CAJ | 학술논문

微拟球藻 Nannochloropsis 被认为是具有作为生物柴油原料开发潜力的微藻。为了能够实现工业化生产，有效地利用基因工程或遗传操作手段改造微藻，提高产量，建立稳定有效的遗传转移方法十分必要。本研究以微拟球藻本源β-tublin基因启动子和三角褐指藻Phaeodactylum tricornutm fcpA终止子驱动和终止来源于细菌的sh ble抗性选择基因，构建了一个转化载体 pHB4857。将 pHB4857以电转移的方法转化海洋富油微拟球藻 Nannochloropsis gaditana CCAP849/5。结果显示，转化子可以在3μg·mL-1 zeocin的抗性培养基中生长， PCR检测sh ble基因为100%插入率，转化效率为1.25×10-6。DNA印迹杂交结果表明，外源基因是以随机整合的方式一个或多个拷贝插入到宿主核基因组中的，大多数转化子中的外源基因的整合是完整的。转化子在抗性培养基中每10天传一代，连续传代7个月以上，未检测到抗性基因丢失现象，外源抗性基因可以在宿主细胞中稳定存在。
미의구조 Nannochloropsis 피인위시구유작위생물시유원료개발잠력적미조。위료능구실현공업화생산，유효지이용기인공정혹유전조작수단개조미조，제고산량，건립은정유효적유전전이방법십분필요。본연구이미의구조본원β-tublin기인계동자화삼각갈지조Phaeodactylum tricornutm fcpA종지자구동화종지래원우세균적sh ble항성선택기인，구건료일개전화재체 pHB4857。장 pHB4857이전전이적방법전화해양부유미의구조 Nannochloropsis gaditana CCAP849/5。결과현시，전화자가이재3μg·mL-1 zeocin적항성배양기중생장， PCR검측sh ble기인위100%삽입솔，전화효솔위1.25×10-6。DNA인적잡교결과표명，외원기인시이수궤정합적방식일개혹다개고패삽입도숙주핵기인조중적，대다수전화자중적외원기인적정합시완정적。전화자재항성배양기중매10천전일대，련속전대7개월이상，미검측도항성기인주실현상，외원항성기인가이재숙주세포중은정존재。
Nannochloropsis has been believed to be a promising genus of microalgae, which has the potential as the feed stock for biofuels. To achieve the goal for industrial application, use of genetic modifications to improve oil-producing and growth characters is of great significance in reduction of costs. So, it is necessary to develop an efficient transformation method. Here, a vector pHB4857 that has a sh ble gene derived by Nannochloropsis β-tublin promoter and Phaeodactylum tricornutum fcpA terminator was constructed and transformed in N. gaditana CCAP849/5 by electroporation. Transformants were able to grow on 3μg·mL-1 zeocin. PCR detection indicated that 100%of the selected colonies were positive transformants. The efficiency was 1.25×10ˉ6. A Southern blot analysis verified that the sh ble gene with single or multiple copies was randomly inserted into the geneome and most of the transformants owned the intact heterologous genes. The transformants were inoculated in fresh zeocin-resistant medium every 10 days for more than seven months. The integration of heterologous gene in the host nuclear genome appeared to be stable since no sh ble gene was lost.