Molecular Analysis of transgenic tobacco (hairy root and leave) containing chimeric chitinase 42 with ChBD in N-terminal

Chitin is the most abundant organic material after cellulose in nature. Chitinases have the ability of chitin digestion that constitutes a main compound of the fungal cell wall, insect exoskeletons, and crustacean shells. Hence chitinases have many usages such as chitooligosacharides production, chitin waste treatment and protoplasts isolated from fungi and yeasts, so the production of chitinase enzymes will be economical. The benefits of producing recombinant proteins in plants relative to other expression systems such as bacteria, yeast and animal systems has made the use of plants for the production of recombinant proteins as a first option, advantages such as low cost, high expression in a short time, correct post-translational modifications such as glycosylation, more security for no transmission of diseases to the humans from a proteinproducing host. Transient expression compared with stable expression of genes, has several advantages for specific applications and is competitive with traditional methods of recombinant protein production. In this study, chimeric chitinase 42 containing introns and also ChBD in its N-terminaed was cloned into an eukaryotic expression vector pARM2 including His tag. His tag was used for purification. The cloning was confirmed by PCR and digestion pattern using appropriate restriction enzymes. This expression construct used for transformation of tobacco using agrobacterium tumefaciens for leave and agrobacterium rhizogenes for hairy root. After confirmation of transgenic hairy root and leave by PCR method, protein expression analysis was achieved by SDS-PAGE and Western blotting methods.

کلیدواژه ها:

Chitin ، Chimeric Chitinase 42 ، Chitin Binding Domain ، Tobacco ، Hairy root.