Genetic transformation of <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> with a bacterial phleomycin resistance gene as dominant selective marker |
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| Authors: | Xiaolei Ma Kehou Pan Lin Zhang Baohua Zhu Guanpin Yang Xiangyang Zhang |
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| Institution: | 1.Foundation College,Jining Medical University,Jining,P. R. China;2.Laboratory of Applied Microalgae Biology, Key Laboratory of Aquaculture of Ministry of Education,Ocean University of China,Qingdao,P. R. China |
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| Abstract: | 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. |
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