A stable and efficient nuclear transformation system for the diatom Chaetoceros gracilis
A stable and efficient nuclear transformation system for the diatom Chaetoceros gracilis
复制标题
DOI：
10.1007/s11120-014-0048-y
复制
发表时间：
2015-02-01
影响因子：
3.7
通讯作者：
Kashino, Yasuhiro
中科院分区：
文献类型：
--
作者：
Ifuku, Kentaro;Yan, Dongyi;Kashino, Yasuhiro
纤细角毛藻属于中心硅藻，近年来被用于光合作用的基础研究。此外，它已被商业化用于渔业，并作为生物燃料生产和生物精炼的原料也引起了人们的兴趣。本研究建立了一套高效的转基因系统。股薄肌使用含有各种启动子的质粒通过多脉冲电穿孔转化硅藻细胞，以驱动诺尔丝菌素乙酰转移酶基因（nat）作为选择标记的表达。转化效率达到类似于每10（8）个受体细胞400个阳性转基因克隆，这是在中心硅藻物种中利用电穿孔成功转化的第一个实例。我们进一步制备了两种表达载体：载体pCgLhcr 5 p含有岩藻黄质叶绿素a/c结合蛋白基因的光依赖性启动子，载体pCgNRp含有硝酸还原酶基因的诱导型启动子以驱动导入基因的表达。在两种载体中，乙酰辅酶A乙酰转移酶启动子驱动nat基因表达用于抗生素选择。荧光素酶基因和绿色荧光蛋白Azami-绿色基因等报告基因在转化的C.股薄肌细胞建立了一个高效、稳定的C. gracilis将能够对特定基因进行功能分析，并为进一步的生物技术应用进行菌株改良。
Chaetoceros gracilis belongs to the centric diatoms, and has recently been used in basic research on photosynthesis. In addition, it has been commercially used in fisheries and is also attracting interest as a feedstock for biofuels production and biorefinery. In this study, we developed an efficient genetic transformation system for C. gracilis. The diatom cells were transformed via multi-pulse electroporation using plasmids containing various promoters to drive expression of the nourseothricin acetyltransferase gene (nat) as a selectable marker. The transformation efficiency reached similar to 400 positive transgenic clones per 10(8) recipient cells, which is the first example of successful transformation with electroporation in a centric diatom species. We further produced two expression vectors: the vector pCgLhcr5p contains the light-dependent promoter of a fucoxanthin chlorophyll a/c binding protein gene and the vector pCgNRp contains the inducible promoter of a nitrate reductase gene to drive the expression of introduced genes. In both vectors, an acetyl-CoA acetyltransferase promoter drives nat gene expression for antibiotic selection. Stable integration and expression of reporter genes, such as the firefly luciferase and green fluorescent protein Azami-Green genes, were observed in transformed C. gracilis cells. This efficient and stable transformation system for C. gracilis will enable both functional analysis of diatom-specific genes and strain improvement for further biotechnological applications.