stable expression of antibiotic-resistant gene ble from streptoalloteichus hindustanus in the mitochondria of chlamydomonas reinhardtii耐药基因的稳定表达祝福从streptoalloteichus hindustanus线粒体的衣藻reinhardtii.pdf

Stable Expression of Antibiotic-Resistant Gene ble from Streptoalloteichus hindustanus in the Mitochondria of Chlamydomonas reinhardtii 1 . 1. 1,2. 1 1 Zhangli Hu * , Zhun Fan , Zhonglin Zhao , Jun Chen , Jiancheng Li 1 Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Science, Shenzhen University, Shenzhen, People’s Republic of China, 2 College of Sciences, Henan Agricultural University, Zhengzhou, People’s Republic of China Abstract 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 ıg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern anal