Genetic Transformation of Nannochloropsis oculata with a Bacterial Phleomycin Resistance Gene as Dominant Selective Marker
Genetic Transformation of Nannochloropsis oculata with a Bacterial Phleomycin Resistance Gene as Dominant Selective Marker
复制标题
以细菌腐草霉素抗性基因作为显性选择标记的微拟球藻的遗传转化
DOI：
10.1007/s11802-016-2715-4
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发表时间：
2016-04-01
影响因子：
1.6
通讯作者：
Zhang Xiangyang
中科院分区：
文献类型：
--
作者：
Ma Xiaolei;Pan Kehou;Zhang Xiangyang
The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2×107 cells mL−1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS188. The construct was transferred into the protoplasts through electroporation (1 kV, 15 μS). The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL−1 zeocin. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.
