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Plasmid construction and arabidopsis transformation

NE Nardana Esmaeili
XY Xiaojie Yang
YC Yifan Cai
LS Li Sun
XZ Xunlu Zhu
GS Guoxin Shen
PP Paxton Payton
WF Weiping Fang
HZ Hong Zhang
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The p35S::AVP1/pUbi::OsSIZ1 construct harboring NptII (kanamycin resistance gene) was cloned into the pBI121 based binary vector, and introduced into Arabidopsis (ecotype Columbia) using Agrobacterium-mediated floral dip method44. The rice SUMO E3 ligase gene OsSIZ1 and the Arabidopsis vacuolar pyrophosphatase gene AVP1 were under the control of the dual 35S promoter and the maize ubiquitin gene promoter, respectively. Transgenic plants were selected by screening seeds on Murashige and Skoog58 (MS) media supplemented with 30 µg/ml of kanamycin. A total of 40 independent putative transgenic plants were obtained on kanamycin plates and were verified by using PCR with AVP1 and OsSIZ1 gene-specific primers. Homozygous transgenic plants were obtained at T3 generation and then used for all experiments.

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