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

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

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 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 --------------------------------------------------------------------------------------------------

• Maize genome database (www.maizegdb.org)

• Aspartate kinase (ask2)

• Arizona Genomics Institute ZM_BFc003612C cDNA library• pCMV Sport 6.1 – sequencing with 35S and T7 primers

Epitope tagging

• HA and FLAG tag addition using PCR reactionHA tag:

HA tag Gene5´-AGC GTA ATC TGG AAC ATC GTA TGG GTA ATG GCT GTG GAT TGT GCC ATT-3´FLAG tag:

FLAG tag Linker5´-CTA TTT GTC ATC GTC GTC CTT GTA GTC TCT GAA

HA tagCTG AGC GTA ATC TGG AAC ATC GTA-3´

Cloning strategy

• Gateway cloning (Invitrogen, USA)• Donor vector - pENTR™/SD/D-TOPO®,

(Invitrogen, USA)• Destination vector- pANIC 6A, University of Tennessee

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)

• Selection of transformants on kanamycin (50μg/mL) plates minipreparation

• PCR, electrophoresis

• Vector suited for monocot transformation – pANIC 6A

TOPO pANIC M

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

• 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

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