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ERF/AP2 Subfamily A3 and ERF/AP2 Subfamily A6 Genes: AT1G64380 and AT4G39780

ERF/AP2 Subfamily A3 and ERF/AP2 Subfamily A6 Genes: AT1G64380 and AT4G39780. ERF/AP2 DREB Subfamily. Characterized by AP2 domain AP2 family genes-shown to participate in regulation of embryo development Encodes putative transcription factors (DNA binding motif)

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ERF/AP2 Subfamily A3 and ERF/AP2 Subfamily A6 Genes: AT1G64380 and AT4G39780

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  1. ERF/AP2 Subfamily A3 and ERF/AP2 Subfamily A6 Genes: AT1G64380 and AT4G39780

  2. ERF/AP2 DREB Subfamily • Characterized by AP2 domain • AP2 family genes-shown to participate in regulation of embryo development • Encodes putative transcription factors (DNA binding motif) • ERF family: CBF and DREB subfamilies • DREB subfamilies contain AP2 and DREB motifs • DREB transcription factors control expression of drought-inducible genes

  3. Where is gene AT1G64380? 976 bp 5’ 3’ 742 bp 1,528 bp 2,247 bp AT1G64370 AT1G64380 AT1G64385 7,861 bp

  4. What is gene AT1G64380’s structure? 185 bp UTR UTR Coding Region (1,008 bp) Not part of gene 335 bp 359 bp 5’ 3’

  5. Where is the T-DNA insert located? LBb1 T-DNA insert: 300 bp RV 185 bp UTR UTR Coding Region (1,008 bp) Not part of gene 335 bp 359 bp 5’ FW 3’ Predicted T-DNA insertion site: 1,206th bp of the gene WT expected length: 1,195 bp T-DNA expected length: 830 bp

  6. Where is gene AT1G64380 expressed? Gene Chip Data Expression Levels 471.6 937.1 406.05 105.45 Chalazal heart stage Chalazal seed coat/ linear cotyledon stage Chalazal seed coat/ Mature green stage Floral bud/ reproductive Leaf/Vegetative

  7. What are the plant genotypes? Homozygous T-DNA (4, 6, 9) 2 T-DNA bands: Top band matches expected amplified T-DNA size (830bp) Lower band approx. 750 bp Wild type Expected WT size: 1,195 bp

  8. What are the possible T-DNA configurations? LBb1 LBb1 T-DNA insert T-DNA insert: 300 bp RV 185 bp UTR UTR Coding Region (1,008 bp) Not part of gene 335 bp 359 bp 5’ FW 3’ A concatamer!

  9. What are the possible T-DNA configurations? LBb1 T-DNA insert: 300 bp RV 185 bp UTR UTR Coding Region (1,008 bp) Not part of gene 335 bp 359 bp 5’ FW 3’ T-DNA insert Two inserts located very close to each other; sequencing inconclusive LBb1

  10. What do these results mean? Presence of homozygous mutant plants: Knockout of gene AT1G64380 does NOT lead to seed lethality

  11. Where in Arabidopsis Thaliana is gene AT1G64380 transcribed? Use RT-PCR to detect AT1G64380 mRNA levels 04/28/09 //120 V // 1 hour // 1% agarose Tubulin control Genomic DNA Gene AT1G64380 is expressed in both Arabidopsis leaf and silique tissues; supports Gene Chip data water g DNA Silique -RT Siliqe +RT Leaf –RT Leaf +RT Ladder

  12. How can we visualize where gene AT1G64380 is transcribed? Promoter Cloning -no recombinant plasmids found in 42 bacteria colonies screened ASCI restriction digest and PCR colony screening Expected size of recombinant: 4.3 kb • WHY? • Initial ligation was not successful • Isolate plasmids from transformed E-coli • PCR to amplify promoter region No promoter region detected Negative control Positive control Pentr only Pentr + gene

  13. Observation of mutant siliques *Location of T-DNA insert!

  14. What is gene AT4G39780’s structure? 3’ 5’ orientation 256 bp RV 146 bp UTR UTR Coding Region (819 bp) 5’ 3’ FW

  15. Where is the T-DNA insert located? LBb1 T-DNA insert: 169 bp 256 bp RV 146 bp UTR UTR Coding Region (819 bp) 5’ FW 3’ Predicted T-DNA insertion site: 695th bp of the gene WT expected length: 741 bp T-DNA expected length: 569 bp

  16. Where is gene AT4G39780 expressed? Gene Chip Data Chalazal seed coat/ globular stage Floral Bud/ reproductive General Seed Coat/ Globular Stage General Seed coat/ Heart stage General seedcoat/ pre-globular stage Leaf/Vegetative

  17. What are the plant genotypes? All plants are wild type Expected WT size: 741 bp

  18. What do these results mean? Enough plants were screened T-DNA was not inserted into the gene Fatality of knockout to seed development is inconclusive No further analysis can be done on these plants

  19. Future Research AT1G64380 *No phenotypic difference observed in mutant siliques Look at Arabidopsis mutant leaves Check protein production levels Obtain different SALK lines for this gene • AT4G39780 • No T-DNA inserts found from screening 24 plants • Obtain a different SALK line for this gene • --Repeat previous methods

  20. Acknowledgements Thank you to Anhthu, Kristen, Daisy, Brandon, Min, Tomo, Kelli, Ingrid and Dr. Goldberg for making this lab experience happen!! This has been an incredible quarter.

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