A novel plant vacuolar Na+/H+ antiporter gene evolved by DNA shuffling confers improved salt tolerance in yeast Source: Journal of Biological Chemistry Authors: Kai Xu, Tao Xia 報告者 : 吴广霞

ABSTRACT Plant vacuolar Na+/H+ antiporters play important roles in maintaining cellular ion homeostasis and mediating the transport of Na+ out of the cytosol and into the vacuole. Vacuolar antiporters have been shown to play significant roles in salt tolerance, however the relatively low Vmax of the Na+/H+ exchange of the Na+/H+ antiporters identified could limit its application in the molecular breeding of salt tolerant crops. In this study, we applied DNA shuffling methodology to generate and recombine the mutations of Arabidopsis thaliana vacuolar Na+/H+ antiporter gene AtNHX1. Screening using a large scale yeast complementation system identified AtNHXS1, a novel Na+/H+ antiporter. Expression of AtNHXS1 in yeast showed that the antiporter localized to the vacuolar membrane and that its expression improved the tolerance of yeast to NaCl, KCl, LiCl and hygromycin B. Measurements of the ion transport activity across the intact yeast vacuole demonstrated that the AtNHXS1 protein showed higher Na+/H+ exchange activity and a slightly improved K+/H+ exchange activity.

DNA 改组 DNA 改组是 DNA 分子的体外重组，是基因在分子水平上有性重组 ( sexual recombination) 。通过改变单个基因 ( 或基因家族， gene family) 原有核苷酸序 列, 创造新基因, 并赋予表达产物以新功能。实际上, 该技术是一种分子水平上 的定向进化 (directed evolution) 。因此也称为 子 育 种 ( molecular-breeding) 。 在创造新基因的过程中, 要设法产生各种变异。 DNA 改组的原理 DNA 改组实际上是依赖于 PCR 的体外诱变技术。它是将单个基因或相关基因家 族的靶序列通过物理或化学方法随机片段化, 由于这些小片段之间具有一定的 同源性, 通过无引物 PCR 和有引物 PCR 组装成全长的嵌合体基因即嵌合体文库。 然后对嵌合体文库进行高通量或超高通量的筛选, 选择具有改进功能或全新功 能的突变体作为下轮 DNA 改组的模板, 重复上述步骤, 进行多轮改组和高通量 的筛选, 直到获得理想的突变体。

Schematic of DNA shuffling method

EXPERIMENTAL PROCEDURES TY001 W303-1B Δena1-2 (Δena1-2::HIS3) NHX1 deletion mutant TY001 (Δnhx1::TRP1) Δena1-2 Δena1-4Δnhx1 (Δena1-4::HIS3, Δnhx1::TRP1) Δena1-4Δnhx1 Yeast strains 1 Δnhx1::TRP1

pGM-T-AtNHX1 AtNHX1 2µg products 100 and 200 bp fragments purified fragments (1µg) Reassemble full-length sequences a single band with the correct size PCR amplification DNase Idigestion 2 min primerless PCR primer PCR DNA shuffling of AtNHX1 2

3 The construction of shuffled gene library and screening for high salt tolerance yeast clones The reassembled fragment of the correct size ligation pYPGE15 transformationlithium acetate method Δena1-4::HIS3,Δnhx1::TRP1 APG selective medium 100mM NaCl OD600=0.5, 5µl cells Early screening 30 ℃ for 4 days APG, 125mM NaCl the second selection 30 ℃ for 4 days

Yeast Transformation, drop tests and intracellular ion content determination 4 pYPGE15- AtNHX1/ AtNHXS1 Δena1-4::HIS3 ， Δnhx1::TRP1 pYPGE15 W303-1B, Δena1-2::HIS3 Δena1-4::HIS3 ， Δnhx1::TRP1 transformation For drop tests, the cells of different strains were harvested by centrifugation and resuspended into APG medium and adjusted the OD600 to 0.5. Serial 5 times dilutions were made and 5µl of the cells were loaded onto APG medium with different salt supplements.

AtNHX1-GFP localization and con-focal microscopy 5 pYPGE15-GFP3 TY001 pYPGE15- AtNHX1-GFP3 TY001 pYPGE15- AtNHXS1-GFP3 TY001

Transport assay 5

Sequence analysis of shuffled AtNHX1 6

L29P, S158P, Y241P, F242L

Salt tolerance conferred by mutagenesis AtNHX1 and truncated AtNHX1 7

DISCUSSION An appropriate digested fragment length is crucial for a successful shuffling. A suitable selective model and expression system are key factors for successful mutant screening.

DISCUSSION Y241P and F242L would not play any role in cation or proton transport. As proline is an helix breaker which can disrupt the alpha-helices’ structure, it is possible that the L29P or S158P substitutions resulted in the alteration of Na+/H+ antiporter structure, favoring Na+/H+ exchange.

CONCLUSIONS 1.This shuffled clone displayed enhanced tolerance to NaCl, KCl, LiCl and hygromycin B. 2. The amino acids mutations and C-terminus partial deletion in AtNHXS1 did not alter the localization of the antiporter, and increased the antiporter Na+/H+ exchange activity, thus enabling a higher NaCl tolerance.

CONCLUSIONS 3.AtNHXS1 displayed a lesser increment in the Vmax of K+/H+ exchange and there was some increase in yeast KCl tolerance. 4.The C-terminus deletion and the mutations in the putative cation transport path and the mutations in the cation binding domain of the Na+/H+ antiporter increased the Na+/H+ exchange activity and the Na+ selectivity, whereas decreased the Na+ and K+ affinity.