海洋富油微拟球藻Nannochloropsis gaditana CCAP849/5 的转化及外源基因整合方式

doi:10.11978/j.issn.1009-5470.2014.02.010

热带海洋学报 ›› 2014, Vol. 33 ›› Issue (2): 72-77.doi: 10.11978/j.issn.1009-5470.2014.02.010cstr: 32234.14.j.issn.1009-5470.2014.02.010

海洋富油微拟球藻Nannochloropsis gaditana CCAP849/5 的转化及外源基因整合方式

余桂兰^{1, 2}, 胡晗华¹, 李秀波¹, 孔任秋¹

1. 中国科学院水生生物研究所, 淡水生态国家重点实验室, 湖北武汉 430072; 2. 中国科学院大学, 北京 100049

收稿日期:2013-04-15 修回日期:2013-05-11 出版日期:2014-04-02 发布日期:2014-04-02
通讯作者: 孔任秋, 副研究员, 主要从事生物技术和藻类分子遗传学研究。E-mail: kongr@ihb.ac.cn
作者简介:余桂兰(1987~), 江西省九江市人, 硕士研究生, 研究方向为海洋藻类生物技术。E-mail:13667289794 @163.com

Transformation of marine oleaginous alga Nannochloropsis gaditana CCAP849/5 and heterologous gene integration

YU Gui-lan^{1, 2}, HU Han-hua¹, LI Xiu-bo¹, KONG Ren-qiu¹

1. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; 2. University of Chinese Academy of Science, Beijing 100049, China

Received:2013-04-15 Revised:2013-05-11 Online:2014-04-02 Published:2014-04-02
Contact: 中国科学院知识创新工程重要方向项目(KSCX2-YW-G-060)

摘要/Abstract

摘要： 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。

关键词: 微拟球藻Nannochloropsis gaditana CCAP849/5, 转化, 外源基因, 整合

Abstract: 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 3 μg·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.

Key words: Nannochloropsis gaditana CCAP849/5, transformation, heterologous gene, integration

中图分类号:

P735

余桂兰, 胡晗华, 李秀波, 孔任秋. 海洋富油微拟球藻Nannochloropsis gaditana CCAP849/5 的转化及外源基因整合方式[J]. 热带海洋学报, 2014, 33(2): 72-77.

YU Gui-lan, HU Han-hua, LI Xiu-bo, KONG Ren-qiu. Transformation of marine oleaginous alga Nannochloropsis gaditana CCAP849/5 and heterologous gene integration[J]. Journal of Tropical Oceanography, 2014, 33(2): 72-77.

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海洋富油微拟球藻Nannochloropsis gaditana CCAP849/5 的转化及外源基因整合方式

Transformation of marine oleaginous alga Nannochloropsis gaditana CCAP849/5 and heterologous gene integration

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