期刊
JOURNAL OF BIOTECHNOLOGY
卷 203, 期 -, 页码 1-8出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2015.02.033
关键词
DNA family shuffling; Na+/H+ antiporter; Salt tolerance; Yeast
资金
- National Natural Science Foundation of China [31271419, 31271793]
- National Science and Technology Key Project of China on GMO Cultivation for New Varieties [2014ZX08003-002B]
The vacuolar Na+/H+ antiporter plays an important role in maintaining ionic homeostasis and the osmotic balance of the cell with the environment by sequestering excessive cytoplasmic Na+ into the vacuole. However, the relatively low Na+/H+ exchange efficiency of the identified Na+/H+ antiporter could limit its application in the molecular breeding of salt tolerant crops. In this study, DNA family shuffling was used to create chimeric Na+/H+ antiporters with improved transport activity. Two homologous Na+/H+ antiporters from halophytes Salicornia europaea (SeNHX1) and Suaeda salsa (SsNHX1) were shuffled to generate a diverse gene library. Using a high-throughput screening system of yeast complementation, a novel chimeric protein SseNHX1 carrying 12 crossover positions and 2 point mutations at amino acid level was selected. Expression of SseNHX1 in yeast mutant exhibited approximately 46% and 22% higher salt tolerance ability in yeast growth test than that of SsNHX1 and SeNHX1, respectively. Measurements of the ion contents demonstrated that SseNHX1 protein in yeast cells accumulated more Na+ and slightly more K+ than the parental proteins did. Furthermore, this chimera also conferred increased tolerance to LiC1 and a similar tolerance to hygromycin B compared with the parental proteins in yeast. (C) 2015 Elsevier B.V. All rights reserved.
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