梨果实钾转运体基因PbKT12的克隆与功能验证  

Cloning and Function Identification of a Potassium Transporter Gene PbKT12 in Pear Fruit

作　　者：阚丽平 石晓倩 杨晗 金雨濛 陈丽妍 张丽娟[1] 徐阳春[1] 沈其荣[1] 董彩霞[1] KAN Liping;SHI Xiaoqian;YANG Han;JIN Yumeng;CHEN Liyan;ZHANG Lijuan;XU Yangchun;SHEN Qirong;DONG Caixia(Jiangsu Provincial Key Laboratory for Organic Solid Waste Utilization,National Engineering Research Center for Organic-based Fertilizers of Ministry of Education,Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization,College of Resources and Environmental Sciences,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学资源与环境科学学院,江苏省固体有机废弃物资源化研究重点实验室,江苏省有机固体废弃物协同创新中心,教育部资源节约型肥料工程技术研究中心,南京210095

出　　处：《园艺学报》2023年第4期713-723,共11页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(32272809);国家现代农业产业技术体系建设专项资金项目(CARS-28)。

摘　　要：PbKT12是基于梨果实转录组分析筛选出的对施钾有强烈响应的钾转运体基因,本研究中进一步验证其功能。以‘黄冠’(PyruspyrifoliaNakai)梨果实为材料克隆得到PbKT12全长序列,分析表明该基因序列共2730 bp,编码909个氨基酸;序列对比表明PbKT12与苹果中同源蛋白氨基酸序列相似性高达97%。亚细胞定位结果显示,PbKT12定位于细胞质膜上。PbKT12转化钾吸收缺陷型酵母R5421在低于5 mmol·L^(-1)K^(+)的培养基上能够恢复生长。qRT-PCR结果表明,PbKT12在梨果实和叶片中的相对表达量随盆栽土壤施钾水平增加而升高。使用农杆菌侵染花序法将PbKT12导入拟南芥,与野生型拟南芥相比,过表达PbKT12拟南芥植株的生长速度比野生型更快,抽薹、开花时间更早,植株葡萄糖积累量更高。采用X射线荧光光谱仪无损检测K^(+)在拟南芥植株中的分布发现,在缺钾胁迫下过表达PbKT12拟南芥中K^(+)向花序运输的量增加。因此推断,PbKT12在梨果实中具有促进K^(+)转运和葡萄糖积累的作用。PbKT12 is a potassium(K)transporter gene identified from transcriptome analysis of pear fruit in response to K supply.In order to verify the function of PbKT12,full length PbKT12 gene from‘Huangguan’pear fruit was cloned and analyzed its amino acid sequence by bioinformatics.The total length of the gene sequence is 2730 bp,encoding 909 amino acids.The amino acid sequence similarity between pear PbKT12 protein and apple homologous protein was up to 97%.Subcellular localization showed that PbKT12 protein was found on the plasma membrane.The yeast mutant strain R5421 transformed by PbKT12 gene recovery growth in the medium containing less than 5 mmol·L^(-1)K^(+).qRT-PCR showed that the relative expression level of PbKT12 in pear fruit and leaves increased under high K^(+)level.The PbKT12 was transformed in Arabidopsis by Agrobacterium-mediated method.Compared with the wild-type,the PbKT12 transgenic plants grew faster with earlier bolting and flowering,and they had higher glucose accumulation.The distribution of K^(+)in Arabidopsis plants was detected by X-ray fluorescence spectrometer.It was found that K^(+)was preferentially transported to inflorescence of Arabidopsis under potassium deficiency.In conclusion,PbKT12 protein may play an important role in transport of potassium and promotion of glucose accumulation in pear fruit.

关 键 词：梨 果实 钾转运体基因 功能验证 拟南芥 X射线荧光光谱 

分 类 号：S661.2[农业科学—果树学]