Phytoextraction of cadmium using recombinant DNA technology in maize

1. Phytoextraction of cadmium using recombinant DNA technology in maize Mario Franić1, Hrvoje Fulgosi2, Lea Vojta2, Domagoj Šimić1 1 Department for breeding and genetics of maize, Agricultural institute Osijek, Osijek, Croatia. 2 Department of molecular biology, Ruđer 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 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 -------------------------------------------------------------------------------------------------- IBM _____________________________________________________________ QTL Chrom. Pos Left_MarkSupp.IV LOD R^2% add dom -------------------------------------------------------------------------------------------------- 1 chrom2 372 umc1028 368- 376 20.01 35.9 0.193 -11.483 --------------------------------------------------------------------------------------------------- • WinQTL Cartographer: QTL - chrom2 for IBM:

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

5. Epitope tagging • HA and FLAG tag addition using PCR reaction HA tag: HA tag Gene 5´-AGC GTA ATC TGG AAC ATC GTA TGG GTA ATG GCT GTG GAT TGT GCC ATT-3´ FLAG tag: FLAG tag Linker 5´-CTA TTT GTC ATC GTC GTC CTT GTA GTCTCT GAA HA tag CTGAGC 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), DH5αE.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 sequenceonce (1517) • Sequencing • Future steps: expression cloneAgrobacterium maize R pANIC M

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