大蒜水通道蛋白基因<i>AsPIP1;1</i>和<i>AsTIP2;1</i>的克隆、鉴定及其对盐胁迫的响应

doi:10.7668/hbnxb.20195933

华北农学报 ›› 2026, Vol. 41 ›› Issue (1): 64-70. doi: 10.7668/hbnxb.20195933

所属专题： 盐碱胁迫；生物技术；蔬菜专题；

• 作物遗传育种·种质资源·生物技术 • 上一篇下一篇

大蒜水通道蛋白基因AsPIP1;1和AsTIP2;1的克隆、鉴定及其对盐胁迫的响应

吴嘉萁¹, 许玉洁¹, 陈洋洋¹, 任旭琴¹^,², 周瑾¹, 熊爱生³, 王广龙¹^,²

¹ 淮阴工学院生命科学与食品工程学院,江苏淮安 223003
² 江苏省农业绿色低碳生产技术工程研究中心,江苏淮安 223003
³ 南京农业大学园艺学院,作物遗传与种质创新利用全国重点实验室,农业农村部华东地区园艺作物生物学与种质创制重点实验室,江苏南京 210095

收稿日期:2025-04-20 出版日期:2026-02-28
通讯作者:
王广龙(1989—),男,江苏连云港人,副教授,博士,硕士生导师,主要从事蔬菜生理与分子生物学研究。
作者简介:
吴嘉萁(2001—),女,湖北孝感人,硕士,主要从事蔬菜生理与分子生物学研究。
基金资助:
青海省科技厅重点实验室项目(2020-ZJ-Y02); 青海省蔬菜遗传与生理重点实验室开放课题(2020-SCSYS-01); 淮阴工学院博士科研启动基金项目(Z301B16531)

Cloning and Characterization of Two Aquaporin Genes,AsPIP1;1 and AsTIP2;1,and Their Response to Salt Stress in Garlic

WU Jiaqi¹, XU Yujie¹, CHEN Yangyang¹, REN Xuqin¹^,², ZHOU Jin¹, XIONG Aisheng³, WANG Guanglong¹^,²

¹ Faculty of Life Science and Food Engineering,Huaiyin Institute of Technology,Huaian 223003,China
² Jiangsu Provincial Agricultural Green and Low Carbon Production Technology Engineering Research Center, Huaian 223003,China
³ College of Horticulture,Nanjing Agricultural University,Key Laboratory of Biology and Germplasm Enhancement of Horticulture Crops in East China,Ministry of Agriculture and Rural Affairs, State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization,Nanjing 210095,China

Received:2025-04-20 Published:2026-02-28

摘要/Abstract

摘要：

水通道蛋白(AQPs)主要负责水分子的跨膜运输,参与植物生长发育和响应环境胁迫等过程。旨在从大蒜中克隆编码质膜内在蛋白(PIP)和液泡膜内在蛋白(TIP)的基因AsPIP1;1和AsTIP2;1,分析其序列特征,并探究它们在盐胁迫条件下的表达模式,以评估其在大蒜耐盐性中的潜在功能。利用RT-PCR技术从大蒜中克隆了AsPIP1;1和AsTIP2;1基因,利用生物信息学方法对克隆基因的开放阅读框、编码氨基酸序列及保守结构域等进行分析,并通过荧光定量PCR检测AsPIP1;1和AsTIP2;1基因在不同组织和盐胁迫条件下的表达特性。结果发现,AsPIP1;1和AsTIP2;1基因的开放阅读框分别为867,747 bp,编码288,248个氨基酸,具有NPA保守基序。AsPIP1;1和AsTIP2;1在大蒜叶片和根部高表达,盐胁迫环境能强烈诱导这2个基因转录水平的改变。且AsPIP1;1和AsTIP2;1可能与其他AQPs蛋白、细胞壁相关蛋白、转运蛋白相互作用,以适应盐胁迫环境。这些结果表明,AsPIP1;1和AsTIP2;1基因可能参与大蒜植株响应盐胁迫的过程。

关键词: 大蒜, 盐胁迫, 水通道蛋白, 表达分析, AsPIP1;1, AsTIP2;1

Abstract:

Aquaporins(AQPs)are primarily responsible for the transmembrane transport of water molecules and are involved in processes such as plant growth,development, and responses to environmental stress. Garlic is an important vegetable crop widely cultivated worldwide, but there are currently few reports on salt-tolerance genes in garlic.It aimed to clone the genes AsPIP1;1 and AsTIP2;1, encoding plasma membrane intrinsic protein (PIP) and tonoplast intrinsic protein (TIP) respectively, from garlic, analyze their sequence characteristics, investigate their expression patterns under salt stress conditions, and evaluate their potential roles in garlic salt tolerance. We cloned the AsPIP1;1 and AsTIP2;1 genes from garlic using RT-PCR. Bioinformatics methods were employed to analyze the open reading frames, encode amino acid sequences, and conserve domains of the cloned genes. Furthermore, the expression profiles of AsPIP1;1 and AsTIP2;1 in different tissues and under salt stress conditions were detected using Quantitative Real-time PCR. The results showed that the open reading frames of the AsPIP1;1 and AsTIP2;1 genes were 867, 747 bp in length, encoding 288,248 amino acids, respectively, with the conserved NPA motifs. AsPIP1;1 and AsTIP2;1 were highly expressed in garlic leaves and roots, and salt stress strongly induced changes in the transcriptional levels of both genes. Furthermore,AsPIP1;1 and AsTIP2;1 may interact with other AQP proteins, cell wall-associated proteins, and transporter proteins to adapt to the salt stress environment. These results indicate that the AsPIP1;1 and AsTIP2;1 genes may be involved in the garlic plant's response to salt stress.

Key words: Garlic, Salt stress, Aquaporins, Expression analysis, AsPIP1;1, AsTIP2;1

中图分类号:

吴嘉萁, 许玉洁, 陈洋洋, 任旭琴, 周瑾, 熊爱生, 王广龙. 大蒜水通道蛋白基因AsPIP1;1和AsTIP2;1的克隆、鉴定及其对盐胁迫的响应[J]. 华北农学报, 2026, 41(1): 64-70. doi: 10.7668/hbnxb.20195933.

WU Jiaqi, XU Yujie, CHEN Yangyang, REN Xuqin, ZHOU Jin, XIONG Aisheng, WANG Guanglong. Cloning and Characterization of Two Aquaporin Genes,AsPIP1;1 and AsTIP2;1,and Their Response to Salt Stress in Garlic[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(1): 64-70. doi: 10.7668/hbnxb.20195933.

http://www.hbnxb.net/CN/Y2026/V41/I1/64

图/表 8

表1 AsPIP1;1和AsTIP2;1基因克隆和荧光定量PCR引物

Tab.1 Primers used for AsPIP1;1 and AsTIP2;1 gene cloning and Quantitative Real-time PCR

基因名称 Gene name	基因克隆引物(上游/下游) Primers for gene cloning (Forward/Reverse)	荧光定量PCR引物(上游/下游) Primers for Quantitative Real-time PCR (Forward/Reverse)
AsPIP1;1	ATGGAGGGTAAAGAAGAAGATGT/TTAAGATCT GCTTTTGAATGGAA	CGTCGTTAAGTCTCCGTCCAAGT/ACAACATTAGCACCGC CACCTT
AsTIP2;1	ATGGTAAAGCTAGCTTTTGGTAGCA/TTAAGGAT AATCCTCAGCGACAGGT	GGCGACTCTCTCAGCAAAGCAT/ACAGGCAAGCGACGGA GGAT

图1 大蒜AsPIP1;1和AsTIP2;1的RT-PCR扩增图谱 M.DNA分子质量标准;1.AsPIP1;1;2.AsTIP2;1。

Fig.1 RT-PCR amplification patterns of AsPIP1;1 and AsTIP2;1 from garlic M.DNA Marker;1.AsPIP1;1;2.AsTIP2;1.

图2 大蒜AsPIP1;1和AsTIP2;1基因的核苷酸序列及其编码的氨基酸序列 A.AsPIP1;1;B.AsPIP2;1。灰色阴影区域表示MIP大家族所特有的保守基序Asn-Pro-Ala(NPA)序列。

Fig.2 Nucleotide acid and deduced amino acid sequences of AsPIP1;1 and AsTIP2;1 from garlic A.AsPIP1;1;B.AsPIP2;1.The grey shaded areas indicate the conserved motif,Asn-Pro-Ala (NPA),peculiar to MIP superfamily.

图3 大蒜与拟南芥PIPs和TIPs氨基酸序列的系统进化树

Fig.3 Phylogenetic tree of PIPs and TIPs from garlic and Arabidopsis

图4 大蒜和拟南芥中PIP和TIP家族蛋白的保守基序

Fig.4 Conserved motifs of PIP and TIP family proteins in garlic and Arabidopsis

图5 大蒜和拟南芥中PIP和TIP结构域的序列标识堆叠柱的整体高度反映序列保守性程度;柱内各残基的高度显示该位点上对应氨基酸的出现频率。

Fig.5 Sequence logos of PIP and TIP domains in garlic and Arabidopsis The overall height of the stack represents the level of sequence conservation;heights of residues within a stack indicate the relative frequency of each residue at that position.

图6 基于拟南芥同源序列鉴定大蒜AsPIP1;1和AsTIP2;1的互作调控网络

Fig.6 The interaction networks of AsPIP1;1 and AsTIP2;1 in garlic according to the orthologs in Arabidopsis

图7 盐胁迫下大蒜AsPIP1;1 和 AsTIP2;1基因的表达分析同一组织内不同小写字母表示在P<0.05水平差异显著。

Fig.7 Expression analysis of AsPIP1;1 and AsTIP2;1 genes in garlic under salinity stress Different lowercase letters in the same tissue indicate significant differences at the 0.05 level.

参考文献 36

[1]	Vaziriyeganeh M, Khan S, Zwiazek J J. Analysis of aquaporins in northern grasses reveal functional importance of Puccinellia nuttalliana PIP2;2 in salt tolerance[J]. Plant,Cell & Environment, 2023, 46(7):2159-2173.doi:10.1111/pce.14589. URL
[2]	Zhu C L, Lin Z M, Yang K B, Lou Y F, Liu Y, Li T K, Li H, Di X L, Wang J F, Sun H Y, Li Y, Li X P, Gao Z M. A bamboo'PeSAPK4-PeMYB99-PeTIP4-3' regulatory model involved in water transport[J]. New Phytologist, 2024, 243(1):195-212.doi:10.1111/nph.19787. URL
[3]	Ding L, Fox A R, Chaumont F. Multifaceted role and regulation of aquaporins for efficient stomatal movements[J]. Plant,Cell & Environment, 2024, 47(9):3330-3343.doi:10.1111/pce.14942. URL
[4]	Ahmed S, Kouser S, Asgher M, Gandhi S G. Plant aquaporins:a frontward to make crop plants drought resistant[J]. Physiologia Plantarum, 2021, 172(2):1089-1105.doi:10.1111/ppl.13416. URL
[5]	Li X T, Guo Y R, Ling Q P, Guo Z J, Lei Y W, Feng X M, Wu J Y, Zhang N N. Advances in the structure,function,and regulatory mechanism of plant plasma membrane intrinsic proteins[J]. Genes, 2025, 16(1):10.doi:10.3390/genes16010010. URL
[6]	Kudoyarova G, Veselov D, Yemelyanov V, Shishova M. The role of aquaporins in plant growth under conditions of oxygen deficiency[J]. International Journal of Molecular Sciences, 2022, 23(17):10159.doi:10.3390/ijms231710159. URL
[7]	Ozu M, Alvear-Arias J J, Fernandez M, Caviglia A, Peña-Pichicoi A, Carrillo C, Carmona E, Otero-Gonzalez A, Garate J A, Amodeo G, Gonzalez C. Aquaporin gating:a new twist to unravel permeation through water channels[J]. International Journal of Molecular Sciences, 2022, 23(20):12317.doi:10.3390/ijms232012317. URL
[8]	Wan Q, Li Y X, Cheng J J, Wang Y, Ge J, Liu T L, Ma L Y, Li Y, Liu J N, Zhou C L, Li H C, Sun X, Chen X L, Li Q X, Yu X Y. Two aquaporins,PIP1;1 and PIP2;1,mediate the uptake of neonicotinoid pesticides in plants[J]. Plant Communications, 2024, 5(5):100830.doi:10.1016/j.xplc.2024.100830. URL
[9]	Yu X S, Wang H R, Lei F F, Li R Q, Yao H P, Shen J B, Ain N U, Cai Y. Structure and functional divergence of PIP peptide family revealed by functional studies on PIP1 and PIP2 in Arabidopsis thaliana[J]. Frontiers in Plant Science, 2023, 14:1208549.doi:10.3389/fpls.2023.1208549. URL
[10]	Bots M, Feron R, Uehlein N, Weterings K, Kaldenhoff R, Mariani T. PIP1 and PIP2 aquaporins are differentially expressed during tobacco anther and stigma development[J]. Journal of Experimental Botany, 2005, 56(409):113-121.doi:10.1093/jxb/eri009. pmid: 15520027
[11]	Fetter K, Van Wilder V, Moshelion M, Chaumont F. Interactions between plasma membrane aquaporins modulate their water channel activity[J]. The Plant Cell, 2004, 16(1):215-228.doi:10.1105/tpc.017194. URL
[12]	Zhang S, Feng M, Chen W, Zhou X F, Lu J Y, Wang Y R, Li Y H, Jiang C Z, Gan S S, Ma N, Gao J P. In rose,transcription factor PTM balances growth and drought survival via PIP2;1 aquaporin[J]. Nature Plants, 2019, 5(3):290-299.doi:10.1038/s41477-019-0376-1.
[13]	Bienert M D, Diehn T A, Richet N, Chaumont F, Bienert G P. Heterotetramerization of plant PIP1 and PIP2 aquaporins is an evolutionary ancient feature to guide PIP1 plasma membrane localization and function[J]. Frontiers in Plant Science, 2018, 9:382.doi:10.3389/fpls.2018.00382. pmid: 29632543
[14]	Liu L H, Ludewig U, Gassert B, Frommer W B, von Wirén N. Urea transport by nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis[J]. Plant Physiology, 2003, 133(3):1220-1228.doi:10.1104/pp.103.027409. URL
[15]	Song L, Nguyen N, Deshmukh R K, Patil G B, Prince S J, Valliyodan B, Mutava R, Pike S M, Gassmann W, Nguyen H T. Soybean TIP gene family analysis and characterization of GmTIP1;5 and GmTIP2;5 water transport activity[J]. Frontiers in Plant Science, 2016, 7:1564.doi:10.3389/fpls.2016.01564. pmid: 27818669
[16]	Footitt S, Clewes R, Feeney M, Finch-Savage W E, Frigerio L. Aquaporins influence seed dormancy and germination in response to stress[J]. Plant,Cell & Environment, 2019, 42(8):2325-2339.doi:10.1111/pce.13561. URL
[17]	Rodrigues M I, Takeda A A S, Bravo J P, Maia I G. The Eucalyptus tonoplast intrinsic protein (TIP)gene subfamily:genomic organization,structural features,and expression profiles[J]. Frontiers in Plant Science, 2016, 7:1810.doi:10.3389/fpls.2016.01810. pmid: 27965702
[18]	Wang Y, Zhang Y Q, An Y C, Wu J Y, He S B, Sun L R, Hao F S. Wheat TaTIP4;1 confers enhanced tolerance to drought,salt and osmotic stress in Arabidopsis and rice[J]. International Journal of Molecular Sciences, 2022, 23(4):2085.doi:10.3390/ijms23042085. URL
[19]	Yi X F, Sun X C, Tian R, Li K X, Ni M, Ying J L, Xu L, Liu L W, Wang Y. Genome-wide characterization of the aquaporin gene family in radish and functional analysis of RsPIP2-6 involved in salt stress[J]. Frontiers in Plant Science, 2022, 13:860742.doi:10.3389/fpls.2022.860742. URL
[20]	Zhang H M, Zhu J H, Gong Z Z, Zhu J K. Abiotic stress responses in plants[J]. Nature Reviews Genetics, 2022, 23(2):104-119.doi:10.1038/s41576-021-00413-0.
[21]	Pandey S P, Chen C, Singh S, Maniar J N, Mishra A, Bakshi S, Mishra V K, Sharma S. Transcriptional response of cultivated peanut (Arachis hypogaea L.) roots to salt stress and the role of DNA methylation[J]. Plant Cell Reports, 2025, 44(6):124.doi:10.1007/s00299-025-03515-9. pmid: 40397176
[22]	Arshad K, Wang Y, Han S Q, Zheng M Y, Xie M Y, Song Y M, Hu L F, Ou R, Gu M Y, Ouyang C, Zhao S C, He J B, Li Y, Fang X D, Gai J Y, Jin S C, Zhang J P. GWAS on HTP-enabled dynamic traits unravels novel genetic architecture of salt tolerance in soybean[J]. The Plant Journal, 2025, 122(4):e70177.doi:10.1111/tpj.70177. URL
[23]	Seo H U, Jang C S. Mutation of a gene with PWWP domain confers salt tolerance in rice[J]. Plant Molecular Biology, 2025, 115(3):63.doi:10.1007/s11103-025-01581-x. pmid: 40327136
[24]	Jain M, Patil N, Mohammed A, Hamzah Z. Valorization of garlic (Allium sativum L.) byproducts:bioactive compounds,biological properties,and applications[J]. Journal of Food Science, 2025, 90(3):e70152.doi:10.1111/1750-3841.70152. URL
[25]	周倩怡, 黄思杰, 田洁. 大蒜2个中性/碱性转化酶基因AsNI的克隆与表达分析[J]. 华北农学报, 2023, 38(4):54-64.doi:10.7668/hbnxb.20193859.
	Zhou Q Y, Huang S J, Tian J. Cloning and expression analysis of two neutral/alkaline invertase gene in garlic[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(4):54-64. doi: 10.7668/hbnxb.20193859
[26]	Wang G L, Ren X Q, Liu J X, Yang F, Wang Y P, Xiong A S. Transcript profiling reveals an important role of cell wall remodeling and hormone signaling under salt stress in garlic[J]. Plant Physiology and Biochemistry, 2019, 135:87-98.doi:10.1016/j.plaphy.2018.11.033. URL
[27]	Gasteiger E. ExPASy:the proteomics server for in-depth protein knowledge and analysis[J]. Nucleic Acids Research, 2003, 31(13):3784-3788.doi:10.1093/nar/gkg563. URL
[28]	Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J]. Molecular Biology and Evolution, 2011, 28(10):2731-2739.doi:10.1093/molbev/msr121. URL
[29]	Wang G L, Tian C, Wang Y P, Wan F X, Hu L B, Xiong A S, Tian J. Selection of reliable reference genes for quantitative RT-PCR in garlic under salt stress[J]. PeerJ, 2019, 7:e7319.doi:10.7717/peerj.7319.
[30]	Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCtmethod[J]. Methods, 2001, 25(4):402-408.doi:10.1006/meth.2001.1262. pmid: 11846609
[31]	Zhou H P, Shi H F, Yang Y Q, Feng X X, Chen X, Xiao F, Lin H H, Guo Y. Insights into plant salt stress signaling and tolerance[J]. Journal of Genetics and Genomics, 2024, 51(1):16-34.doi:10.1016/j.jgg.2023.08.007. URL
[32]	Wang J J, Yang L L, Chai S S, Ren Y F, Guan M, Ma F W, Liu J Y. An aquaporin gene MdPIP1;2 from Malus domestica confers salt tolerance in transgenic Arabidopsis[J]. Journal of Plant Physiology, 2022, 273:153711.doi:10.1016/j.jplph.2022.153711. URL
[33]	Gambetta G A, Fei J, Rost T L, Knipfer T, Matthews M A, Shackel K A, Walker M A, McElrone A J. Water uptake along the length of grapevine fine roots:developmental anatomy,tissue-specific aquaporin expression,and pathways of water transport[J]. Plant Physiology, 2013, 163(3):1254-1265.doi:10.1104/pp.113.221283. pmid: 24047863
[34]	Guo A H, Hao J F, Su Y, Li B, Zhao N, Zhu M, Huang Y, Tian B M, Shi G Y, Hua J P. Two aquaporin genes,GhPIP2;7 and GhTIP2;1,positively regulate the tolerance of upland cotton to salt and osmotic stresses[J]. Frontiers in Plant Science, 2022, 12:780486.doi:10.3389/fpls.2021.780486. URL
[35]	Martins C P S, Neves D M, Cidade L C, Mendes A F S, Silva D C, Almeida A F, Coelho-Filho M A, Gesteira A S, Soares-Filho W S, Costa M G C. Expression of the Citrus CsTIP2;1 gene improves tobacco plant growth,antioxidant capacity and physiological adaptation under stress conditions[J]. Planta, 2017, 245(5):951-963.doi:10.1007/s00425-017-2653-4. URL
[36]	Rahman A, Kawamura Y, Maeshima M, Rahman A, Uemura M. Plasma membrane aquaporin members PIPs act in concert to regulate cold acclimation and freezing tolerance responses in Arabidopsis thaliana[J]. Plant and Cell Physiology, 2020, 61(4):787-802.doi:10.1093/pcp/pcaa005. pmid: 31999343

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大蒜水通道蛋白基因AsPIP1;1和AsTIP2;1的克隆、鉴定及其对盐胁迫的响应

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