Nourseothricin N-acetyl transferase (NAT), a new selectable marker for nuclear gene expression in Chlamydomonas
Nourseothricin N-acetyl transferase (NAT), a new selectable marker for nuclear gene expression in Chlamydomonas
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DOI：
10.1186/s13007-019-0526-5
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发表时间：
2019-11-19
期刊：
影响因子：
5.1
通讯作者：
Pan, Junmin
中科院分区：
文献类型：
--
作者：
Yang, Xinjia;Peng, Jialin;Pan, Junmin
背景资料：莱茵衣藻是一种单细胞绿色真菌，是基础研究和生物技术应用中最常用的模式生物。通常需要选择标记的转基因菌株的产生在此类研究/应用中是有用的。与其他生物相比，该生物的选择标记数量有限。诺尔丝菌素（NTC）N-乙酰转移酶（NAT）已被报道为多种生物体中的选择标记，但不包括C。莱因哈德氏菌因此，我们研究了NAT是否是有用的和有效的选择转基因菌株在C。莱因哈德氏菌NAT的成功应用将为该微藻以及其他微藻的选择标记提供新的选择。莱茵衣藻对浓度低至5 μ g/ml的NTC敏感。在已用潮霉素B和/或巴龙霉素抗性基因转化的菌株中，对诺尔丝菌素没有交叉抗性。合成来自诺尔斯链霉菌的密码子优化的NAT，并组装到不同的表达载体中，随后转化到衣原体中。用50 ng DNA进行10 μ g/ml NTC筛选，可获得约500个转化子。转化体表现出正常的生长速率，并且即使在没有选择的条件下也稳定至少10个月。我们成功地测试了NAT可以用作在具有巴龙霉素和潮霉素B抗性标记的菌株中异位表达IFT 54-HA的选择标记。进一步证明了将NAT与IPT 54-HA融合，再用FMDV 2A肽段处理，可显著提高IPT 54-HA阳性克隆的筛选率。reinhardtii，并提供证据表明NAT可用作转基因菌株的有效选择标记。为C.莱因哈德氏菌NAT与巴龙霉素和潮霉素B抗性基因相容，这允许多重选择。
Background: Chlamydomonas reinhardtii is a unicellular green alga, which is a most commonly used model organism for basic research and biotechnological applications. Generation of transgenic strains, which usually requires selectable markers, is instrumental in such studies/applications. Compared to other organisms, the number of selectable markers is limited in this organism. Nourseothricin (NTC) N-acetyl transferase (NAT) has been reported as a selectable marker in a variety of organisms but not including C. reinhardtii. Thus, we investigated whether NAT was useful and effective for selection of transgenic strains in C. reinhardtii. The successful use of NAT would provide alterative choice for selectable markers in this organism and likely in other microalgae.Results: C. reinhardtii was sensitive to NTC at concentrations as low as 5 mu g/ml. There was no cross-resistance to nourseothricin in strains that had been transformed with hygromycin B and/or paromomycin resistance genes. A codon-optimized NAT from Streptomyces noursei was synthesized and assembled into different expression vectors followed by transformation into Chlamydomonas. Around 500 transformants could be obtained by using 50 ng DNA on selection with 10 mu g/ml NTC. The transformants exhibited normal growth rate and were stable at least for 10 months on conditions even without selection. We successfully tested that NAT could be used as a selectable marker for ectopic expression of IFT54-HA in strains with paromomycin and hygromycin B resistance markers. We further showed that the selection rate for IFT54-HA positive clones was greatly increased by fusing IFT54-HA to NAT and processing with the FMDV 2A peptide.Conclusions: This work represents the first demonstration of stable expression of NAT in the nuclear genome of C. reinhardtii and provides evidence that NAT can be used as an effective selectable marker for transgenic strains. It provides alterative choice for selectable markers in C. reinhardtii. NAT is compatible with paromomycin and hygromycin B resistance genes, which allows for multiple selections.