Overexpression of OsCam1-1 gene in transgenic rice Oryza sativa L. 'KDML105' and tobacco Nicotiana tabacum L. 'Virginia Coker'

Abstract

Calmodulin (CaM) proteins, members of the EF-hand family of Ca²⁺ -binding proteins are important relays in calcium signals that mediate stress response in plants. In previous study, expression of a Cam gene from Oryza sativa L. (OsCam1-1) was highly induced by osmotic stress and wounding. To generate transgenic rice and tobacco plants overexpressing OsCam1-1 gene, a gene sequence consisting of the OsCam1-1 gene driven by the 35SCaMV promoter was introduced into rice (Oryza sativa L.) and tobacco (Nicotiana tabacum L. cv. Virginia Coker) by Agrobacterium-mediated transformation. The transformation of all potential transgenic lines was confirmed by the assay of GUS activity using X-Gluc as a substrate and by PCR amplification of the 35S CaMV promoter-OsCam1-1 gene cassette using their genomic DNA as template. At least one transgenic rice line and two independent control rice lines, harboring pCAMBIA1301 alone were obtained from the regeneration of hygromycin resistant rice calli and confirmed by Southern blot analysis. The OsCam1-1 mRNA was detected in all transgenic rice and transgenic tobacco lines by RT-PCR using oligonucleotide primers designed based on its transcript but not found in the control plants harboring pCAMBIA1301 vector used for the introduction of the OsCam1-1 gene alone. By northern blot analysis transgenic rice plants showed the overexpression of the OsCam1-1 transcript as compared with the wild type plants. Since, the 35S CaMV promoter-OsCam1-1 gene cassette was constructed to encode OsCam1-1 fused with a His tag at its carboxyl terminal, blot analyses of crude proteins from the transgenic rice and transgenic tobacco plants using His antibodies were conducted. All transgenic rice and transgenic tobacco plants did not produce the bands of the recombinant OsCaM1-1 protein and of GUS protein fused with a His tag.