title: A chimeric vacuolar Na(+)/H(+) antiporter gene evolved by DNA family shuffling confers increased salt tolerance in yeast.
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.
authors.affiliation: School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, People's Republic of China; School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China.
authors.forename: Guangxia
authors.initials: G
authors.lastname: Wu
authors.affiliation: School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, People's Republic of China. Electronic address: gangwang@tju.edu.cn.
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.affiliation: School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, People's Republic of China.
authors.forename: Jing
authors.initials: J
authors.lastname: Ji
authors.affiliation: Key Laboratory of Ecosystem Network Observation and Modeling, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Science, Beijing 100101, People's Republic of China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Li
authors.affiliation: School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, People's Republic of China.
authors.forename: Hailing
authors.initials: H
authors.lastname: Gao
authors.affiliation: School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China; Department of Agronomy, Tianjin Agricultural University, Tianjin 300384, People's Republic of China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Wu
authors.affiliation: School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, People's Republic of China.
authors.forename: Wenzhu
authors.initials: W
authors.lastname: Guan
mesh_terms: D019663:Chenopodiaceae / Q000235:genetics*; D032621:DNA Shuffling; D018744:DNA, Plant / Q000235:genetics; D017343:Genes, Plant; D010940:Plant Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D055049:Salt Tolerance / Q000235:genetics*; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics*; D014617:Vacuoles
chemical_list: D018744:DNA, Plant; D010940:Plant Proteins; D017923:Sodium-Hydrogen Exchangers
keywords: DNA family shuffling; Na(+)/H(+) antiporter; Salt tolerance; Yeast