Phytoextraction of cadmium using recombinant DNA technology in
Phytoextraction of cadmium using recombinant DNA technology in maize Mario Franić1, Hrvoje Fulgosi 2, Lea Vojta 2, Domagoj Šimić1 1 Department for breeding and genetics of maize, Agricultural institute 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 IBM _______________________________ QTL Chrom. Pos Left_Mark Supp. IV LOD R^2% add dom -------------------------------------------------1 chrom 2 372 umc 1028 368 - 376 20. 01 35. 9 0. 193 -11. 483 -------------------------------------------------- B 84 x. Os 6 -2 _______________________________ QTL Chrom. Pos Left_Mark Supp. IV LOD R^2% add dom -------------------------------------------------1 chrom 2 36 Z 1359 34 - 38 32. 51 40. 3 -0. 348 -0. 134 ------------------------------------------------- • Win. QTL Cartographer: QTL - chrom 2 for IBM:
• Maize genome database (www. maizegdb. org) • Aspartate kinase (ask 2) • Arizona Genomics Institute ZM_BFc 003612 C c. DNA library • p. CMV Sport 6. 1 – sequencing with 35 S and T 7 primers
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 CTT GTA GTC TCT GAA HA tag CTG AGC GTA ATC TGG AAC ATC GTA-3´
Cloning strategy • Gateway cloning (Invitrogen, USA) • Donor vector - p. ENTR™/SD/D-TOPO®, (Invitrogen, USA) • Destination vector- p. ANIC 6 A, 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 (ask 2 gene with HA and FLAG tag) into a TOPO entry vector (p. ENTR™/SD/D-TOPO®, Invitrogen, USA) • Transformation of One Shot® TOP 10 Chemically Competent E. coli cells (Invitrogen, USA)
• Selection of transformants on kanamycin (50μg/m. L) plates minipreparation TOPO p. ANIC M • PCR, electrophoresis • Vector suited for monocot transformation – p. ANIC 6 A
LR reaction • LR reaction was done according to manufacturers protocol (Invitrogen), DH 5α E. coli cells were transformed and plated on kanamycin plates (50μg/m. L) • TOPO entry vector • p. ANIC 6 A destination vector
• Minipreparation of overnight cultures • Restriction using Eco. RV – cleaves the p. ANIC 6 A vector once (16937) and ask 2 sequence once (1517) R p. ANIC M • Sequencing • Future steps: expression clone Agrobacterium maize
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