Cloning, expression and functional analysis of PbNHX1 gene in Pyrus betulaefolia

Astract:【Objective】Pyrus betulaefolia is one of the main rootstocks for pear P. betulaefolia as a rootstock can effectively reduce Na+accumulation in the root, inhibit the Na+transportation to the scion, and improve the salt tolerance of pear varieties. In order to reveal the molecular mechanism of salt tolerance and provide experimental basis for the further research, we researched the Na+/H+antiporter protein in P.betulaefolia. One PbNHX1 gene was cloned from this species and its sequence characteristics and expression characteristics were analyzed.【Methods】The sodium/hydrogen exchanger 1-like gene (XM018651314.1) in Pyrus×bretschneideri (Chinese white pear) was used as the electronic probe to search the transcriptome database of P. betulaefolia Na Cl-treated seedlings. Then, one transcript Pbr017120 was obtained for designing gene-specific primers. Indeed, its c DNA and DNA sequences were isolated using RT-PCR and PCR techniques. And the physical and chemical properties of PbNHX1 protein were analyzed by Protparam online software. The introns and exons of PbNHX1 genes were analyzed using Gene Structure Display Server. The signature motifs were analyzed by MEME software and the phylogenetic tree was built by MEGA 6.0. The expression condition of PbNHX1 under the abiotic stresses, such as 200 mmol·L^-1 Na Cl, 10% (φ) PEG6000 and 100 μmol·L^-1 ABA, were analyzed by quantitative PCR (q RT-PCR) . Finally, yeast complementary experiments with a salt sensitive yeast mutant AXT3 were performedto verify the functions of PbNHX1 gene. After solid and liquid cultivation, the growth status of transgenicyeast was detected. Their total Na+and K+contents were also tested by a flame-graphite furnace atomicabsorption spectrometer.【Results】The c DNAs of PbNHX1 was 1 704 bp, and its DNA was 3 594 bp, which included 13 exons and 12 introns. This gene encoded a protein containing 567 amino acids, its rela-tive molecular weight and isoelectric point (p I) was 62.179 ku and 5.55, respectively. Moreover, the PbNHX1 elements consisted of C2 861 H4 437 N669 O805 S21. The phylogenetic tree showed that PbNHX1 was located inthe branch of vacuolar Na+/H+antiporter, which was far from the plasma membrane Na+/H+antiporter geneof Arabidopsis At NHX7 or rice Os NHX8, and was closely related to poplar vacuolar Na+/H+antiporter gene Pt NHX1.3 or Arabidopsis At NHX2. The expression level of the PbNHX1 was higher in the leaves than thatin its roots under normal growth conditions. After treatment with 200 mmol·L^-1 Na Cl, PbNHX1 transcrip-tional level obviously increased both in the roots and in the leaves. For example, PbNHX1 expression levelincreased firstly and then decreased in the roots once the seedlings were treated with salt. Its expressionpeak appeared at 6 h. At that time, the amount of PbNHX1 transcription was 2.4 times higher than that ofthe control. Then, its expression level began to decrease closely to the original level at 24 h. On the onehand, the expression level of PbNHX1 kept on rising in the leaves after the treatment of salt. In the case of10% PEG6000, PbNHX1 expression level increased firstly and then decreased in the roots. Its expressionpeak appeared at 3 h, which was 1.2 times higher than that of the control. After that time, its expressionlevel turned to fall down and recovered closely to the original level at 24 h. In the leaves, the expressionlevel of PbNHX1 continued to rise when the salt existed and it was 22.6 times higher than that of the con-trol at 24 h. After 100 μmol·L^-1 ABA treatment, PbNHX1 expression level increased firstly and then de-creased in the roots. Its expression peak appeared at 6 h, which was 1.6 times higher than that of the con-trol. Then its transcription declined closely to the original level at 24 h. In the leaves, the expression levelof PbNHX1 increased during the treatment period, which was 8.0 times higher than that of the control at 24 h. These results indicated that PbNHX1 was regulated significantly in the leaves under different abiotic stress. The results of YPD solid culture showed that transform of PbNHX1 could recover the growth inhi-bition of Na Cl, KCl and hygromycin B to the nhx1 mutant yeast strain AXT3 when the cells were treatedwith 20 mg·L^-1 hygromycin B, 20-50 mmol·L^-1 Na Cl or 0.5-1.00 mol·L^-1 KCl. Meanwhile, the results ofliquid culture showed that transform of PbNHX1 reduced the growth inhibition of the mutant strain AXT3 to Na Cl and KCl when the cells were treated with 50-100 mmol·L^-1 Na Cl or 0.5-1.0 mol·L^-1 KCl. Fur-thermore, the contents of Na+and K+significantly increased in PbNHX1 transgenic yeast cells comparedwith the mutant strain without this gene when 20 mmol·L^-1 Na Cl presence.【Conclusion】Our results haveshowed that PbNHX1 gene belong to the NHX gene family of P. betulaefoli, which has the inherent charac-teristics of plant NHXs family. This gene response to Na Cl, osmotic and ABA stresses. Transfer of the PbNHX1 gene can increase the salt tolerance of the nhx1 mutant yeast strain AXT3 and partly recover its iontransport capacity and facilitate the accumulation of the Na+and K+ions.