杜梨PbNHX1基因的克隆、表达分析及功能验证

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

【目的】克隆1个杜梨Na^+/H^+逆向转运蛋白基因PbNHX1,并对其序列特征、表达特点及功能进行研究。【方法】采用RT-PCR和PCR克隆PbNHX1的c DNA和DNA序列,利用生物信息学方法进行序列分析,定量PCR检测其在非生物胁迫下转录水平变化,酵母互补试验检测PbNHX1基因的离子转运能力。【结果】杜梨PbNHX1基因c DNA编码区长1 704 bp,对应基因组DNA序列长3 594 bp,由13个外显子和12个内含子组成,编码蛋白含567个氨基酸。系统进化树显示,PbNHX1位于液泡膜型Na^+/H^+逆向转运蛋白分支上,与杨树液泡膜型Na^+/H^+逆向转运蛋白Pt NHX1.3基因亲缘关系较近。正常生长条件下,PbNHX1在杜梨叶片中表达量高于根。施加200 mmol·L-1Na Cl、10%(φ)PEG6000或100μmol·L-1ABA后,PbNHX1在叶片中的转录水平持续上升;其在根中的表达量先升后降,表达高峰依次出现在处理后6、3和6 h。PbNHX1的转入可恢复Na Cl、KCl和潮霉素B对nhx1缺失酵母菌株AXT3的生长抑制,转基因酵母细胞中Na^+和K^+含量显著增加。【结论】PbNHX1具有植物NHXs基因家族的固有特征,对盐碱、渗透胁迫和ABA处理均存在转录响应,转入该基因能够提高酵母nhx1缺失突变体AXT3对盐胁迫的耐受能力,部分恢复其对阳离子的转运功能,从而促进Na^+、K^+积累。

[Objective]Pyrus betulaefolia is one of the main rootstocks for pear P. betulaefolia as a root- stock 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 expres- sion characteristics were analyzed. [Methods] The sodium/hydrogen exchanger 1-like gene （XM_ 018651314.1） in Pyrus^bretschneideri （Chinese white pear） was used as the electronic probe to search the transcriptome database of P. betulaefolia NaCl-treated seedlings. Then, one transcript Pbr017120 was obtained for designing gene-specific primers. Indeed, its cDNA and DNA sequences were isolated using RT-PCR and PCR techniques. And the physical and chemical properties of PbNHX1 protein were ana- lyzed 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 NaCl, 10% （φ） PEG6000 and 100 μmol. L-1 ABA, were analyzed by quantitative PCR （qRT- PCR）. Finally, yeast complementary experiments with a salt sensitive yeast mutant AXT3 were performed to verify the functions of PbNHX1 gene. After solid and liquid cultivation, the growth status of transgenic yeast was detected. Their total Na＋ and K＋ contents were also tested by a flame-graphite furnace atomic absorption spectrometer. [Results] The cDNAs 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 （pI） was 62.179 ku and 5.55, respectively. Moreover, the PbN- HX1 elements consisted of C2 861H4437 N6690805S2b The phylogenetic tree showed that PbNHX1 was located in the branch of vacuolar Na＋/H＋ antiporter, which was far from the plasma membrane Na＋/H＋ antiporter gene of Arabidopsis AtNHX7 or rice OsNHX8, and was closely related to poplar vacuolar Na＋/H＋ antiporter gene PtNHX1.3 or Arabidopsis AtNHX2. The expression level of the PbNHX1 was higher in the leaves than that in its roots under normal growth conditions. After treatment with 200 mmol. L-1 NaC1, PbNHX1 transcriptional level obviously increased both in the roots and in the leaves. For example, PbNHX1 expression level increased firstly and then decreased in the roots once the seedlings were treated with salt. Its expression peak appeared at 6 h. At that time, the amount of PbNHX1 transcription was 2.4 times higher than that of the control. Then, its expression level began to decrease closely to the original level at 24 h. On the one hand, the expression level of PbNHX1 kept on rising in the leaves after the treatment of salt. In the case of 10% PEG6000, PbNHX1 expression level increased firstly and then decreased in the roots. Its expression peak appeared at 3 h, which was 1.2 times higher than that of the control. After that time, its expression level turned to fall down and recovered closely to the original level at 24 h. In the leaves, the expression level of PbNHX1 continued to rise when the salt existed and it was 22.6 times higher than that of the control at 24 h. After 100 p, mol. L-1 ABA treatment, PbNHX1 expression level increased firstly and then decreased 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 level of 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 inhibition of NaC1, KC1 and hygromycin B to the nhxl mutant yeast strain AXT3 when the cells were treated with 20 mg. L-1 hygromycin B, 20-50 mmol. L-1 NaC1 or 0.5-1.00 tool- L-1 KC1. Meanwhile, the results of liquid culture showed that transform of PbNHX1 reduced the growth inhibition of the mutant strain AXT3 to NaC1 and KC1 when the cells were treated with 50-100 retool-L-＇ NaC1 or 0.5-1.0 mol. L-＇ KC1. Fur- thermore, the contents of Na＋ and K＋ significantly increased in PbNHX1 transgenic yeast cells compared with the mutant strain without this gene when 20 mmol. L-1 NaC1 presence. [Conclusion] Our results have showed that PbNHX1 gene belong to the NHX gene family of P. betulaefoli, which has the inherent characteristics of plant NHXs family. This gene response to NaC1, osmotic and ABA stresses. Transfer of the PbN- HX1 gene can increase the salt tolerance of the nhxl mutant yeast strain AXT3 and partly recover its ion transport capacity and facilitate the accumulation of the Na＋ and K＋ ions.