Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

miRNA Activity in Arabidopsis thaliana

633 views

Published on

Research performed by Andrew Tsai and Helen Oh in early 2011. Identifies potential genes that regulate miRNA activity, especially in regards to growth and development.

  • Be the first to comment

  • Be the first to like this

miRNA Activity in Arabidopsis thaliana

  1. 1. miRNA activity inArabidopsis thaliana plantsHelen OhAndrew Tsai
  2. 2. Purpose To identify potential genes that regulate miRNA activity, especially in regards to growth and development.
  3. 3. Overview To identify those potential genes:  Mutagenize with T-DNA insertions  Screen using BASTA  Screen using ABA  Screen for typical mutant miRNA phenotypes  Find location of T-DNA insertion  Determine whether miRNA target genes are affected  Germination assay  BASTA assay
  4. 4. either too slow (ABA hypersensitive) or too fast (ABA hyposensitive), these collection ofMutagenize with T-DNA mutant plants will be highly enriched in mutations that affect miRNA activity. In sum, we will direct our studies on screening for mutations that enhance or diminish the response of Arabidopsis plants to the plant hormone abscisic acid (ABA). This approach has insertion already resulted in the identification of a number of interesting genes, and will hopefully lead to identification of a number more within this semesters class.
  5. 5. Overview To identify those potential genes:  Mutagenize with T-DNA insertions  Screen using BASTA  Screen using ABA  Screen for typical mutant miRNA phenotypes  Find location of T-DNA insertion  Determine whether miRNA target genes are affected  Germination assay  BASTA assay
  6. 6. Screen using BASTA I1. Mutagenesis strategy using T-DNA containing three tandem strong rview of procedure Brian Gregory used to prepare the mutagenized seed th screen. B) Cartoon depicting thegerminated is inserted into the plant genom  Ten plants T-DNA that on BASTA.ns. Shown is a random gene that is induced by the insertion (green arrow, r borders of the T-DNA (RB, LB, orange), the herbicide resistance gene exp rDNA that we will use to select mutagenized seedlings (BASTA , purple), and enhancers (35S enhancers, red).
  7. 7. Overview To identify those potential genes:  Mutagenize with T-DNA insertions  Screen using BASTA  Screen using ABA  Screen for typical mutant miRNA phenotypes  Find location of T-DNA insertion  Determine whether miRNA target genes are affected  Germination assay  BASTA assay
  8. 8. Screen using ABA Six plants displayed mutated responses to ABA , specifically they displayed hypersensitive root growth.
  9. 9. Overview To identify those potential genes:  Mutagenize with T-DNA insertions  Screen using BASTA  Screen using ABA  Screen for typical mutant miRNA phenotypes  Find location of T-DNA insertion  Determine whether miRNA target genes are affected  Germination assay  BASTA assay
  10. 10. Screen for typical mutant miRNA phenotype HOAT2-2
  11. 11. Screen for typical mutant miRNA phenotypes HOAT3-2
  12. 12. Screen for typical mutant miRNA phenotype HOAT5-2
  13. 13. Overview To identify those potential genes:  Mutagenize with T-DNA insertions  Screen using BASTA  Screen using ABA  Screen for typical mutant miRNA phenotypes  Find location of T-DNA insertion  Determine whether miRNA target genes are affected  Germination assay  BASTA assay
  14. 14. Find location of T-DNA insertion TAIL PCR was performed. AD1/AD2 This gel shows that AD primers AD2 are about 100 bps.
  15. 15. Find location of T-DNA insertion TAIL PCR product for HOAT2-2AD1 HOAT2-2AD2 HOAT2-2AD3 HOAT2-2AD2 and HOAT3-2AD1 HOAT3-2AD2 HOAT3-2AD3 HOAT5-2AD1 HOAT3-2AD2 are about 750 bps. Lower bands are AD primers.
  16. 16. Find location of T-DNA insertion Heavy upper bands could be HOAT5-2AD2 HOAT5-2AD3 due to precipitated DNA. Lower bands are AD primers.
  17. 17. Find location of T-DNA insertion DNA sequence analysis performed. HOAT3-2:  Had greater or equal to 200 as alignment score.  Expected value was 0.0.
  18. 18. Find location of T-DNA insertion AT3G09720 codes for a upstream protein with a p-loop.  Binds nucleotides and upstream ATP  Helicase functions downstream AT3G09730 has no known function.
  19. 19. Overview To identify those potential genes:  Mutagenize with T-DNA insertions  Screen using BASTA  Screen using ABA  Screen for typical mutant miRNA phenotypes  Find location of T-DNA insertion  Determine whether miRNA target genes are affected  Germination assay  BASTA assay
  20. 20. Determine whether miRNA target genes are affected  GRF3 is a known target of miRNA. Look at amount of mRNA transcribed from GRF3 to determine if miRNA activity is potentially altered.
  21. 21. Determine whether miRNA target genes are affected HOAT2-2 HOAT5-2 HOAT3-2 Col-0 Non-degraded RNA has the following bands: 28s rRNA and 18s rRNA. 28s rRNA Create cDNA for HOAT3-2 18s rRNA and HOAT5-2.
  22. 22. Determine whether miRNA target genes are affected 26 cycles HOAT3-2 HOAT5-2  sqPCR was Col-0 DDL performed.• Actin primers (above). 26 cycles HOAT3-2 HOAT5-2 Col-0 DDL Col-0• GRF3 primers (below).
  23. 23. Determine whether miRNA target genes are affected qPCR was performed. qPCR 1200 1000 800Percentages 600 400 200 0 Col-0 DDL HOAT3-2 HOAT5-2 Plant Name
  24. 24. HOAT3-2 HOAT5-2 DDL 26 cycles Col-0 HOAT3-2 Actin primers HOAT5-2 DDL 30 cycles Col-0HOAT3-2, 30 26 cyclesHOAT3-2, 30 30 cycles Col-0, 30 26 cycles Col-0, 30 30 cyclesHOAT3-2, 20 26 cycles target genes are affectedHOAT3-2, 20 30 cycles Determine whether miRNA Col-0, 20 26 cycles ATG09730 and ATG09720 primers Col-0, 20 30 cycles
  25. 25. Overview To identify those potential genes:  Mutagenize with T-DNA insertions  Screen using BASTA  Screen using ABA  Screen for typical mutant miRNA phenotypes  Find location of T-DNA insertion  Determine whether miRNA target genes are affected  Germination assay  BASTA assay
  26. 26. Germination Assay plated onto ABA plates of concentrations: 0 μM 0.5 μM 1 μM 1.5 μM
  27. 27. Germination Assay 0 μM ABA • Baseline 100 90 • note 3-2 low 80 70 Percent 60 germination 50Germination 40 30 20 10 0 wt 2-2 3-2 5-2 Experimental
  28. 28. Germination Assay 0.5 μM ABA • Mimics 0 ABA control 100 90 • T-value of 0.5275 80 70 Percent 60 • Don’t see too much 50Germination 40 30 from this plate 20 10 0 wt 2-2 3-2 5-2 Experimental
  29. 29. Germination Assay 1 μM ABA • Start seeing trends 100 90 80 • 3-2, 5-2 hypersensitive 70 Percent 60 50 • 2-2 ambiguousGermination 40 30 20 10 0 wt 2-2 3-2 5-2 Experimental
  30. 30. Germination Assay 1.5 μM ABA • Similar to 1.0 μM ABA 100 90 • 3-2, 5-2 80 70 60 Percent 50Germination 40 30 20 10 0 wt 2-2 3-2 5-2 Experimental
  31. 31. Germination Assay Seeds from HOAT 2-2 may have a T-DNAinsertion but are not hyper/hyposensitive to ABA!
  32. 32. Overview To identify those potential genes:  Mutagenize with T-DNA insertions  Screen using BASTA  Screen using ABA  Screen for typical mutant miRNA phenotypes  Find location of T-DNA insertion  Determine whether miRNA target genes are affected  Germination assay  BASTA assay
  33. 33. BASTA Assay - Theory HYPOTHESIS: ABA hyper/hyposensitivity caused by T-DNA insertion.GIVEN: T-DNA insertion confers BASTA resistance ABA-hypersensitive plants on BASTA – should all have T-DNA insertionPlated surviving plants from ABA treatment ontoBASTA plates
  34. 34. BASTA Assay Survival on BASTA • Data does not confirm 100 80 theory 60Percent Col-0 • Most surviving plantsSurvival 40 HOAT 2-2 HOAT 3-2 20 have BASTA resistance HOAT 5-2 0 0.5 1 1.5 [ABA] in μM
  35. 35. BASTA Assay - ConclusionsSeeds from HOAT 2-2 HAVE a T-DNA insertion but are not hyper/hyposensitive to ABA!  Contrary to original screen  2-2 originally identified as hypersensitive
  36. 36. Possible Explanations  Chance from first hyper/hyposensitivity assay (n = 1)  NOT chance from this experiment n = large (despite contamination)  Possibly growth conditionsBOTTOM LINE: We have a lot of factors at work here that we don’t know about!
  37. 37. Possible Explanations Possible interaction Survival on BASTA 100 between ABA and 80Percent 60 Col-0 BASTASurvival 40 HOAT 2-2 HOAT 3-2 20 HOAT 5-2 0 0.5 1 1.5 [ABA] in μM
  38. 38. ConclusionsHOAT 2-2, HOAT 3-2, HOAT 5-2  Root growth assay  potential to have miRNA mutations  Observable “miRNA phenotypes”
  39. 39. ConclusionsHOAT 3-2, HOAT 5-2  Increase in GRF3 expression – KNOWN miR396- REGULATED GENE  Decrease in ATG09720 gene expression  T-DNA insertion inside gene.Future work: How do changes in these geneexpressions explain original phenotype?
  40. 40. References Earley, et al., An endogenous F-box protein regulates ARGONAUTE1 in Arabidopsis thaliana Silence 2010, 1:15 Rodriguez RE, Mecchia MA, Debernardi JM, Schommer C, Weigel D, Palatnik JF. 2010. Control of cell proliferation in Arabidopsis thaliana by microRNA miR396. Development 137, 103–112. Dr. John Wagner, personal communication. Allison Lesher, personal communication. NCBI BLAST database: gene identification TAIR database: gene information

×