Biochemistry Laboratory, Experimental Sciences Faculty, University of Huelva, Huelva 27071, Spain.
Mar Drugs. 2012 Dec 4;10(12):2749-65. doi: 10.3390/md10122749.
The lack of highly active endogenous promoters to drive the expression of transgenes is one of the main drawbacks to achieving efficient transformation of many microalgal species. Using the model chlorophyte Chlamydomonas reinhardtii and the paromomycin resistance APHVIII gene from Streptomyces rimosus as a marker, we have demonstrated that random insertion of the promoterless marker gene and subsequent isolation of the most robust transformants allows for the identification of novel strong promoter sequences in microalgae. Digestion of the genomic DNA with an enzyme that has a unique restriction site inside the marker gene and a high number of target sites in the genome of the microalga, followed by inverse PCR, allows for easy determination of the genomic region, which precedes the APHVIII marker gene. In most of the transformants analyzed, the marker gene is inserted in intragenic regions and its expression relies on its adequate insertion in frame with native genes. As an example, one of the new promoters identified was used to direct the expression of the APHVIII marker gene in C. reinhardtii, showing high transformation efficiencies.
缺乏高效的内源性启动子来驱动转基因的表达是实现许多微藻物种高效转化的主要障碍之一。我们使用模式绿藻莱茵衣藻和来自棘孢小单孢菌的博莱霉素抗性 APHVIII 基因作为标记，已经证明，标记基因的无启动子随机插入和随后最稳健转化体的分离，可以鉴定出微藻中新型强启动子序列。用一种内切酶消化基因组 DNA，该内切酶在标记基因内有一个独特的限制位点，在微藻基因组中有大量的靶位点，然后进行反向 PCR，就可以轻松确定 APHVIII 标记基因之前的基因组区域。在分析的大多数转化体中，标记基因插入到基因内区域，其表达依赖于与内源基因适当的框内插入。例如，鉴定出的一个新启动子被用于指导 APHVIII 标记基因在莱茵衣藻中的表达，显示出很高的转化效率。
