Wu Guangxia, Wang Gang, Ji Jing, Li Yong, Gao Hailing, Wu Jiang, Guan Wenzhu
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.
School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, People's Republic of China.
J Biotechnol. 2015 Jun 10;203:1-8. doi: 10.1016/j.jbiotec.2015.02.033. Epub 2015 Mar 14.
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.
液泡型Na(+)/H(+)逆向转运蛋白通过将过量的细胞质Na(+)隔离到液泡中，在维持细胞离子稳态和细胞与环境的渗透平衡方面发挥着重要作用。然而，已鉴定的Na(+)/H(+)逆向转运蛋白相对较低的Na(+)/H(+)交换效率可能会限制其在耐盐作物分子育种中的应用。在本研究中，采用DNA家族改组技术创建具有更高转运活性的嵌合Na(+)/H(+)逆向转运蛋白。将盐生植物欧洲海蓬子（SeNHX1）和碱蓬（SsNHX1）的两个同源Na(+)/H(+)逆向转运蛋白进行改组，以产生一个多样化的基因文库。利用酵母互补高通量筛选系统，筛选出一种在氨基酸水平上具有12个交叉位置和2个点突变的新型嵌合蛋白SseNHX1。在酵母突变体中表达SseNHX1，在酵母生长试验中，其耐盐能力分别比SsNHX1和SeNHX1高出约46%和22%。离子含量测定表明，酵母细胞中的SseNHX1蛋白比亲本蛋白积累了更多的Na(+)和略多的K(+)。此外，与酵母中的亲本蛋白相比，这种嵌合体对LiCl的耐受性也有所提高，对潮霉素B的耐受性相似。
