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. Specific
restriction 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 rbcL
gene 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 the
default core sequences