杜梨响应低钾和盐胁迫基因PbHAK17的克隆及功能分析

Cloning and functional analysis of PbHAK17 from Pyrus betulifolia in response to low potassium and salt stress

【目的】杜梨(Pyrus betulaefolia)是栽培中常用的耐盐胁迫砧木。对前期杜梨转录组数据筛选出的可能同时响应钾胁迫和盐胁迫的钾转运体基因PbHAK17进行生物信息学、时空表达和遗传转化分析,旨在明确PbHAK17响应钾营养吸收转运及耐盐的表达特征及作用机理。【方法】通过克隆PbHAK17基因,分析该蛋白结构以及系统进化关系。通过亚细胞定位和组织表达分析明确该基因的时空表达特征,采用钾缺陷型酵母和转基因拟南芥进行异源功能验证。在缺钾与适钾(0、3 mmol/L K^(+))、正常与盐胁迫(50、200 mmol/L Na^(+))条件下,培养超表达拟南芥株系与野生型拟南芥株系,分析其生长状况、K^(+)和Na^(+)积累量。【结果】PbHAK17基因长度为2370 bp,编码789个氨基酸。亚细胞定位结果表明,PbHAK17蛋白定位于细胞膜上。实时荧光定量PCR结果表明,PbHAK17主要在杜梨根系中表达,缺钾和盐胁迫诱导其表达量显著上调。酵母异源验证结果表明,在低钾条件下PbHAK17酵母转化子回补钾缺陷型酵母生长效果明显,同时在盐胁迫下仍可恢复缺陷型酵母生长。缺钾处理下,超表达PbHAK17拟南芥叶片和根部的K^(+)积累量均高于野生型拟南芥。同一盐浓度处理下,PbHAK17转基因拟南芥比野生型拟南芥莲座叶更大、更密,根系的K^(+)/Na^(+)值显著高于野生型拟南芥。【结论】杜梨钾转运体基因PbHAK17定位于细胞质膜上,缺钾和盐胁迫可以诱导杜梨PbHAK17基因在根部的过表达,提升植物中钾积累量和K^(+)/Na^(+)比例,进而提高杜梨的耐低钾和盐胁迫能力。

【Objectives】Pyrus betulifolia is a commonly cultivated rootstock with high salt tolerance.A potassium transporter gene,PbHAK17 that was screened out from Pyrus betulaefolia,seems respond to both K and salt stress.We studied its expression and function characteristics through bioinformatics,gene expression,and genetic transformation analysis.【Methods】Firstly,the PbHAK17 gene was cloned,and the protein structure and phylogenetic relationship were analyzed.Subcellular localization,K-deficient yeast,and transgenic Arabidopsis thaliana experiments were then employed to determine the function of the gene.The growth status,K^(+)and Na^(+)contents of overexpressed Arabidopsis lines and wild-type Arabidopsis lines were analyzed under K deficiency and normal K level(0 and 3 mmol/L K^(+)),and normal condition and salt stress(50 and 200 mmol/LNa^(+)).【Results】The PbHAK17 is 2370 bp in length and encodes 789 amino acids,and the PbHAK17 protein was localized on the cell membrane.K deficiency and salt stress induced a significant up-expression of PbHAK17 in the root of Pyrus betulifoliaand.In conditions of potassium deficiency,the PbHAK17 yeast transformant showed significant promotion effect on the growth of deficient yeast through supplementing K,and restored the growth of deficient yeast under salt stress.The K^(+)content in leaves and roots of Arabidopsis overexpressing PbHAK17 was higher than that of wild-type Arabidopsis under low K treatment.Under the same Na^(+)concentration,PbHAK17 transgenic Arabidopsis had larger and denser leaves than wild-type Arabidopsis,and the K^(+)/Na^(+)ratio of roots was significantly higher than that of wild-type Arabidopsis.【Conclusions】The pear potassium transporter gene PbHAK17 was localized in the plasma membrane of the cell,K deficiency and salt stress could induce the overexpression of PbHAK17 in the root system,increase K^(+)accumulation in root and leaves,and regulate the K^(+)/Na^(+)ratio in plant,bring about stronger tolerance of Pyrus betulifolia to K deficiency and salt stress.