Efficiency of Different Heterologous Promoters in the Unicellular Microalga Chlamydomonas reinhardtii
Efficiency of Different Heterologous Promoters in the Unicellular Microalga Chlamydomonas reinhardtii
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DOI：
10.1002/btpr.1690
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发表时间：
2013-03-01
影响因子：
2.9
通讯作者：
Leon, Rosa
中科院分区：
文献类型：
--
作者：
Diaz-Santos, Encarnacion;de la Vega, Marta;Leon, Rosa
尽管微藻具有生物技术方面的兴趣，但目前还没有可靠和稳定的方法来对大多数微藻菌株进行遗传转化。现有研究中关于异源启动子在微藻中的效率的数据很少且分散，而且使用了不同的转化方法、DNA数量和报告基因，这使得对它们的效率进行真正的比较非常困难。以莱茵衣藻为寄主，评价了花椰菜花叶病毒35S(CaMV 35S)和农杆菌诺帕林合成酶(NOS)基因的异源启动子的效率。将这些启动子与帕罗霉素抗性氨基糖苷3-磷酸转移酶编码基因(APHVIII)融合，用玻璃微珠搅拌法转化莱茵葡萄球菌。对每一种启动子的转化效率、APHVIII转录本和蛋白水平进行了评价。用HSP70A/RBCS2嵌合启动子和无启动子的APHVIII标记基因进行比较。我们发现，含有NOS启动子的转化子的转化效率和APHVIII的表达水平显著高于含有CaMV 35S启动子的转化子。一氧化氮合酶启动子被广泛应用于高等植物的遗传操作，但很少用于微藻的转化。这些结果表明，这种异源启动子有可能成为微藻基因操作的有效系统。(C)2013年美国化学工程师协会生物技术研究所。程序，29：319328,2013年
Despite the biotechnological interest of microalgae, no robust and stable methods for genetic transformation of most microalgal strains exist. The scanty and disperse data about the efficiency of heterologous promoters in microalgae and the use of different transformation methods, DNA quantities and reporter genes in the existing studies makes very difficult a real comparison of their efficiency. Using Chlamydomonas reinhardtii as a host, we have evaluated the efficiency of the heterologous promoters of cauliflower mosaic virus 35S (CaMV 35S) and Agrobacterium nopaline synthase (NOS) genes. These promoters were fused to the paromomycin conferring-resistance aminoglycoside 3-phosphotransferase encoding gene (APHVIII), and C. reinhardtii was transformed by the glass beads agitation method. The transformation efficiency and the APHVIII transcript and protein levels were evaluated in a series of transformants for each promoter. The chimeric promoter HSP70A/RBCS2 and the promoter-less APHVIII marker gene were used for comparison. We found significantly higher transformation efficiencies and higher level of APHVIII expression in those transformants harboring the NOS promoter than in those transformed with CaMV 35S promoter. The NOS promoter, widely used for genetic manipulation of higher plants, has been very rarely used for the transformation of microalgae. The results shown here suggest the possibilities of this heterologous promoter as an efficient system for the genetic manipulation of microalgae. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 319328, 2013