Vector construction for transformation of tobacco with a NAC-type transcription factor, TaNAC69-1

NAC type transcription factors are a large family of plant transcription factors related to defense, development, biotic and abiotic responses of plants. To construct vectors for tobacco transformation, traditional and Gateway cloning technology were used. For traditional cloning, firstly TaNAC69-1 gene was isolated from Triticum aestivum L. cv. Yuregir-89. After cDNA synthesis, TaNAC69-1 gene was cloned into pJET1.2 cloning vector. To transfer the gene into dicot expression vector, pORE-E3, both pJET1.2 and pORE-E3 vector were cut with ClaI and NotI enzymes forming sticky ends on both vectors. TaNAC69-1 gene was bound to pORE-E3 vector carrying same sticky ends like the gene. T4 DNA ligase was used for ligation of TaNAC69-1 gene into the vector. After transferring obtained vector into empty Escherichia coli DH5α strain, positive colonies growing on LB media including kanamycin were selected and colony PCR was performed. pORE-E3 vector carrying TaNAC69-1 gene was transferred to Agrobacterium tumefaciens EHA 105 strain via electroporation. For Gateway cloning, pENTR™/D-TOPO cloning vector and pEarleyGate 100 expression vector were used. After isolation, TaNAC69-1 gene was amplified using wheat cDNA samples and cloned into pENTR™/D-TOPO cloning vector. After cloning into entry vector, TaNAC69-1 gene was transferred into Gateway compatible binary destination vector, pEarleyGate 100, through LR reaction. A. tumefaciens EHA105 strain was transformed with destination vector via electroporation. To check the insertion of TaNAC69-1 gene, colony PCR was performed using gene specific primers