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A chimeric vacuolar Na+/H+ antiporter gene evolved by DNA family shuffling confers increased salt tolerance in yeast

Author:
Wu, Guangxia, Wang, Gang, Ji, Jing, Li, Yong, Gao, Hailing, Wu, Jiang, Guan, Wenzhu
Source:
Journal of biotechnology 2015 v.203 pp. 1-8
ISSN:
0168-1656
Subject:
DNA, DNA libraries, Salicornia europaea, Suaeda, amino acids, breeding, cellular microenvironment, chimerism, crops, genes, halophytes, homeostasis, hygromycin B, lithium chloride, mutants, point mutation, potassium, recombinant fusion proteins, salt tolerance, screening, sodium, sodium-hydrogen antiporter, vacuoles, yeasts
Abstract:
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 SsNHX1and 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 LiCl and a similar tolerance to hygromycin B compared with the parental proteins in yeast.
Agid:
5521162