The C3HC4-Type RING Zinc Finger and MYB Transcription Factor Families Matthew Taube June 5, 2008 HC70AL What was the first gene studied with knockout analysis? AT3G23060 Chromosome 3 Location: base pairs 2598 base pairs long Forward orientation What is the genes structure and where is the T-DNA insert? 5 UTR, 7 exons, 6 introns, 3 UTR UTRexon intron What is the genes structure and where is the T-DNA insert? 5 UTR, 7 exons, 6 introns, 3 UTR SALK_148145: Expected insertion site: 255 bp What is the genes structure and where is the T-DNA insert? 5 UTR, 7 exons, 6 introns, 3 UTR SALK_148145: Expected insertion site: 255 bp Actual insertion site: 156 bp UPSTREAM What protein does this gene encode? C3HC4-Type RING Zinc Finger protein 480 amino acids Cys3HisCys4 amino acid motif Diverse functionality Binds one or more zinc ions DNA binding: transcription factor Also RNA, protein, and lipid binding Was T-DNA actually inserted into the plant samples? Multiplex PCR to test the genotypes What do we expect to see on the genotyping gel? Two possible bands: WT (542 bp) and T-DNA (1.1 kb) 1 WT to 2 hemizygote to 1 T-DNA homozygote (1:2:1) Was T-DNA actually inserted into the plant samples? T-DNA 5 hemizygous 9 WT WT The knockout appears to be seed lethal Were there any phenotypic differences in the siliques and seeds? No, the siliques and seeds display WT phenotype WT Hemizygous Were there any phenotypic differences during embryo and seed development? Nomarski microscopy of the mature green stage reveals no apparent phenotypic differences WT Hemizygous In what tissues is the gene active? The mRNA accumulation in the silique suggests that the gene is active in seed development Gene specific bands, indicating accumulation in that sample In what tissues is the gene active? Overview of AT3G23060 The knockout was seed lethal but did not cause any apparent phenotypic changes in the hemizygous plants Active in seed development, especially important during the globular stage The T-DNA insert was found in the upstream region What did we learn about this gene? What steps should be taken for further analysis? The upstream region should be studied and used to perform GUS analysis Genotyping more plants to verify seed lethality What was the second gene studied with knockout analysis? AT5G62470 Chromosome 5 Location: bp 2111 base pairs long Reverse orientation What is the genes structure and where is the T-DNA insert? 5 UTR, 3 exons, 2 introns, 3 UTR UTRexon intron What is the genes structure and where is the T-DNA insert? 5 UTR, 3 exons, 2 introns, 3 UTR SALK_ Expected insertion site: 761 bp What protein does this gene encode? MYB96: MYB domain protein 96 (R2R3) 352 amino acids 203 identified MYB family transcription factors, 126 of which are R2R3 MYB Large sized gene family with similar binding specificity DNA binding transcription factor Response to salt stress and dehydration Was T-DNA actually inserted into the plant samples? What do we expect to see on the genotyping gel? Two possible bands: WT (995 bp) and T-DNA (853 bp) 1:2:1 ratio Was T-DNA actually inserted into the plant samples? WT No T-DNA was inserted into the sampled plants. Why? SALK error Sowing error Dominant mutation (seed lethal) Not enough plants tested In what tissues is the gene active? Gene specific bands, indicating accumulation in both the silique and leaf Gene appears to be active in both the seed and leaf In what tissues is the gene acitve? Why is the upstream region important and what work was done with it? The promoter region was isolated via PCR and ligated into the TOPO-vector E. coli cells were transformed with the recombinant plasmid The authenticity of the promoter insert was verified by restriction enzyme digestion and sequencing analysis Culture harboring recombinant plasmid Expected fragment size: 2.6 kb kb = 5.4 kb Overview of AT5G62470 New seeds should be ordered, grown, and genotyped Experiments (like those for gene one) should be carried out accordingly Perform GUS analysis with upstream region What did we learn about this gene? What further steps should be taken to complete the analysis? No T-DNA inserts were found, and not much can be said without performing the appropriate knockout experiments Active in seed and leaf tissues in roughly equal amounts The upstream region is amplified and ready for GUS analysis Conclusions Even though no mutant phenotypes were observed, that does not mean that these two genes are not important in seed development Redundant gene function There is still much to learn about these genes and the proteins they encode Further experimentation and analysis might reveal interesting results Acknowledgements I would like to thank Dr. Goldberg, Anhthu, Daisy, Bekah, the HC70AL class, and every one else in the Goldberg Lab for all of your help and guidance throughout the course.