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The mitochondrial expression of exogenous antibiotic resistance genes has not been demonstrated successfully to date, which has limited the development of antibiotic resistance genes as selectable markers for mitochondrial site-directed transformation in  Chlamydomonas reinhardtii . In this work, the plasmid pBSLPNCB was constructed by inserting the gene  ble  of  Streptoalloteichus hindustanus  ( Sh ble ), encoding a small (14-kilodalton) protective protein into the site between TERMINVREP-Left repeats and the  cob  gene in a fragment of mitochondrial DNA (mtDNA) of  C. reinhardtii . The fusion DNA-construct, which contained TERMINVREP-Left,  Sh ble ,  cob , and partial  nd4  sequence, were introduced into the mitochondria of the respiratory deficient  dum-1  mutant CC-2654 of  C. reinhardtii  by biolistic particle delivery system. A large number of transformants were obtained after eight weeks in the dark. Subsequent subculture of the transformants on the selection TAP media containing 3 ìg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern analyses on the mitochondrial genome of the different MT-B lines revealed that  Sh ble  gene had been integrated into the mitochondrial genome of  C. reinhardtii . Both Western blot, using the anti-BLE monoclonal antibody, and Zeomycin tolerance analysis confirmed the presence of BLE protein in the transgenic algal cells. It indicates that the  Sh ble  gene can be stably expressed in the mitochondria of  C. reinhardtii .

Stable Expression of Antibiotic-Resistant Gene ble from Streptoalloteichus hindustanus in the Mitochondria of Chlamydomonas reinhardtii