplied GA ABA mutants Seed dormancy Stomatal closure ABA mutants ABA-deficient mutants: aba1, aba2, and aba3 precocious germination viviparous germinate in the presence of paclobutrazol (GA inhibitor) high zeaxanthin (ABA precursor) ABA-deficient mutants: aba1 droopy stem under low humidity lack of stomatal aperture control Auxin mutants IAA synthesis: Trp-independent pathway Little labeled trp converted to Iabeled IAA Trp-deficient mutant accumulated IAA Several pathways for IAA biosynthesis Signal transduction Effects of hormone depending on type concentration mode of application developmental stage Specific information pathway From extrinsic signal to specific response Signal transduction Specific receptor: membrane protein with high affinity binding upon binding with hormone conformation change Activated receptor to Signal cascade intermediate steps Signaling molecule Positive or Negative regulator Phosphorylation Dephosphorylation Hydrolysis of guanine nucleotide Rapid and Reversible Redundancy Signaling mutant Mutation to signaling components Gain of Function Loss of Function Signaling molecule Positive regulator without hormone no/inactivated signaling molecule no response Signaling molecule Positive regulator with hormone activated signaling molecule response Mutation? Signaling molecule Negative regulator without hormone activated signaling molecule no response Signaling molecule Negative regulator with hormone deactivated signaling molecule response Mutation? Identification of signaling mutant Basic mutant screen Rule out synthesis mutant Define complementation groups Epistatic analysis of components Identification of signaling mutant With hormone application: (+) oversensitive response (0) insensitive response Without hormone application: (-) auxotrophic / deficient phenotype GA insensitive mutants gai: fail to respond to applied GA elevated endogenous GA normal seed germination poor stem elongation delayed flowering GA insensitive mutants gai: gai mutation on chromosome1 semidominant GAI gene product inhibits stem elongation GA de-represses GAI action GA oversensitive mutants spy mutants: longer hypocotyl spindly early flowering resistant to paclobutrazol GA oversensitive mutants spy mutants: normal GA synthesis Resistant to ga1 mutation Suppress all phenotypes associated with GA deficiency GA oversensitive mutants Wild type SPY gene product a negative regulator of GA signaling flux Mutation affects GA signal transduction pathway in a GA-independent manner ABA mutants ABA insensitive mutants: abi1, abi2, abi3, abi4, and abi5 Germination with exogenous ABA application Decreased seed dormancy Elevated endogenous ABA level ABA mutants ABA insensitive mutants: Other phenotypes similar to aba mutants abi1 and abi2: wilty and viviparous abi3: viviparous seed maturation processes Auxin insensitive mutants: resistant to toxic precursor (2,4-D) toxic level of applied auxin Auxin insensitive mutants: axr: reduced hypocotyl elongation reduced fertility increased lateral branch reduced root gravitropism aux: not responsive to gravitropism Signaling component interaction Epistatic analysis of components shared or separate pathway acting order of gene products Epistasis of phenotypes Double mutant: combined mutant loci Signaling component interaction Consider 2 loci: A and B A activate / inhibit B B activate / inhibit A Single mutant a: aa BB Single mutant b: AA bb Double mutant ab: aa bb Signaling component interaction Opposite responses Epistatic phenotype: downstream component Same responses Epistatic phenotype: upstream component Signaling component interaction eg. Opposite responses activated A component = + response activated B component = - response Hormone A B response double mutant phenotype: b mutant (downstream) B epistatic to A Signaling component interaction Suppression analysis mutation of mutant Intragenic suppressor same gene / new allele Extragenic suppressor second site revertant Transgene technology Breakdown species barrier Transfer genes from any source to plants Transgene technology Initiate changes in specific Tissues certain developmental stage Manipulate hormone level or hormone sensitivity Using transgenes Transgenic plants for hormone study Mechanism of hormone biosynthesis Role of hormone in development Control of hormone function and monitor responses Gene structure Coding sequence Regulatory sequence promoter terminator Agrobacterium Soil bacteria A. tumefaciens: crown gall tumor A. rhizogenes: hairy root disease Chromosomal DNA Plasmid DNA Agrobacterium Tumor-inducing plasmid Ti plasmid Transferred DNA T-DNA genes involved in synthesis of amino acid derivatives tumor formation Agrobacterium Gene transfer Activated by plant substances phenolics acetosyringone coniferyl alcohol from wound For production of amino acid derivatives : opine octopine and nopaline Agrobacterium Opines synthesized only in plant cells Used by bacteria For transformation: replace bacterial DNA between T-DNA borders with desired genes System selection for gene transfer Transformation technique established easy available System selection for gene transfer Hormone-related mutants Background information on hormone level Physiological work on hormone effect growth & development Transgenic plants Effect of gene expression on overall development Interaction among various hormones Auxin/CK Auxin/C2H4 GA/ABA Apical dominance High auxin inhibits dormancy release of lateral buds High CK stimulates lateral bud growth Apical dominance Transgenic plant with auxin overproduction CK application to a dormant lateral bud branching Apical dominance High auxin activates C2H4 production Transgenics w/ iaaM: reduced internode elongation Transgenics w/ iaaM and ACCD: normal height No difference on apical dominance Vascular differentiation Auxin and xylem formation quality and quantity Petunia w/ high auxin: xylem cells w/ increased numbers smaller and more lignified Tobacco w/ low auxin: larger and less-lignified xylem cells Vascular differentiation Auxin: cell division in vascular cambium secondary wall formation lignin synthesis Auxin content: rate of cell division Fruit ripening C2H4 as a catalyst: involved in some aspects of ripening Ripening occurs with lower than a significant level (slow but not delay) chlorophyll breakdown fruit softening Fruit ripening C2H4 as a coordinator: activates a large number of genes for rapid and uniform ripening *************** Senescence Transgenic tomato (P35S:ACCS) Vegetative tissues remain green until setting fruit Most flowers abort prior to fertilization due to premature induction of abscission in pedicel Senescence Transgenic tomato with antisense ACCS antisense ACCO expressing ACCD Delayed senescence