Genetic Engineering of Plant Nuclear Genome for Specific gene Expression in Chloroplast Using Design and Transformation of Hybrid Sigma Factor

A system was designed using E. coli heat shock promoter (groE) in plastid vector and a hybrid plant/bacteria sigma factor was constructed under control of a tissue specific promoter. This system was designed for overcome to deleterious effects on plant growth and fertility that may be caused by transgene overexpression. So that hybrid sigma factors contained N- terminal motives of tobacco sigma factors including chloroplast signal peptide and RNA polymerase interaction domains, composed by C-terminal motif of E. coli sigma32 that able to recognition and binding to groE promoter. Then this gene, HSig, was cloned in Agrobacterium vector after adding regulatory elements. The result vector was used for transformation of an Iranian variety of tobacco. Detection of transgenic plants was performed by PCR, southern blot and RT-PCR analysis. The Hsig gene expression and its targeting to plastid was confirmed after transformation of tobacco chloroplast using gene gun technique for targeting of green florescent protein (GFP) under control of groE promoter using pFNGi vector into transgenic HSig explants. We hope that the system that was designed and constructed in this study for GFP expression in chloroplast genome, be able to apply in molecular farming for expression of any other desired genes instead of GFP for specific gene expression in chloroplast.