A nitrogen source-dependent inducible and repressible gene expression system in the red alga Cyanidioschyzon merolae.
A nitrogen source-dependent inducible and repressible gene expression system in the red alga Cyanidioschyzon merolae.
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DOI：
10.3389/fpls.2015.00657
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发表时间：
2015
影响因子：
5.6
通讯作者：
Miyagishima SY
中科院分区：
文献类型：
--
作者：
Fujiwara T;Kanesaki Y;Hirooka S;Era A;Sumiya N;Yoshikawa H;Tanaka K;Miyagishima SY
单细胞红藻Cyanidioschyzonmerolae是研究光合生物基础生物学的模式生物。梭merolae细胞由一组非常简单的细胞器组成。基因组已完全测序。最近建立了基因靶向和热休克诱导的基因表达系统。然而，条件基因敲低系统尚未建立，这是必要的细胞活力和原发性突变缺陷的基因的功能检查。在目前的研究中，我们首先评估了来自两个染色体中性位点的转基因的表达，所述两个染色体中性位点位于CMD 184 C和CMD 185 C之间的基因间区域以及URA5.3基因上游的区域。这两个基因座的表达量无显著差异，提示两者均可作为中性位点。然后，我们设计了一个诱导型和抑制型的基因表达，通过使用硝酸盐同化基因的启动子。硝酸盐同化基因如NR（硝酸还原酶）、NIR（亚硝酸还原酶）和NRT（硝酸盐/亚硝酸盐转运蛋白）的表达受氮源依赖性的可逆调节。我们构建了稳定的菌株，其中含有NR、NIR或NRT启动子和sfGFP基因的盒插入URA5.3上游区域，并检查了启动子的功效。NR、NIR和NRT启动子在硝酸盐培养基中组成型激活，而在铵存在下它们的活性极低。每个启动子的激活立即抑制在1小时内通过添加铵。因此，C.成功地建立了Merolae。启动子之间的活性不同，并且每种启动子可根据通过RNA测序估计的靶基因的表达水平进行选择。该方法适用于光合生物中目的基因的缺陷。
The unicellular red alga Cyanidioschyzon merolae is a model organism for studying the basic biology of photosynthetic organisms. The C. merolae cell is composed of an extremely simple set of organelles. The genome is completely sequenced. Gene targeting and a heat-shock inducible gene expression system has been recently established. However, a conditional gene knockdown system has not been established, which is required for the examination of function of genes that are essential to cell viability and primary mutant defects. In the current study, we first evaluated the expression of a transgene from two chromosomal neutral loci located in the intergenic region between CMD184C and CMD185C, and a region upstream of the URA5.3 gene. There was no significant difference in expression between them and this result suggests that both may be used as neutral loci. We then designed an inducible and repressible gene expression by using promoters of nitrate-assimilation genes. The expression of nitrate-assimilation genes such as NR (nitrate reductase), NIR (nitrite reductase), and NRT (the nitrate/nitrite transporter) are reversibly regulated by their dependence on nitrogen sources. We constructed stable strains in which a cassette containing the NR, NIR, or NRT promoter and sfGFP gene was inserted in a region upstream of URA5.3 and examined the efficacy of the promoters. The NR, NIR, and NRT promoters were constitutively activated in the nitrate medium, whereas their activities were extremely low in presence of ammonium. The activation of each promoter was immediately inhibited within a period of 1 h by the addition of ammonium. Thus, a conditional knockdown system in C. merolae was successfully established. The activity varies among the promoters, and each is selectable according to the expression level of a target gene estimated by RNA-sequencing. This method is applicable to defects in genes of interest in photosynthetic organism.