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

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

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (4): 713-723.doi: 10.16420/j.issn.0513-353x.2022-0100

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

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

Received:2022-11-30 Revised:2023-02-02 Online:2023-04-25 Published:2023-04-27
Contact: *（E-mail：cxdong@njau.edu.cn）

Abstract

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

CLC Number:

S661.2

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.

Figures/Tables 12

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

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

Fig. 2 Prediction of transmembrane structure of PbKT12 protein

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

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

Fig. 5 Subcellular localization of PbKT12 protein

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.

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

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

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

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.

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

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

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