Cloning and sequencing of the promoter and terminator regions of the rbcL gene from Gunnera perpensa L.

Abstract

Primers were designed based on the chloroplast genome sequence from the cotton Gossypium barbadense L. Specificrestriction sites were included in the primers so as to facilitate the process of vector assembly for future chloroplast transformation. Polymerase chain reaction (PCR) was applied to amplify the promoter and terminator regions of the rbcLgene from the chloroplast DNA (cpDNA) of G. perpensa. These promoter and terminator regions of the rbcL gene were around 0.25 and 0.5 kilobase pair (kb) in length, respectively. The amplicons were purified and cloned into pGEM®-T Easy Vector System I, then transformed into DH5α strain of E. coli bacteria. Transformed bacteria were propagated and recombinant plasmids were extracted and sequenced. The resulted sequences were aligned and compared with the sequenced promoter and terminator of rbcL from cotton as the reference plant. The promoter region of rbcL gene from G. perpensa showed 81.5% identity with that of the cotton plant, while the terminator region of rbcL gene expressed 69.9% identity with that of cotton plant. The core sequences at -35 and -10 of the promoter from G. perpensa scored 54.44% of thedefault core sequences