1. Phytoextraction of cadmium using recombinant DNA technology in maize Mario Franić 1, Hrvoje Fulgosi 2, Lea Vojta 2, Domagoj Šimić 1 1 Department for breeding and genetics of maize, Agricultural institute Osijek, Osijek, Croatia. 2 Department of molecular biology, Ru đ er Bošković Institute, Zagreb, Croatia

2. Cadmium Heavy metal Toxic at low concentrations Water soluble, high bioavailability  accumulation in tissues Health concern Accumulation of Cd in soil Expensive remediation techniques Adverse reactions on soil fertility Phytoremediation  phytoextraction

3. Candidate gene for cadmium accumulation in maize leaf WinQTL Cartographer: QTL - chrom2 for IBM: IBM _____________________________________________________________ QTL Chrom. Pos Left_Mark Supp.IV LOD R^2% add dom -------------------------------------------------------------------------------------------------- 1 chrom2 372 umc1028 1 chrom2 372 umc1028 368- 376 20.01 35.9 0.193 -11.483 --------------------------------------------------------------------------------------------------- B84xOs6-2 _____________________________________________________________ QTL Chrom. Pos Left_Mark Supp.IV LOD R^2% add dom --------------------------------------------------------------------------------------------------- 1 chrom2 36 Z1359 34- 38 32.51 40.3 -0.348 -0.134 --------------------------------------------------------------------------------------------------

4. Maize genome database (www.maizegdb.org)www.maizegdb.org Aspartate kinase (ask2) Arizona Genomics Institute  ZM_BFc003612C cDNA library pCMV Sport 6.1 – sequencing with 35S and T7 primers

5. Epitope tagging HA and FLAG tag addition using PCR reaction HA tag: HA tagGene 5´-AGC GTA ATC TGG AAC ATC GTA TGG GTA ATG GCT GTG GAT TGT GCC ATT-3´ FLAG tag: FLAG tagLinker 5´-CTA TTT GTC ATC GTC GTC CTT GTA GTC TCT GAA HA tag CTG AGC GTA ATC TGG AAC ATC GTA-3´

6. Cloning strategy Gateway cloning (Invitrogen, USA) Donor vector - pENTR™/SD/D-TOPO®, (Invitrogen, USA) Destination vector- pANIC 6A, University of Tennessee

7. TOPO cloning Purification of PCR products - GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences, UK) Cloning the purified DNA construct (ask2 gene with HA and FLAG tag) into a TOPO entry vector (pENTR™/SD/D-TOPO®, Invitrogen, USA) Transformation of One Shot® TOP10 Chemically Competent E. coli cells (Invitrogen, USA)

8. Selection of transformants on kanamycin (50μg/mL) plates  minipreparation PCR, electrophoresis Vector suited for monocot transformation – pANIC 6A TOPO pANIC M

9. LR reaction LR reaction was done according to manufacturers protocol (Invitrogen), DH5α E.coli cells were transformed and plated on kanamycin plates (50μg/mL) TOPO entry vector pANIC 6A destination vector

10. Minipreparation of overnight cultures Restriction using EcoRV – cleaves the pANIC 6A vector once (16937) and ask2 sequence once (1517) Sequencing Future steps: expression clone  Agrobacterium  maize R pANIC M

11. THANK YOU