Directed Evolution of Dunaliella salina Ds-26-16 and Salt-Tolerant Response in Escherichia coli.
Directed Evolution of Dunaliella salina Ds-26-16 and Salt-Tolerant Response in Escherichia coli.
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盐生杜氏藻 Ds-26-16 的定向进化和大肠杆菌的耐盐反应
DOI：
10.3390/ijms17111813
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发表时间：
2016-10-29
影响因子：
5.6
通讯作者：
Chen X
中科院分区：
文献类型：
--
作者：
Guo Y;Dong Y;Hong X;Pang X;Chen D;Chen X
耐盐基因的鉴定和进化是利用生物技术开发耐盐作物或微生物的关键步骤。 Ds-26-16 是从杜氏盐藻中分离出来的耐盐基因，它编码的转录因子可以赋予多种生物体耐盐性，包括大肠杆菌 (E. coli)、雨生红球藻和烟草。为了进一步提高其耐盐性，利用脱氧肌苷三磷酸介导的易错PCR技术构建随机诱变文库，然后利用基于其广谱耐盐性的大肠杆菌表达系统进行筛选。获得了七个具有增强耐盐性的变体。与野生型基因的 1.03 M 相比，含有突变 S32P 的变体 EP-5 显示出最大的改进，大肠杆菌转化体耐受盐浓度高达 1.54 M。此外，Ds-26-16和EP-5还赋予大肠杆菌转化体对冷冻、冷、热、Cu2+和碱的耐受性。同源模型显示EP-5中的突变S32P引起N端和C端α螺旋的构象变化。 Ds-26-16和EP-5的表达维持正常的细胞形态，增加细胞内抗氧化酶活性，降低丙二醛含量，并刺激一氧化氮合成，从而增强大肠杆菌转化体的耐盐性。
Identification and evolution of salt tolerant genes are crucial steps in developing salt tolerant crops or microorganisms using biotechnology. Ds-26-16, a salt tolerant gene that was isolated from Dunaliella salina, encodes a transcription factor that can confer salt tolerance to a number of organisms including Escherichia coli (E. coli), Haematococcus pluvialis and tobacco. To further improve its salt tolerance, a random mutagenesis library was constructed using deoxyinosine triphosphate-mediated error-prone PCR technology, and then screened using an E. coli expression system that is based on its broad-spectrum salt tolerance. Seven variants with enhanced salt tolerance were obtained. Variant EP-5 that contained mutation S32P showed the most improvement with the E. coli transformant enduring salt concentrations up to 1.54 M, in comparison with 1.03 M for the wild type gene. Besides, Ds-26-16 and EP-5 also conferred E. coli transformant tolerance to freezing, cold, heat, Cu2+ and alkaline. Homology modeling revealed that mutation S32P in EP-5 caused the conformational change of N- and C-terminal α-helixes. Expression of Ds-26-16 and EP-5 maintained normal cellular morphology, increased the intracellular antioxidant enzymatic activity, reduced malondialdehyde content, and stimulated Nitric Oxide synthesis, thus enhancing salt tolerance to E. coli transformants.
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影响因子：
3.6
作者：
Koshkin, AA
通讯作者：
Koshkin, AA
影响因子：
3.5
作者：
Mangano, Silvina;Silberstein, Susana;Santa-Maria, Guillermo E.
通讯作者：
Santa-Maria, Guillermo E.
蛋白质组学分析揭示杜氏盐藻 Ds-26-16 基因增强大肠杆菌耐盐性的机制
影响因子：
3.7
作者：
Wang Y;Hu B;Du S;Gao S;Chen X;Chen D
通讯作者：
Chen D
影响因子：
3.7
作者：
Bazakos C;Manioudaki ME;Therios I;Voyiatzis D;Kafetzopoulos D;Awada T;Kalaitzis P
通讯作者：
Kalaitzis P
影响因子：
4.3
作者：
Liang, YC;Chen, Q;Ding, RX
通讯作者：
Ding, RX