Construction of modular tandem expression vectors for the green alga Chlamydomonas reinhardtii using the Cre/lox-system
Construction of modular tandem expression vectors for the green alga Chlamydomonas reinhardtii using the Cre/lox-system
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DOI：
10.2144/000112556
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发表时间：
2007-09-01
期刊：
影响因子：
2.7
通讯作者：
Zschoernig, Barbara
中科院分区：
文献类型：
--
作者：
Heitzer, Markus;Zschoernig, Barbara
外源基因的成功表达主要取决于可靠的转化方法和合适的启动子序列。我们构建了一系列模块化质粒，在莱茵衣藻不同启动子序列的控制下，便于快速构建大型串联载体进行转基因表达。通过Cre/lox位点特异性重组，将两个质粒在体外融合，制备了携带Renilla荧光素酶和代谢选择标记（ARG7）表达盒的串联载体。使用超螺旋质粒和线性质粒转化精氨酸营养缺陷衣藻菌株，并监测常用启动子（HSP70A、LHCB1、PSAD和嵌合HSP70A/RBCS2）的共表达率和荧光素酶活性水平。与标准的共转化方案相比，线性化串联载体通常增加了共表达频率（高达77%）。大多数转化子表现出单一且完整的整合事件，证实了核基因组内活性选择标记与报告基因的密切联系。荧光素酶活性分析显示，所使用的启动子的表达水平在三个数量级以内，其中人工HSP70A/RRBCS2是最活跃的。在所有携带HSP70A启动子的发光转化子中，69%的荧光素酶表达在热休克下增强，这表明在转基因环境下启动子具有生理功能。
The successful expression of,foreign genes mainly depends on both a reliable method for tranformation and a suitable promoter sequence. We created a series of modular plasmids that facilitate the rapid construction of large tandem vectors for transgene expression under the control of different promoter sequences in Chlamydomonas reinhardtii. Tandem vectors carrying expression cassettes for Renilla luciferase and a metabolic selection marker (ARG7) were manufactured by fusing two plasmids in vitro using Cre/lox site-specific recombination. Supercoiled and linear plasmids were used to transform an arginine auxotrophic Chlamydomonas strain, and rates of co-expression as well as levels luciferase activity were monitored for frequently) used promoters (HSP70A, LHCB1, PSAD, and the chimeric HSP70A/RBCS2). Linearized tandem vectors generally increased the co-expression frequency (up to 77%) compared with standard cotransformation protocols. Most transformants showed a single and complete integration event confirming the close linkage of active selectable marker and reporter gene within the nuclear genome. The analysis of luciferase activity showed expression levels within three orders of magnitude for the promoters used, with the artificial HSP70A/RRBCS2 being the most active. For 69% of all luminescent tranformants carrying the HSP70A promoter luciferase expression was enhanced by heatshock, indicating physiological promoter function in a transgenic context.