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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
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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
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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 --------------------------------------------------------------------------------------------------
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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
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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´
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Cloning strategy Gateway cloning (Invitrogen, USA) Donor vector - pENTR™/SD/D-TOPO®, (Invitrogen, USA) Destination vector- pANIC 6A, University of Tennessee
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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)
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Selection of transformants on kanamycin (50μg/mL) plates minipreparation PCR, electrophoresis Vector suited for monocot transformation – pANIC 6A TOPO pANIC M
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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
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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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THANK YOU
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