For successful plastid transformation, a foreign DNA and endogenous regulatory sequences on the transgene cassette are flanked with plastid DNA fragments homologous to a specific site on plastome. To clone the intergene between rbcL and AtpB of Glycine max plastome along with portions of rbcL and AtpB genes as flanking sequences, forward and reversed primers were designed using Primer-Blast in NCBA. Restriction sites and over-hanged AA were added to 5' of either primer. A pre-test gradient temperature was used to identify the best annealing temperature for multiple primer sets during polymerase chain reaction, which was 62.7°C in this case. Flanking sequences as long as 195 Nucleotides from 3' late part of RbcL and 306 Nucleotides from 5' region of AtpB were amplified. Results showed that RbcL part of the fragment carries 10 gaps out of 195 nucleotide pairs, which accounts for 5%, but there is no gap in AtpB part.

AtpB, flanking sequence, gradient temperature, Plastid transformation, RbcL,

For successful plastid transformation, a foreign DNA and endogenous regulatory sequences on the transgene cassette are flanked with plastid DNA fragments homologous to a specific site on plastome. To clone the intergene between rbcL and AtpB of Glycine max plastome along with portions of rbcL and AtpB genes as flanking sequences, forward and reversed primers were designed using Primer-Blast in NCBA. Restriction sites and over-hanged AA were added to 5' of either primer. A pre-test gradient temperature was used to identify the best annealing temperature for multiple primer sets during polymerase chain reaction, which was 62.7°C in this case. Flanking sequences as long as 195 Nucleotides from 3' late part of RbcL and 306 Nucleotides from 5' region of AtpB were amplified. Results showed that RbcL part of the fragment carries 10 gaps out of 195 nucleotide pairs, which accounts for 5%, but there is no gap in AtpB part.

Amir Sam Pordel, Bahram Baghban Kohne-Rooz and Parviz Soleimani (2016). Cloning of Flanking Regions of Plastidal Vectors in Soybean (Glycine max L.): partial AtpB and RbcL , Biological Forum – An International Journal 8(1): 397-403.