1. Arabidopsis thealiana AT3G56230 AT4G18650 What are the functions of them? Are they important for seed development? Spring 2004 Mariko Onozawa

2. What is AT3G56230? ・ About 1200bps. ・ 5’UTR. ・ Three SALK lines. ・ This protein containing BTB/POZ domain. What is AT4G18650? ・ About 1400bps. ・ 5’ and 3’UTR. ・ No SALK line. ・ This protein is related to transcription factor and has weak similarity to TGAGG-sequence specific DNA- binding protein(TGA-2.1). FV RV FV RV

3. mRNA *Find the pattern of mRNA concentration RT-PCR (SRB) Genechip database Gene * Find KOs of my gene Madison lines SALK lines Phenotype *Study a phenotype of plants which containing the KO Check genotypes of the plants (SALK) mRNAGenePhenotype Reverse Genetics

4. RT-PCR result using SRB (AT3G56230) Genechip data *RT-PCR result says that the gene which is homologous toAT3G56230 is expressed at all stages and organs of SRB. *Genechip data says that AT3G56230 is expressed only at Mature green seed, Seedling (3DAI), Roots and stem. A border between presence and absence Leaves +RT -RT Stem +RTFlower +RTOvules +RTEmbryo +RTAxes +RTLeaves +RTStem +RT control gene specific

5. Is there any KO of my gene in Madison lines? *AT3G56230 I couldn’t find any KO of AT3G56230. The insertion site is upstream of the gene. (-594bp) *AT4G18650 I couldn’t find any KO of AT4G18650. The insertion site is downstream of the gene. FVRV R19 FVRV R15

6. Checking phenotypes of the plants T-DNA,homo 1,4 WT, homo 2,3,5,6 ① WT ① T-DNA ② WT, T-DNA Only tubilan’s bands nothing Some samples don’t have any band. ① WT ① T-DNA ② WT, T-DNA Only tubulin’s bands Nothing Some seedlings don’t have any band. ③ WT, T-DNA ④ WT, T-DNA smears 1 2 3 4 5 6WC ③ WT, T-DNA smears ④ WT T-DNA 1 2 3 4 5 6 WC

7. I couldn’t find any significant difference between WT and mutant plants. T-DNAWild Type

8. What should be done next to study the functions? *Get more plants which have a insertion in the genes. *Observe and study the knockout plants. *Study with other knockout lines. The final goal is to answer these questions. *What is the function of the protein? *What is the structure of the protein? *How it is regulated? What we did in this class is only the beginning of the research.