Cloning and Expression Analysis of Six Genes of the NPF Family in Tea Plants and Functional Verification of CsNPF7.3

doi:10.13560/j.cnki.biotech.bull.1985.2025-0353

Abstract

Abstract:

Objective Nitrate transporter protein 1/peptide transporter (NRT1/PTR, NPF) family plays a pivotal role in plant nitrogen uptake, hormone transport and stress response mechanisms. Analysis of CsNPFs gene characteristics and expression patterns in tea plants provides a theoretical basis for efficient nitrogen utilization and stress-resistant molecular breeding. Method Members of the CsNPFs gene family were identified by gene cloning and bioinformatics analysis. Tissue-specific expression profiles and transcriptional responses to hormones (IAA, ABA, GA₃) and nitrate (NO₃^-) treatments were investigated using RT-qPCR. The function of CsNPF7.3 was verified by heterologous expression in Arabidopsis thaliana. Result Full-length coding sequences (CDS) of six CsNPFs genes were successfully cloned, encoding hydrophobic transmembrane proteins. Phylogenetic analysis revealed the closest evolutionary relationship between CsNPFs and ClNPFsfrom Camellia lanceoleosa. Expression pattern analysis demonstrated root-specific dominance of CsNPF5.5 and CsNPF7.3, while other family members CsNPF2.13/2.7/3.1/7.1 showed preferential accumulation in the leaves. Hormonal treatments significantly modulated gene expression. CsNPF7.3 demonstrated marked upregulation in the roots, suggesting a primary role in root-based hormone signaling. CsNPF5.5 showed leaf-specific induction, indicating tissue-dependent regulatory mechanisms. Under NO₃⁻ treatment, leaf-expressed CsNPFs responded dynamically, with general down-regulation observed across all genes except CsNPF2.7 and CsNPF3.1. Notably, the expressions of CsNPF5.5 and CsNPF7.3 in the root were significantly up-regulated. The overexpression of CsNPF7.3 in A. thaliana showed that CsNPF7.3 significantly increased the biomass accumulation of Arabidopsis, enhanced the plant’s response to IAA, ABA and GA₃, and thus promoted the development of lateral roots, root elongation and the increased in petiole length. Conclusion CsNPFs play a significant role in the nitrogen absorption and hormone response processes of tea plants. Among them, CsNPF7.3 is a key functional gene in the root of tea plants, which can participate in regulating the growth and stress adaptability of tea plants.

Key words: Camellia sinensis, CsNPF gene family, gene cloning, plant hormones, NO₃?, expression analysis, CsNPF7.3, functional verification

HAN Yu, YUAN Qing-yun, ZHANG Qing-ping, WU Chun-lai, HE Wei, ZHANG Fen. Cloning and Expression Analysis of Six Genes of the NPF Family in Tea Plants and Functional Verification of CsNPF7.3[J]. Biotechnology Bulletin, 2025, 41(10): 264-276.

Figures/Tables 12

Table 1 Primers’ sequences for gene cloning and RT-qPCR analysis

目的 Purpose	引物名称 Primer name	前引物序列 Forward primer sequence （5′-3′）	后引物序列 Reverse primer sequence （5′-3′）
基因克隆 Gene cloneing	CsNPF2.13	CCCAAGATAAGGAAAAATGGAGGTA	CATAGATTTGTCAAAGTGACTTCAT
	CsNPF2.7	AGACTCACACTCGGAGGCATAAACT	ACAAACAACACCACAACAACAGATG
	CsNPF3.1	ATCCGACCGCCTCTACTCT	AGTTACAAACATATCGTTTAAGCTA
	CsNPF5.5	GTGAGAGTGGAAAAGACAACGCATA	CCTAACAGCAGCAGCAACCCT
	CsNPF7.1	TGGGGCTGTTTTATTAGTTGAG	AATATAGCCCGAAATAAGCCTGCA
	CsNPF7.3	CCCAAACACCAACTTCTCTTCTCTC	CCTCACACAAATGCCTACTGTCCTT
表达分析 RT-qPCR analysis	CsGAPDH	TTGGCATCGTTGAGGGTCT	CAGTGGGAACACGGAAAGC
	CsNPF2.13-q	AGGAAGGAAAGGAATCAACAGC	AACCTCGTCCCAACAAAGAAC
	CsNPF2.7-q	CGAACGCTGACACCTCTACA	GAGCCGCTTTAACTCCACCA
	CsNPF3.1-q	GCCACCTAATCCGAGGGATG	TGCCCAACTCAAGTGGACTG
	CsNPF5.5-q	GGCTAACTTTCATCGCCTGC	CAAGCTTGCGGTTCATGTGG
	CsNPF6.1-q	CGATAGCAGCGTGTTTGTGG	GGCTGCCTTGTCCAAACATC
	CsNPF6.3-q	GCCTCACTGACTGTTTTCTTCG	AACCGCTTGATTTCGCCTA
	CsNPF7.1-q	TAGCCACATTCGGAGCAGAC	CCGACGTTGAGTGCAGAGTA
	CsNPF7.3-q	GCCAACCGGCAATACGAGAA	GACATTATTAGCTGCGTCGGC
	Atβ-tubulin-q	ACCACTCCTAGCTTTGGTGATCTG	AGGTTCACTGCGAGCTTCCTCA

Fig. 1 Electrophoresis results of clone of CsNPFs in tea plant

Table 2 Sequence information of CsNPFs in tea plants

基因名称 Gene name	开放阅读框长度 Open reading frame (ORF) (bp)	编码氨基酸数目 Number of amino acids
CsNPF2.13	1 923	640
CsNPF2.7	1 470	489
CsNPF3.1	1 815	604
CsNPF5.5	1 659	552
CsNPF7.1	1 734	577
CsNPF7.3	1 800	599

Table 3 Basic characteristics of CsNPFs in tea plants

蛋白 Protein	蛋白分子量 Molecular weight (kD)	蛋白等电点 Theoretical pI	总原子数 Total number of atom	不稳定系数 Instability index	脂肪系数 Aliphatic index	亲疏水性平均系数 Grand average of hydropathicity (GRAVY)	信号肽 Signal peptide	亚细胞定位 Sub-cellular localization	跨膜螺旋预测 Transmembrane helices prediction
CsNPF2.13	70.26	8.82	9 950	46.14	97.66	0.228	0.001 3	质膜（8.48）	12
CsNPF2.7	53.62	8.81	7 633	42.13	108.4	0.332	0.287 5	质膜（9.4）	12
CsNPF3.1	67.10	6.11	9 523	32.75	104.59	0.183	0.000 5	质膜（8.3）	11
CsNPF5.5	61.38	8.70	8 713	32.29	102.46	0.375	0.526 5	质膜（8.5）	9
CsNPF7.1	62.92	6.05	8 849	25.63	97.14	0.337	0.004 6	质膜（8.4）	12
CsNPF7.3	66.51	8.53	9 378	29.64	96.51	0.263	0.002 0	质膜（9.1）	12

Fig. 2 Phylogenetic tree ofCsNPFs proteinsAt: Arabidopsis thaliana; Os: Oryza sativa; Zm: Zea mays; Mt: Medicago truncatula; Md: Malus domestica; Cl: Camellia lanceoleosa; Vv: Vitis vinifera; Si: Sesamum indicum; Pa: Populus alba; Cn: Cocos nucifera; Cr: Catharanthus roseus; Tc: Theobroma cacao; Pp: Pinus pinaster; Bn: Brassica napus; Gm: Glycine max; Na: Nicotiana attenuata;Pt: Populus trichocarpa; Ls: Lactuca sativais; Me: Manihot esculenta; Mr: Morella rubra; Ar: Actinidia rufa; Vr: Vitis riparis; Sl: Solanum lycopersicum; Gs: Glycine soja

Fig. 3 Conserved motif (A) and domain (B) analysis of CsNPFs proteinsMFS_NPF1_2, MFS superfamily, and MFS_NPF7 denote nitrate transport-related domains

Fig. 4 Analysis of the promoter structure of CsNPFs genes

Fig. 5 Organizational characteristics analysis of CsNPFs genesDifferent tea varieties ‘Zhongcha 108’ (ZC108), ‘Longjing 43’ (LJ43), different tissue parts of the young leaf (YL), mature leaf (ML), root (ROOT). NA indicates that data is not detected. The error bars indicate the standard deviation among the three replicates. The data are three biological replicates ± SD. * P < 0.05, ** P < 0.01. The same below

Fig. 6 Expressions of CsNPFs in different tissues under hormone treatmentThe average values of three biological replicates were used to generate a heat map using MeV software. The intensity value bars are shown above the heat map. Blue denotes low expression, and red denotes high expression. The grey color indicates that no data has been detected

Fig. 7 Expressions of CsNPFs genes in the leaves and roots under nitrate treatmentNitrogen starvation treatment for 10 d (-N), nitrogen addition 1 mol/L (low nitrogen, +LN), 4 mol/L (high nitrogen, +HN), respectively, for 2 h and 2 d

Fig. 8 Phenotypic difference analysis of CsNPF7.3 overexpressing lines (OE) compared with WT (Col-0) under different treatment conditionsA: Phenotype of Arabidopsis thaliana growth under 1/4 MS (10 mmol/L NO3-) conditions. B: Positive identification of overexpressing lines. C: Root lengths of different lines under 10 mmol/L NO3- conditions. D: Fresh weights of different lines under 10 mmol/L NO3- conditions. WT refers to the wild-type A. thaliana Col-0, and OE refers to the CsNPF7.3 transgenic overexpressing A. thaliana (OE1, OE2, and OE3 are 3 overexpressing lines). The same below

Fig. 9 Phenotypic difference analysis of CsNPF7.3 overexpressing lines (OE) compared with WT (Col-0) under different hormone treatment conditionsA: Phenotype of plant growth under 5 μmol/L IAA conditions. B: Phenotype of plant growth under 10 μmol/L ABA conditions. C: Phenotype of plant growth under 5 μmol/L GA3 conditions. D: The number of fibrous roots analysis among different lines under 5 μmol/L IAA conditions. E: Analysis of the difference in root length among plants under 10 μmol/L ABA conditions. F: Analysis of the difference in leaf width and leaf length among plants under 5 μmol/L GA3 conditions

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