เทคนิคทางชีว โมเลกุล กับการศึกษาฮอร์โมน

1. เทคนิคทางชีวโมเลกุลกับการศึกษาฮอร์โมนเทคนิคทางชีวโมเลกุลกับการศึกษาฮอร์โมน

2. Why at Molecular Level? 1 hormone  many responses  different responses Different hormones  same responses Interaction / Induction / Inhibition Molecular strategies for better understanding

3. Hormone Studies physiology biochemistry molecular biology

4. Molecular Studies gene gene product gene expression control of gene expression

5. molecular biology & hormone study synthesis signal transduction action/response

6. Approach for molecular study genetics reverse genetics Advantages/Disadvantages

7. Genetic Approach Mutagenesis: making mutants Mutant: organisms with altered arrangement or altered amount of genetic materials Phenotypic changes: yes / no

8. Genetic Approach Mutation: inversion translocation deletion duplication point

9. Point mutation base substitution • Ethylmethane sulfonate (EMS) • GC to AT transition • Sodium azide (NaN3) • AT to GC transition

10. Point mutation base substitution Results: no change (GGG/GGA = glycine) amino acid substitution (effects on function: yes/no) stop codon(TGG tryp  TGA stop)

11. 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

12. Deletion mutation (ionizing radiation) Large scale deletion resulting in loss of entire coding sequence or chromosome rearrangement

13. 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)

14. Effect of Mutation Mostly recessive to wild type allele Heterozygote with normal phenotype Homozygous mutant  effect of mutation

15. Effect of Mutation Loss of function mutation: inadequate gene product of mutant allele usually Recessive

16. Effect of Mutation Gain of function mutation: overproduction of normal gene product production of novel/toxic gene product usually Dominant

17. Mutant Screen Visible screen: morphology anatomy development Biochemical screen: hormone precursor intermediate

18. Mutant check • Heritability (selfing): • mutation of germ cells • Pattern of inheritance (crossing): • dominant/recessive trait • single/multiple gene • Allelic test: • complementation group • phenotypic epistasis

19. x x x x x x x  Allelic test Crossing of homozygous mutants Phenotypes of F1 compared to parents Mutation at the same locus same complementation group phenotype of F1 = ?

20. Allelic test Mutation at different loci Different complementation group dominant x dominant  F1? recessive x recessive  F1? dominant x recessive  F1?

21. x x Allelic test dominant x dominant  F1? x x  x x x Check for phenotypic epistasis

22. Hormone mutant With hormone no response Without hormone responsive phenotype

23. Hormone mutant • changes in • Synthesis: synthetic pathway • Sensitivity: perception / signaling • Regulation: responsive phenotype

24. Synthesis mutant Analysis of hormone level Use of hormone hormone inhibitor Rescue WT phenotypes

25. Synthesis mutant Reproducible Clear Complete penetrance

26. Ethylene mutants • Triple response • Ethylene overproduction: • ctr1 and eto2 • chromosome 5

27. GA mutants seed germination stem elongation flowering

28. 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

29. GA-deficient mutants Mutant phenotypes complemented by chemical compounds ga1, ga2, and ga3 dwarf plants tall with exogenous/supplied GA

30. ABA mutants Seed dormancy Stomatal closure

31. ABA mutants • ABA-deficient mutants: • aba1, aba2, and aba3 • precocious germination • viviparous • germinate in the presence of • paclobutrazol (GA inhibitor) • high zeaxanthin(ABA precursor)

32. ABA-deficient mutants: aba1 droopy stem under low humidity lack of stomatal aperture control

33. Auxin mutants IAA synthesis: Trp-independent pathway Little labeled trp converted to Iabeled IAA Trp-deficient mutant accumulated IAA Several pathways for IAA biosynthesis

34. Signal transduction Effects of hormone depending on type concentration mode of application developmental stage Specific information pathway From extrinsic signal to specific response

35. Signal transduction Specific receptor: membrane protein with high affinity binding upon binding with hormone conformation change Activated receptor to Signal cascade intermediate steps

36. Signaling molecule Positive or Negative regulator Phosphorylation Dephosphorylation Hydrolysis of guanine nucleotide Rapid and Reversible Redundancy

37. Signaling mutant Mutation to signaling components Gain of Function Loss of Function

38. Signaling molecule Positive regulator without hormone no/inactivated signaling molecule no response

39. Signaling molecule Positive regulator with hormone activated signaling molecule response Mutation?

40. Signaling molecule Negative regulator without hormone activated signaling molecule no response

41. Signaling molecule Negative regulator with hormone deactivated signaling molecule response Mutation?

42. Identification of signaling mutant Basic mutant screen Rule out synthesis mutant Define complementation groups Epistatic analysis of components

43. Identification of signaling mutant With hormone application: (+) oversensitive response (0) insensitive response Without hormone application: (-) auxotrophic / deficient phenotype

44. GA insensitive mutants • gai: fail to respond to applied GA • elevated endogenous GA • normal seed germination • poor stem elongation • delayed flowering

45. GA insensitive mutants • gai:gai mutation on chromosome1 • semidominant • GAI gene product • inhibits stem elongation • GA de-represses GAI action

46. GA oversensitive mutants spy mutants: longer hypocotyl spindly early flowering resistant to paclobutrazol

47. GA oversensitive mutants spy mutants: normal GA synthesis Resistant to ga1 mutation Suppress all phenotypes associated with GA deficiency

48. 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

49. ABA mutants • ABA insensitive mutants: • abi1, abi2, abi3, abi4, and abi5 • Germination with • exogenous ABA application • Decreased seed dormancy • Elevated endogenous ABA level

50. ABA mutants ABA insensitive mutants: Other phenotypes similar to aba mutants abi1 and abi2: wilty and viviparous abi3: viviparous seed maturation processes