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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文摘
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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。 |
其他语种文摘
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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 1.25×10~(-6). 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. |
来源
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热带海洋学报
,2014,33(2):72-77 【核心库】
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关键词
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微拟球藻Nannochloropsis gaditana CCAP849/5
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转化
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外源基因
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整合
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地址
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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
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语种
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中文 |
文献类型
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研究性论文 |
ISSN
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1009-5470 |
学科
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海洋学 |
基金
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中国科学院知识创新工程重要方向项目
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文献收藏号
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CSCD:5106549
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参考文献 共
23
共2页
|
1.
黄伟超. 微拟球藻属对盐度的耐受及其产油特性分析.
水生生物学报,2013,37(2):384-387
|
被引
1
次
|
|
|
|
2.
李秀波. 五种微绿球藻产油和产多不饱和脂肪酸的研究.
水生生物学报,2010,34(5):893-897
|
被引
20
次
|
|
|
|
3.
刘飞飞. 三角褐指藻产油突变株的筛选.
水生生物学报,2013,37(4):799-802
|
被引
6
次
|
|
|
|
4.
Brennan L. Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products.
Renewable and Sustainable Energy Reviews,2010,14:557-577
|
被引
105
次
|
|
|
|
5.
Chen H L. Conditional production of a functional fish growth hormone in the transgenic line of Nannochloropsis oculata (Eustigmatophyceae).
J Phycol,2008,44:768-776
|
被引
8
次
|
|
|
|
6.
Gong Y. Microalgae as platforms for production of recombinant proteins and valuable compounds: Progress and prospects.
J Ind Microbiol Biotechnol,2011,38:1879-1890
|
被引
1
次
|
|
|
|
7.
Guillard R R L. Culture of phytoplankton for feeding marine invertebrates.
Culture of marine invertebrate animals,1975:29-60
|
被引
64
次
|
|
|
|
8.
Hempel F. Microalgae as bioreactors for bioplastic production.
Microbial Cell Factories,2011,10:81
|
被引
2
次
|
|
|
|
9.
Hibberd D J. Notes on taxonomy and nomenclature of the algal classes Eustigmatophyceae and Tribophyceae (Synonym Xanthophyceae).
Bot J Linn Soc,1981,82:93-119
|
被引
18
次
|
|
|
|
10.
Jinkerson R E. Genomic insights from the oleaginous model alga Nannochloropsis gaditana.
Bioengineered,2013,4(1):37-43
|
被引
5
次
|
|
|
|
11.
Kilian O. High-efficiency homologous recombination in the oilproducing alga Nannochloropsis sp.
Proc Nat Acad Sci,2011,108:20859-20860
|
被引
20
次
|
|
|
|
12.
Larkum A W D. Selection, breeding and engineering of microalgae for bioenergy and biofuel production.
Trends in Biotechnology,2012,30(4):198-205
|
被引
6
次
|
|
|
|
13.
Leon R. Nuclear transformation of eukaryotic microalgae.
Adv Exp Med Biol,2007,616:1-11
|
被引
2
次
|
|
|
|
14.
Li J. The nitrate reductase gene-switch: A system for regulated expression in transformed cells of Dunaliella salina.
Gene,2007,4031:32-142
|
被引
1
次
|
|
|
|
15.
Li S S. Transgenic microalgae as a non-antibiotic bactericide producer to defend against bacterial pathogen infection in the fish digestive tract.
Fish Shellfish Immunol,2009,26:316-325
|
被引
7
次
|
|
|
|
16.
Murray M G. Rapidisolation of high molecular weight plant DNA.
Nucleic Acids Research,1980,8:4321-4325
|
被引
1535
次
|
|
|
|
17.
Pan K. Nuclear monoploidy and asexual propagation of Nannochloropsis oceanica (Eustigm atophyceae) as revealed by its genome sequence.
J Phycol,2011,47:1425-1432
|
被引
6
次
|
|
|
|
18.
Pawlowski W P. Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA.
Proc Nat Acad Sci,1998,95:12106-12110
|
被引
13
次
|
|
|
|
19.
Radakovits R. Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropsis gaditana.
Nat Commun,2012,3:686
|
被引
18
次
|
|
|
|
20.
Rodofi L. Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor.
Biotech and Bioeng,2009,102(1):100-112
|
被引
99
次
|
|
|
|
|