枳丛枝菌根共生相关硝酸盐转运蛋白基因PtNPF5.2的功能鉴定

doi:10.16420/j.issn.0513-353x.2026-0001

摘要/Abstract

摘要： 为探究柑橘菌根（arbuscular mycorrhiza，AM）途径硝酸盐（NO₃^-）转运的分子机制，以柑橘常用砧木枳（Poncirus trifoliata）为材料，检测了其NO₃^-转运蛋白基因PtNPF5.2的表达模式、亚细胞定位及其同源基因在模式植物蒺藜苜蓿（Medicago truncatula）和番茄（Solanum lycopersicum）丛枝菌根共生中的作用。结果表明，PtNPF5.2受丛枝菌根真菌（arbuscular mycorrhizal fungi，AMF）特异诱导表达，其启动子在含有丛枝的根系细胞中激活，编码蛋白定位于细胞膜。苜蓿同源基因MtNPF5.3的干涉或插入突变均未影响AMF侵染。番茄同源基因SlNPF5.2的CRISPR/Cas9敲除同样未影响AMF侵染，但导致地上部NO₃^-含量显著降低。综上，PtNPF5.2可能参与枳AM共生中NO₃^-在植物体内的分配调控，为解析柑橘菌根氮（N）转运机制提供了线索。

关键词: 枳, 丛枝菌根, 硝酸盐转运蛋白, PtNPF5.2, 氮转运, 基因功能鉴定

Abstract:

To investigate the molecular mechanism of nitrate（NO₃^-）transport via the arbuscular mycorrhiza（AM）pathway in citrus, this study used Poncirus trifoliata，a common citrus rootstock，to examine the expression pattern and subcellular localization of the NO₃^- transporter gene PtNPF5.2，as well as the role of its homologous genes in the AM symbiosis of the model plants Medicago truncatula and Solanum lycopersicum. The results showed that PtNPF5.2 was specifically induced by arbuscular mycorrhizal fungi（AMF），its promoter was activated in root cells containing arbuscules，and the encoded protein was localized to the plasma membrane. Interference or insertion mutation of the homologous gene MtNPF5.3 in Medicago truncatula did not affect AMF colonization. Similarly，CRISPR/Cas9 knockout of the homologous gene SlNPF5.2 in Solanum lycopersicum did not affect AMF colonization but led to a significant decrease in shoot NO₃^- content. In summary，PtNPF5.2 may be involved in regulating the NO₃^- allocation within the plant during AM symbiosis in Poncirus trifoliata，providing insights into the nitrogen transport mechanisms via the AM pathway in citrus.

Key words: Poncirus trifoliata, arbuscular mycorrhiza, nitrate transport protein, PtNPF5.2, nitrogen transport, gene functional identification

于淼, 黄圣宇, 潘志勇. 枳丛枝菌根共生相关硝酸盐转运蛋白基因PtNPF5.2的功能鉴定[J]. 园艺学报, 2026, 53(4): 1025-1037.

YU Miao, HUANG Shengyu, PAN Zhiyong. Functional Identification of a Nitrate Transport Gene PtNPF5.2 Related to Arbuscular Mycorrhizal Symbiosis in Poncirus trifoliata[J]. Acta Horticulturae Sinica, 2026, 53(4): 1025-1037.

图/表 9

表1 本研究中使用的引物序列信息

Table 1 Primer sequence information used in this study

基因 Gene	引物名称 Primer name	引物序列（5′-3′） Primer sequence
Pt3g030260	pPtNPF5.2-GUS-F	TGTGGTCTCAGGAGTACGCCACAGGGAAAAAGCTTCA
	pPtNPF5.2-GUS-R	TGTGGTCTCACATTTAATCAATTATTACTTACTGCTCCCTGGAGAGAG
	PtNPF5.2-GFP-F	TGTGGTCTCAAATGGCCACATTAAAGCTAGGTGAAGAG
	PtNPF5.2-GFP-R	TGTGGTCTCACGAACCGCCGAATCCAGTGCCAGC
	qPtActin-F	CAAGCAGCATGAAGATCAA
	qPtActin-R	ATCTGCTGGAAGGTGCTGAG
	qPtNPF5.2-F	CCAAGCTCACCGTGGATTCT
	qPtNPF5.2-R	GCGCTTGGTATGAGTGTTGC
	qPtPT4-F	ATGCTCTTGACACTGCTAGGAC
	qPtPT4-R	AGCTTTGAGACGGTAGAGATGC
Medtr7g098040	MtNPF5.3-RNAi-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTACACGTCATTATAATGGTCACTGCATG
	MtNPF5.3-RNAi-R	GGGGACCACTTTGTACAAGAAAGCTGGGTTCGAAGTTATCAGCAACCCCTGT
	Tnt1-F	ACAGTGCTACCTCCTCTGGATG
	Tnt1-R	CAGTGAACGAGCAGAACCTGTG
	NF18636-F	TGACATGACCAGTGACTAGAAAGG
	NF18636-R	CATTACCATGAACTATTGCCTTACTCG
	qMtEF1-F	GAGACCCACAGACAAGCC
	qMtEF1-R	ACTGGCACAGTTCCAATACC
	qMtNPF5.3-F	CAATGTGGGTTGGGCTGTTG
	qMtNPF5.3-R	GTCAAAGGGCTTCCTGAGGG
	qMtPT4-F	GACACGAGGCGCTTTCATAGCAGC
	qMtPT4-R	GTCATCGCAGCTGGAACAGCACCG
	qMtBCP1-F	CCGGAAAGGACGTTATTCAA
	qMtBCP1-R	TGGCTACCATGATGACTCCA
	qRiEF1α-F	GCTATTTTGATCATTGCCGCC
	qRiEF1α-R	TCATTAAAACGTTCTTCCGACC
Solyc09g072780	CRI-SlNPF5.2-F	ATATATGGTCTCGTTTGTTCCTTTGCCACAACTTGGGTTTTAGAGCTAGAAATAGC
	CRI-SlNPF5.2-R	ATTATTGGTCTCGAAACGGTCCAACGTTGTCTTGTACCAAACTACACTGTTAGATTC
	qSlEF1-F	GGAACTTGAGAAGGAGCCTAAG
	qSlEF1-R	CAACACCAACAGCAACAGTCT
	qSlNPF5.2-F	AGGCAGCAGTGAAAACAGGA
	qSlNPF5.2-R	GTGCTAGCATTGTGCTTGGT
	qSlPT4-F	ATGGCGGGCAGAATGAGA
	qSlPT4-R	GCACAAGGCGTAGTGATG

图1 枳PtNPF5.2的表达分析 A：PtNPF5.2在不同组织中的表达量；B：PtNPF5.2在接种AMF后不同时间点的表达量；C：PtPT4在接种AMF后不同时间点的表达量。NM：未接种植株，AM：接种植株

Fig. 1 Expression analysis of PtNPF5.2 in Poncirus trifoliate A：Expression levels of PtNPF5.2 in different tissues；B：Expression levels of PtNPF5.2 at different time points after inoculation with AMF；C：Expression levels of PtPT4 at different time points after inoculation with AMF. NM：Non-mycorrhizal plants，AM：Mycorrhizal plants

图2 PtNPF5.2启动子在苜蓿根系中的活性

Fig. 2 Activity of the PtNPF5.2 promoter in medicago roots

图3 PtNPF5.2在本氏烟草叶片中的亚细胞定位

Fig. 3 Subcellular localization of PtNPF5.2 in Nicotiana benthamiana leaves

图4 PtNPF5.2及其在蒺藜苜蓿中同源基因的系统进化树

Fig. 4 Phylogenetic tree of PtNPF5.2 and its homologous genes in Medicago truncatula

图5 蒺藜苜蓿MtNPF5.3干涉植株中基因表达与AMF侵染分析 A：MtNPF5.3在R108和干涉植株中的相对表达量；B：AM共生标记基因MtPT4、MtBCP1、RiEF1α在R108和干涉植株中的相对表达量；C ~ E：AMF侵染率统计。F%：总根段中被菌丝侵染的根段所占比例，M%：整个根中菌丝侵染的强度，A%：整个根中丛枝的丰度。t-test，* P < 0.05。下同

Fig. 5 Analysis of gene expression and AMF colonization in MtNPF5.3 RNAi plants of Medicago truncatula A：Relative expression level of MtNPF5.3 in R108 and RNAi plants；B：Relative expression levels of AM symbiosis marker genes MtPT4，MtBCP1，and RiEF1α in R108 and RNAi plants；C-E：Statistics analysis of AMF colonization. F%：Percentage of root fragments colonized by hyphae，M%：Intensity of hyphal colonization in whole roots，A%：Arbuscule abundance in whole roots. t-test，* P < 0.05. The same below

图6 蒺藜苜蓿MtNPF5.3插入突变体NF18636中基因表达与AMF侵染分析 A：MtNPF5.3在野生型R108和突变体NF18636中的相对表达量；B：AM共生标记基因MtPT4、MtBCP1、RiEF1α在R108和NF18636中的相对表达量；C ~ E：AMF侵染率；F：R108和NF18636根系菌根结构显微图像。Ih：根内菌丝，Arb：丛枝

Fig. 6 Analysis of gene expression and AMF colonization in the MtNPF5.3 insertion mutant NF18636 of Medicago truncatula A：Relative expression level of MtNPF5.3 in wild-type R108 and mutant NF18636；B：Relative expression levels of AM symbiosis marker genes MtPT4，MtBCP1，and RiEF1α in R108 and NF18636；C-E：Statistics of AMF colonization；F：Microscopic images of mycorrhizal structures in roots of R108 and NF18636. Ih：Intraradical hyphae，Arb：Arbuscules

图7 番茄SlNPF5.2敲除植株中基因表达与AMF侵染分析 A ~ D：SlNPF5.2和AM共生标记基因SlPT4在‘Micro-Tom'和2个敲除系（slnpf5.2-1和slnpf5.2-2）中的相对表达量；E ~ G：AMF侵染率统计；H ~ I：‘Micro-Tom'和2个敲除系的根系菌根结构显微图像。* P < 0.05；***P < 0.001

Fig. 7 Analysis of gene expression and AMF colonization in SlNPF5.2 knockout tomato plants A-D：Relative expression levels of SlNPF5.2 and AM symbiosis marker gene SlPT4 in‘Micro-Tom'and two knockout lines（slnpf5.2-1 and slnpf5.2-2）；E-G：Statistics of AMF colonization；H-I：Microscopic images of mycorrhizal structures in the roots of‘Micro-Tom'and the two knockout lines. * P < 0.05；***P < 0.001

图8 番茄SlNPF5.2敲除植株地下部和地上部NO3-含量敲除系slnpf5.2-1和slnpf5.2-2各自野生型对照‘Micro-Tom'

Fig. 8 NO3- content in roots and shoots of SlNPF5.2 knockout tomato plants Knockout lines slnpf5.2-1 and slnpf5.2-2 each have their own wild-type control‘Micro-Tom'

参考文献 40

[1]	Breuillin-Sessoms F, Floss D S, Gomez S K, Pumplin N, Ding Y, Levesque-Tremblay V, Noar R D, Daniels D A, Bravo A, Eaglesham J B, Benedito V A, Udvardi M K, Harrison M J. 2015. Suppression of arbuscule degeneration in Medicago truncatula phosphate transporter 4 mutants is dependent on the ammonium transporter 2 family protein AMT2;3. The Plant Cell,27: 1352-1366.
[2]	Chen A Q, Gu M, Sun S B, Zhu L L, Hong S, Xu G H. 2011. Identification of two conserved cis-acting elements,MYCS and P1BS,involved in the regulation of mycorrhiza-activated phosphate transporters in eudicot species. New Phytologist,189:1157-1169.
[3]	Engler C, Youles M, Gruetzner R, Ehnert T M, Werner S, Jones J D, Patron N J, Marillonnet S. 2014. A golden gate modular cloning toolbox for plants. ACS Synthetic Biology,3:839-843.
[4]	Fan Z Y, Wu Y F, Zhao L Y, Fu L N, Deng L L, Deng J R, Ding D K, Xiao S Y, Deng X X, Peng S, Pan Z Y. 2022. MYB308-mediated transcriptional activation of plasma membrane H⁺-ATPase 6 promotes iron uptake in citrus. Horticulture Research,9:uhac088.
[5]	Govindarajulu M, Pfeffer P E, Jin H, Abubaker J, Douds D D, Allen J W, Bücking H, Lammers P J, Shachar-Hill Y. 2005. Nitrogen transfer in the arbuscular mycorrhizal symbiosis. Nature,435:819-823.
[6]	Guether M, Balestrini R, Hannah M, He J, Udvardi M K, Bonfante P. 2009a. Genome-wide reprogramming of regulatory networks,transport,cell wall and membrane biogenesis during arbuscular mycorrhizal symbiosis in Lotus japonicus. New Phytologist,182:200-212.
[7]	Guether M, Neuhäuser B, Balestrini R, Dynowski M, Ludewig U, Bonfante P. 2009b. A mycorrhizal-specific ammonium transporter from Lotus japonicus acquires nitrogen released by arbuscular mycorrhizal fungi. Plant Physiology,150:73-83.
[8]	Gu R L, Duan F Y, An X, Zhang F S, von Wirén N, Yuan L X. 2013. Characterization of AMT-mediated high-affinity ammonium uptake in roots of maize(Zea mays L.). Plant and Cell Physiology,54:1515-1524.
[9]	Hodge A, Campbell C D, Fitter A H. 2001. An arbuscular mycorrhizal fungus accelerates decomposition and acquires nitrogen directly from organic material. Nature,413:297-299.
[10]	Hu B, Wang W, Ou S J, Tang J Y, Li H, Che R H, Zhang Z H, Chai X Y, Wang H R, Wang Y Q, Liang C Z, Liu L C, Piao Z Z, Deng Q Y, Deng K, Xu C, Liang Y, Zhang L H, Li L G, Chu C C. 2015. Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies. Nature Genetics,47:834-838.
[11]	Hui J, An X, Li Z B, Neuhäuser B, Ludewig U, Wu X N, Schulze W X, Chen F J, Feng G, Lambers H, Zhang F S, Yuan L X. 2022. The mycorrhiza-specific ammonium transporter ZmAMT3;1 mediates mycorrhiza-dependent nitrogen uptake in maize roots. The Plant Cell, 34:4066-4087.
[12]	Hwang C F, Lin Y, D'Souza T, Cheng C L. 1997. Sequences necessary for nitrate-dependent transcription of Arabidopsis nitrate reductase genes. Plant Physiology,113:853-862.
[13]	Javot H, Penmetsa R V, Breuillin F, Bhattarai K K, Noar R D, Gomez S K, Zhang Q, Cook D R, Harrison M J. 2011. Medicago truncatula mtpt4 mutants reveal a role for nitrogen in the regulation of arbuscule degeneration in arbuscular mycorrhizal symbiosis. The Plant Journal,68:954-965.
[14]	Ji C Y, Song F, He C, An J Y, Huang S Y, Yu H M, Lu H, Xiao S Y, Bucher M, Pan Z Y. 2023. Integrated miRNA-mRNA analysis reveals candidate miRNA family regulating arbuscular mycorrhizal symbiosis of Poncirus trifoliata. Plant,Cell & Environment,46:1805-1821.
[15]	Kobae Y, Tamura Y, Takai S, Banba M, Hata S. 2010. Localized expression of arbuscular mycorrhiza-inducible ammonium transporters in soybean. Plant and Cell Physiology,51:1411-1415.
[16]	Koegel S, Ait Lahmidi N, Arnould C, Chatagnier O, Walder F, Ineichen K, Boller T, Wipf D, Wiemken A, Courty P E. 2013. The family of ammonium transporters(AMT)in Sorghum bicolor:two AMT members are induced locally,but not systemically in roots colonized by arbuscular mycorrhizal fungi. New Phytologist,198:853-865.
[17]	Konishi M, Yanagisawa S. 2010. Identification of a nitrate-responsive cis-element in the Arabidopsis NIR1 promoter defines the presence of multiple cis-regulatory elements for nitrogen response. The Plant Journal,63:269-282.
[18]	Krouk G, Lacombe B, Bielach A, Perrine-Walker F, Malinska K, Mounier E, Hoyerova K, Tillard P, Leon S, Ljung K. 2010. Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Developmental Cell,18:927-937.
[19]	Limpens E, Ramos J, Franken C, Raz V, Compaan B, Franssen H, Bisseling T, Geurts R. 2004. RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula. Journal of Experimental Botany,55:983-992.
[20]	Lu S W, Zhang Y, Zhu K J, Yang W, Ye J L, Chai L J, Xu Q X, Deng X. 2018. The citrus transcription factor CsMADS 6 modulates carotenoid metabolism by directly regulating carotenogenic genes. Plant Physiology,176:2657-2676.
[21]	Martín Y, Navarro F J, Siverio J M. 2008. Functional characterization of the Arabidopsis thaliana nitrate transporter CHL 1 in the yeast Hansenula polymorpha. Plant Molecular Biology,68:215-224.
[22]	Morales de Los Ríos L, Pham T D, Perez T, Ben Amar M, Corratgé-Faillie C, Barbier F, Lacombe B. 2026. Functional characterization of NRT1/PTR FAMILY transporters:looking for a needle in a haystack. New Phytologist,249:1124-1144.
[23]	Pumplin N, Harrison M J. 2009. Live-cell imaging reveals periarbuscular membrane domains and organelle location in Medicago truncatula roots during arbuscular mycorrhizal symbiosis. Plant Physiology,151:809-819.
[24]	Rastogi R, Bate N J, Sivasankar S, Rothstein S J. 1997. Footprinting of the spinach nitrite reductase gene promoter reveals the preservation of nitrate regulatory elements between fungi and higher plants. Plant Molecular Biology,34:465-476.
[25]	Smith S E, Smith F A. 2011. Roles of arbuscular mycorrhizas in plant nutrition and growth:new paradigms from cellular to ecosystem scales. Annual Review of Plant Biology,62:227-250.
[26]	Smith S E, Smith F A. 2012. Fresh perspectives on the roles of arbuscular mycorrhizal fungi in plant nutrition and growth. Mycologia,104:1-13.
[27]	Sun J, Bankston J R, Payandeh J, Hinds T R, Zagotta W N, Zheng N. 2014. Crystal structure of the plant dual-affinity nitrate transporter NRT1.1. Nature,507:73-77.
[28]	Trouvelot A, Kough J, Gianinazzi-Pearson V. 1986. Evaluation of VA infection levels in root systems. Research for estimation methods having a functional significance//: Gianinazzi-Pearson V, Gianinazzi S. Physiological and Genetical Aspects of Mycorrhizae. Paris:INRA Press:217-221.
[29]	Tsay Y F, Schroeder J I, Feldmann K A, Crawford N M. 1993. The herbicide sensitivity gene CHL 1 of Arabidopsis encodes a nitrate-inducible nitrate transporter. Cell,72:705-713.
[30]	Wang S S, Chen A Q, Xie K, Yang X F, Luo Z Z, Chen J D, Zeng D C, Ren Y H, Yang C F, Wang L X, Feng H M, López-Arredondo D L, Herrera-Estrella L R, Xu G H. 2020. Functional analysis of the OsNPF4.5 nitrate transporter reveals a conserved mycorrhizal pathway of nitrogen acquisition in plants. Proceedings of the National Academy of Sciences of the United States of America,117:16649-16659.
[31]	Wang W, Hu B, Yuan D Y, Liu Y Q, Che R H, Hu Y C, Ou S J, Liu Y X, Zhang Z H, Wang H R, Li H, Jiang Z M, Zhang Z L, Gao X K, Qiu Y H, Meng X B, Liu Y X, Bai Y, Liang Y, Wang Y Q, Zhang L H, Li L G, Sodmergen, Jing H C, Li J Y, Chu C C. 2018. Expression of the nitrate transporter gene OsNRT1.1A/OsNPF6.3 confers high yield and early maturation in rice. Plant Cell,30:638-651.
[32]	Wang Z H, Zhang S Q, Liang J W, Chen H, Jiang Z J, Hu W T, Tang M. 2024. Rhizophagus irregularis regulates RiCPSI and RiCARI expression to influence plant drought tolerance. Plant Physiology,197:kiae645.
[33]	Wu Q S, Liu C Y, Zhang D J, Zou Y N, He X H, Wu Q H. 2016. Mycorrhiza alters the profile of root hairs in trifoliate orange. Mycorrhiza,26:237-247.
[34]	Wu Q S, Xia R X. 2006. Arbuscular mycorrhizal fungi influence growth,osmotic adjustment and photosynthesis of citrus under well-watered and water stress conditions. Journal of Plant Physiology,163:417-425.
[35]	Xing H L, Dong L, Wang Z P, Zhang H Y, Han C Y, Liu B, Wang X C, Chen Q J. 2014. A CRISPR/Cas 9 toolkit for multiplex genome editing in plants. BMC Plant Biology,14:327.
[36]	Xu G H, Fan X R, Miller A J. 2012. Plant nitrogen assimilation and use efficiency. Annual Review of Plant Biology,63:153-182.
[37]	Xu S, Wei Y F, Zhao P Z, Sun Y W, Gao K X, Yin C C, Wang C M, Fang R X, Ye J. 2025. A nitrate transporter OsNPF6.1 promotes nitric oxide signaling and virus resistance. Plant,Cell & Environment,48:6493-6508.
[38]	Yu H M, Bai F X, Ji C Y, Fan Z Y, Luo J Y, Ouyang B, Deng X X, Xiao S Y, Bisseling T, Limpens E, Pan Z Y. 2023. Plant lysin motif extracellular proteins are required for arbuscular mycorrhizal symbiosis. Proceedings of the National Academy of Sciences of the United States of America,120:e2301884120.
[39]	Zeng T, Holmer R, Hontelez J, Te Lintel-Hekkert B, Marufu L, de Zeeuw T, Wu F, Schijlen E, Bisseling T, Limpens E. 2018. Host- and stage-dependent secretome of the arbuscular mycorrhizal fungus Rhizophagus irregularis. The Plant Journal,94:411-425.
[40]	Zeng Z, Liu Y, Feng X Y, Li S X, Jiang X M, Chen J Q, Shao Z Q. 2023. The RNAome landscape of tomato during arbuscular mycorrhizal symbiosis reveals an evolving RNA layer symbiotic regulatory network. Plant Communications,4:100429.