梨果实钾转运体基因PbKT12的克隆与功能验证

doi:10.16420/j.issn.0513-353x.2022-0100

摘要/Abstract

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’（Pyrus pyrifolia Nakai）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2 730 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。

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

Abstract:

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 2 730 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.

Key words: pear, fruit, potassium transporter gene, function identification, Arabidopsis, X-ray fluorescence spectroscopy

中图分类号:

S661.2

阚丽平, 石晓倩, 杨晗, 金雨濛, 陈丽妍, 张丽娟, 徐阳春, 沈其荣, 董彩霞. 梨果实钾转运体基因PbKT12的克隆与功能验证[J]. 园艺学报, 2023, 50(4): 713-723.

KAN Liping, SHI Xiaoqian, YANG Han, JIN Yumeng, CHEN Liyan, ZHANG Lijuan, XU Yangchun, SHEN Qirong, DONG Caixia. Cloning and Function Identification of a Potassium Transporter Gene PbKT12 in Pear Fruit[J]. Acta Horticulturae Sinica, 2023, 50(4): 713-723.

图/表 12

表1 本试验中使用的引物

Table 1 Primers in this study

用途 Usage	引物 Primer	序列（5′-3′） Sequence
基因克隆Gene cloning	PbKT12-F/R	ATGTCAATAATGTCCGTGTCG TCAGACCATGTAAGTCATTCC
亚细胞定位Subcellular localization	PbKT12N-F/R	AGAGATCTCGAGCTCAAGCTTATGTCAATAATGTCCGTGTCG CCTTGCTCACCATCAGGATCCGGACCATGTAAGTCATTCCGA
亚细胞定位Subcellular localization	PbKT12-2288-F/R	ACCACCCATAATACCCATAAACCGGTCAACATGTGGAGCA GCGATGGCAAACAGCTATTATGCGTCACTGGATTTTGGTTT
qRT-PCR	qRT-PbKT12-F/R	CGTGGTTCGCTCTTGGTGGTT CCTGAATGGCTCGCACAGAGAA
内参基因Reference gene	Actin-F/R	CTCCCAGGGCTGTGTTTCCTA CTCCATGTCATCCCAGTTGCT
酵母表达载体构建pYES2 expression	pYES2-PbKT12-F/R	ACTATAGGGAATATTAAGCTTATGTCAATAATGTCCGTGTCG AGTGGATCCGAGCTCGGTACCTCAGACCATGTAAGTCATTCC
转基因载体的构建 pCAMBIA1305.1-expression	pCAMBIA1305.1- KT12-F/R	TCCTCTAGATCGACCTGCAGATGTCAATAATGTCCGTGTCG ACGACGGCCAGTGCCAAGCTTTCAGACCATGTAAGTCATTCCG

图1 PbKT12在‘黄冠’梨果实中的扩增结果

Fig. 1 The PCR product of PbKT1 in‘Huangguan’pear

图2 PbKT12 蛋白跨结构域预测

Fig. 2 Prediction of transmembrane structure of PbKT12 protein

图3 PbKT12氨基酸序列及其他物种同源序列的系统进化树分析

Fig. 3 The phylogenetic tree of PbKT12 amino acid sequence with other species of homologous sequence

图4 PbKT12 与其他植物 KT12蛋白同源序列比对

Fig. 4 Sequence alignment between PbKT12 and other plant KT12 proteins

图5 PbKT12 蛋白的亚细胞定位

Fig. 5 Subcellular localization of PbKT12 protein

图6 ‘黄冠’梨盆栽土壤施钾处理下PbKT12在叶片和果实中的相对表达量不同小写字母表示相同时期不同处理间差异显著（P < 0.05）。下同。

Fig. 6 The relative expression of PbKT12 gene in leaf and fruit of‘Huangguan’pear under different potassium levels Different lowercase letters indicate significant differences of root growth index（P < 0.05）. The same below.

图7 PbKT12 蛋白对K+ 吸收缺陷型酵母菌株R5421的回补作用

Fig. 7 Functional complementation test of PbKT12 in yeast R5421 for absorbing K+ from culture medium containing different levels of K+

图8 供钾（+ K）和缺钾（-K）拟南芥过表达株系PbKT12-OE及野生型（WT）生根（A）和开花（B）表型

Fig. 8 The rooting（A）and flowering（B）phenotypes of the potassium donor（+ K）and potassium deficient（-K）Arabidopsis thaliana overexpression lines PbKT12-OE and WT

表2 培养液中钾对拟南芥过表达株系PbKT12-OE和野生型（WT）根系生长和开花数的影响

Table 2 Effect of potassium in culture medium on root growth and flowering number of PbKT12-OE and WT

拟南芥株系 Arabidopsis	MS培养液		根平均直径/mm Mean root diameter	根表面积/cm² Root surface area	总根长/cm Total root length	单株开花数 Blossom number
拟南芥株系 Arabidopsis	处理	K⁺/（mmol · L^-1）	根平均直径/mm Mean root diameter	根表面积/cm² Root surface area	总根长/cm Total root length	单株开花数 Blossom number
野生型WT	-K	0	0.18 ± 0.03 b	7.18 ± 1.37 c	69.28 ± 25.51 b	5.00 ± 1.10 c
	+ K	20	0.19 ± 0.02 b	9.69 ± 1.50 b	97.99 ± 18.47 b	3.50 ± 1.05 c
PbKT12-OE	-K	0	0.29 ± 0.09 a	7.17 ± 1.60 c	75.09 ± 28.57 b	8.17 ± 1.33 b
	+ K	20	0.18 ± 0.02 b	12.19 ± 1.70 a	156.63 ± 38.76 a	10.67 ± 2.73 a

图9 供钾（+ K）和缺钾（-K）条件下拟南芥过表达株系PbKT12-OE及野生型（WT）植株中K+的分布图中红色表示K+含量高，蓝色表示K+含量低。

Fig. 9 Distribution of K+ in Arabidopsis PbKT12-OE and WT plants with potassium supply（+ K）and potassium deficiency（-K）conditions Red indicates high K+ content and blue indicates low K+ content.

图10 供钾（+ K）和缺钾（-K）条件下拟南芥过表达株系PbKT12-OE及野生型（WT）植株中葡萄糖的积累量

Fig. 10 Glucose accumulation in Arabidopsis PbKT12-OE and WT with potassium supply（+ K）and potassium deficiency（-K）conditions

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