‘黄冠梨’钾转运体基因PbKT8的克隆与表达分析

Cloning and expression analysis of potassium transporter gene PbKT8 in ‘Huangguan’ pear

[目的]本文旨在克隆‘黄冠梨’钾转运体 PbKT 8 基因,对基因定位及表达特征进行初步分析,为其功能研究提供基础。[方法]从‘黄冠梨’果实中克隆 PbKT 8 基因的cDNA全长序列,利用生物信息学对其同源序列进行分析,构建含GFP载体并通过激光共聚焦显微镜精确定位蛋白位置,实时荧光定量PCR分析不同施钾处理下该基因在果实成熟期的表达特征。[结果]钾转运体 PbKT 8 基因序列全长2 328 bp,编码775个氨基酸,PbKT8蛋白与苹果( Malus domestica )进化关系最相近,同源序列相似性高达99.10%;亚细胞定位结果显示,PbKT8蛋白定位于细胞膜上;PbKT 8 基因转化酵母突变菌株R5421在低于5 mmol · L^-1 K^+培养基上恢复生长;qRT-PCR结果表明, PbKT 8 基因在果实和叶片中相对表达量随施钾水平增加而上升。[结论] PbKT8蛋白定位在质膜上具有转运钾离子的功能,在果实成熟期受高钾响应表达。

[Objectives] The study cloned the potassium transporter gene PbKT 8 from‘Huangguan’pear fruits and analyzed its genetic mapping and expression characteristics,which provided the basis for further research.[Methods] The whole cDNA sequence PbKT 8 gene was cloned from‘Huangguan’pear,and its amino acid sequence was analyzed by bioinformatics,we had constructed the subcellular localization vector GFP and analyzed the spatial expression by confocal laser scanning microscope,and then the real-time fluorescence quantitative PCR technology was used to explore its expression pattern in fruit under different potassium concentrations.[Results] The full length of potassium transporter PbKT 8 cDNA sequence was 2 328 bp,encoding 775 amino acids. The phylogenetic analysis of amino acid homologous showed that the PbKT8 had the highest similarity(99.10%)with the apples( Malus domestica ). PbKT8 was verified to be located in the cytoplasm. The yeast mutant strain R5421 containing PbKT 8 gene resumed growth in the 5 mmol · L^-1 K + mediums. qRT-PCR analysis showed that the expression of PbKT 8 gene increased with the potassium concentration in fruit and leaf.[Conclusions] PbKT8 is located in the cytoplasm and has a significant impact on potassium absorption,and it can be induced by high potassium at the fruit maturity.