植盐胁迫使严重危害作物生产的非生物胁迫因素之一，其引起的离子毒害对植物的多种生命活动都会产生不利影响。植物则通过对离子的选择性吸收、渗透调节物质的积累、活性氧的清除以及激素应答等多种机制维持正常生命活动。植物对盐胁迫耐受性的增强不仅表现在Na+含量的降低，维持最适胞质Na+/K+比值是植物耐盐的关键决定因素，因此寻找植物Na+、K+稳态的典型离子通道和转运体以及特定的信号通路，对解析盐胁迫应答机制至关重要。植物遭受盐害时，会激活一系列的信号通路抵抗盐胁迫，首先通过细胞表面的传感器感知环境盐胁迫信号，随后将信号传递给第二信使（如Ca2+），通过第二信使将信号传递到细胞内，进而引起一系列的应答机制。大豆是人类生产生活中植物蛋白和油脂的重要来源，栽培大豆(Glycine max)是由野生大豆(Glycine soja)驯化而来，与栽培大豆相比，野生大豆具有更丰富的遗传多样性、并具有耐盐、耐旱、耐荫和抗病虫等多种抗性较强的野外生存能力，因此在大豆耐盐机理和品种改良方面具有重要的研究价值。

本研究再野生大豆BB52材料和栽培大豆N23674材料存在耐盐性差异的基础上，通过比较转录组学和生物信息学分析筛选和鉴定了与耐盐性相关的关键功能基因，利用qRT-PCR、亚细胞定位、酵母双杂（Yeast two-hybrid，Y2H）、荧光素酶（Luciferase, LUC）互补实验、双分子荧光互补（Bimo-lecular fluorescence complementation, BiFC）、CRISPR-Cas9基因编辑、拟南芥杂交、拟南芥浸花法转化和大豆发根转化等多种分子生物学技术和遗传学手段，研究了野生大豆耐盐关键基因参与盐胁迫调控的生理功能机制。主要研究结果如下：

盐胁迫下野生大豆BB52材料与栽培大豆N23674材料相比具有更强的盐胁迫适应能力；野生大豆BB52可将过量的Na+主要积累在根部，减少向地上部的运输，降低地上部Na+含量和Na+/K+值，从而缓解NaCl对大豆植株地上部的伤害。从盐胁迫下BB52和N23674幼苗根的转录组测序结果中重点分析了仅在耐盐的野生大豆BB52根中上调的8651个差异表达基因。通过GO注释、KEGG富集等手段筛选到植物病原互作途径中与Ca2+信号相关的调控途径，在该途径上游显著富集了6个编码Ca2+转运相关的通道蛋白——环核苷酸门控通道（Cyclic Nucleotide Gated Channels，CNGCs）家族基因，其中GsCNGC20-d的上调倍数最高；与Ca2+信号直接相关的钙依赖的蛋白激酶（Calcuim Dependent Protein Kinases，CDPKs）家族成员在上述通路中也显著富集，其中注释为GsCDPK29的基因上调倍数最高。由此推测GsCNGC20-d 和GsCDPK29可能为野生大豆BB52材料耐盐的关键候选基因。

野生大豆中共有38个CNGC成员，它们均具有保守的CNBD结构域（L/I-X2-G/A-X1-F/S/V/A-X1-G-D/E-ELL-X1-W/R-X12,13,22-S-X2-T-X7-E-A/S-F/L-X1-L）。38个GsCNGC成员被分为5个亚族，其中GsCNGC20-d聚类在第Ⅳ-A亚族，与拟南芥AtCNGC20高度同源。GsCNGC家族的进化选择压力分析结果显示所有的GsCNGC基因在进化过程中均为负选择，差异候选基因GsCNGC20-d为三倍复制基因。RT-PCR鉴定和GsCNGC20-dpro::GUS转基因拟南芥的GUS染色结果反映了GsCNGC20-d基因及其启动子对盐胁迫的应答特性。GsCNGC20-d蛋白定位于细胞质膜。盐胁迫下，GsCNGC20-d能介导大豆发根组合植株或转基因拟南芥植株根尖细胞Ca2+的内流，从而提高[Ca2+]cyt水平，影响盐胁迫下GsCNGC20-d过表达大豆发根组合植株或拟南芥植株根系对Na+、K+吸收及其向地上部转运，使植株地上部的Na+/K+值显著降低，进而导致植株耐盐性的增强。同时，GsCNGC20-d在大豆发根组合植株中过表达会导致SOS3、SOS1和NHX1的表达量上调，并在NaCl处理后表达量显著高于转空载（Empty Vector, EV）的植株，表明了GsCNGC20-d能提高耐盐相关基因的表达来减轻NaCl对植株的伤害作用。

GsCDPK29与已报道的参与耐盐性调控的CDPK家族基因高度同源，RT-PCR鉴定与GUS染色结果也反映了GsCDPK29及其启动子对盐胁迫的响应。GsCDPK29蛋白定位于细胞质膜，并与GsCNGC20-d在细胞质膜上存在互作效应。GsCNGC20-d和GsCDPK29基因过表达时均可增强OEGsCNGC20-d、OEGsCDPK29大豆发根组合植株和WT-GsCNGC20-d、WT-GsCDPK29转基因拟南芥植株的耐盐性，而二者共表达的大豆发根组合植株（Co-OE）和转基因拟南芥植株（WT-Co-OE）则表现协同增强的盐害缓解效应。而拟南芥atcngc20atcdpk29双突变体植株则表现出与WT、突变体atcngc20和atcdpk29突变体相比对盐胁迫更敏感的表型，说明在拟南芥中两基因之间也存在协同作用，这也从侧面印证了野生大豆GsCNGC20-d和GsCDPK29蛋白之间的协同关系。

综上所述，野生大豆BB52与栽培大豆N23674的耐盐差异基因编码的GsCNGC20-d能够通过介导盐胁迫下植株根尖[Ca2+]cyt水平的变化，并与GsCDPK29协同作用，影响过表达大豆发根组合植株或转基因拟南芥幼苗对Na+、K+离子的吸收及其向地上部的转运，维持地上部较低的Na+/K+值，从而增强植株的耐盐性。

Salt stress is one of the abiotic stress factors that seriously harm the production of crops, and the ion toxicity caused by salt stress will have adverse effects on various life activities of plants. Plants maintain their normal life activities through the selective absorption of ions, accumulation of osmotic regulatory substances, scavenging of reactive oxygen species, and hormone response mechanisms. The increased ability on salt stress tolerance of plants is not only reflected in the reduction of Na+ content but also in the maintenance of the optimal cytosolic Na+/K+ ratio is the key determinant factor of plant tolerance to salt stress. Therefore, it is important to search for typical ion channels and transporters of plant Na+ and K+ homeostasis, and specific signaling pathways to understand salt stress response mechanisms. Meanwhile, plants also can activate a series of signal pathways resistance to salt stress. In order to activate these response pathways, plants first sense salt stress signals through sensors on the cell surface, and then transmit the signals to second messengers (such as Ca2+), finally the second messengers carried the signals into the cell, causing a series of response mechanisms. Soybean is an important source of vegetable protein and oil in human production and life. The cultivated soybean (Glycine max) was domesticated from its wild species (Glycine soja). Compared with cultivated soybean, wild soybean has more abundant genetic diversity, salt tolerance, drought tolerance, shade tolerance, disease and insect resistance and stronger field survival ability. Thus, wild soybean has important research value in the soybean salt tolerance mechanism and variety improvement.

In this study, comparative transcriptomic and bioinformatics analysis methods were used and screened key functional genes were based on the difference in salt tolerance between wild soybean BB52 and cultivated soybean N23674 materials. The qRT-PCR, subcellular localization, Y2H, LUC, BiFC, CRISPR-Cas9, Arabidopsis hybridization, Arabidopsis floral dip and soybean hairy-root transformation technologies were used to investigate the physiological function mechanisms of key genes for salt tolerance in wild soybean involved in salt stress regulation. The main research results are as follows:

Wild soybean BB52 materials showed stronger adaptability to salt stress than cultivated soybean N23674. Wild soybean BB52 could mainly accumulate excessive Na+ in the roots, reduce the transport to the shoots, reduce the transport to the aboveground part, and reduce the content of Na+ and the Na+/K+ ratio in the shoots, thus alleviating the damage of NaCl on the shoots of soybean plants. 8651 differentially expressed genes were analyzed specifically up-regulated in the roots of salt-tolerant wild soybean BB52 according to the transcriptome sequencing results of the roots of BB52 and N23674 seedlings under salt stress. The Ca2+ signal-related regulatory pathways in plant-pathogen interaction pathways were screened by GO annotation and KEGG enrichment, and six Cyclic Nucleotide Gated Channels (CNGCs) encoding Ca2+ transport-related channel proteins were significantly enriched in the upstream of the pathway, among which GsCNGC20-d had the highest expression level. Calcium Dependent Protein Kinases (CDPK) family members directly related to Ca2+ signaling are also significantly enriched in the above plant-pathogen interaction pathways. Among them, the differentially expressed gene annotated as GsCDPK29 has the highest expression level. It is speculated that GsCNGC20-d and GsCDPK29 may be the key candidate genes for salt tolerance of wild soybean BB52.

A total of 38 CNGC members were identified, and all of them were found to have a conserved CNBD domain (L/I-X2-G/A-X1-F/S/V/A-X1-G-D/E-ELL-X1-W/R-X12,13,22-S-X2-T-X7-E-A/S-F/L-X1-L). 38 CNGC were divided into 5 subfamilies, GsCNGC20-d was clustered in subfamily Ⅳ-A, which was highly homologous to AtCNGC20. The results of the evolutionary selection pressure analysis of GsCNGC family showed that all GsCNGC genes were negatively selected during the evolutionary process. And GsCNGC20-d belongs to a triple duplication gene. The results of qRT-PCR identification and GUS staining analysis of GsCNGC20-dpro::GUS transgenic Arabidopsis showed that GsCNGC20-d and its promoter response to salt stress. GsCNGC20-d protein was located on the plasma membrane. GsCNGC20-d could promote Ca2+ influx and increased [Ca2+]cyt levels under salt stress, influence the Na+ and K+ uptake and its transport in the roots of GsCNGC20-d overexpressing soybean hairy-root composite plants or transgenic Arabidopsis plants under salt stress, significantly reduced the Na+/K+ ratio in the shoots of the plants, thus resulting in increased the salt tolerance of the plants. Meanwhile, OEGsCNGC20-d soybean hairy-root composite plants resulted in the upregulation of SOS3, SOS1, and NHX1, showing a significantly higher expression than EV plants under NaCl treatment. These results indicated that GsCNGC20-d could further relieve the damage of NaCl on plants by improving the expression of salt-tolerant-related genes.

GsCDPK29 was highly homologous to the reported CDPK family genes related to salt tolerance, and the results of qRT-PCR and GUS staining also reflected GsCDPK29 and its promoter response to salt stress. GsCDPK29 was located on plasma membrane, and interacted with GsCNGC20-d on the plasma membrane. Overexpressed GsCNGC20-d or GsCDPK29 genes could enhance the salt tolerance of soybean hairy-root composite plants (OEGsCNGC20-d and OEGsCDPK29) and Arabidopsis plants (WT-GsCNGC20-d and WT-GsCDPK29). However, their co-overexpressed soybean hairy-root composite plants (Co-OE) and transgenic Arabidopsis plants (WT-Co-OE) showed synergistic enhanced salt mitigation effects. Arabidopsis atcngc20atcdpk29 double mutant plants showed a more salt-sensitive phenotype than WT, atcngc20 and atcdpk29 mutants. These results indicated that there was a synergistic effect between these two genes in Arabidopsis, and it was also confirmed from the side that the synergistic relationship existed between wild soybean GsCNGC20-d and GsCDPK29 protein.

In conclusion, the GsCNGC20-d protein encoded by salt tolerance differentially expressed gene between BB52 and N23674 can mediate the changes of root tip cells [Ca2+]cyt level and synergistic with GsCDPK29, influenced the process of Na+, K+ absorption and transport to the shoots of transgenic soybean hairy-root composite plants and Arabidopsis seedlings, maintained the lower Na+/K+ values in shoots of plants, thus enhanced the salt tolerance of plants.

[1] 杜莉莉．栽培和滩涂野大豆及其杂交后代耐盐性、农艺性状与籽粒品质研究［D］．南京：南京农业大学，2009：31-37．

[2] 黄璐．海大麦特异耐盐机制及大麦HKT1;5功能比较研究［D］．浙江大学，2019：18-42．

[3] 黄琼．盐胁迫对栽培大豆和野生大豆及其杂交后代幼苗的生理效应比较［D］．南京农业大学，2013：42-49．

[4] 罗庆云．野生大豆和栽培大豆耐盐机理及遗传研究［D］．南京农业大学，2003：24-44．

[5] 於丙军，刘振，卫培培．基于盐逆境下植物CLC同源基因的发掘和功能解析的研究进展［J］.南京农业大学学报, 2017，40(2)：187-194．

[6] 张晓可．利用滩涂野大豆提高栽培大豆耐盐性的生理和细胞学研究［D］．南京农业大学，2009：32-36．

[7] 朱健康，倪建平．植物非生物胁迫信号转导及应答［J］．中国稻米，2016，22（06）：52-60．

[8] Amin I, Rasool S, Mir MA, et al. Ion homeostasis for salinity tolerance in plants: a molecular approach[J]. Physiologia Plantarum, 2020, 171(4):578-594.

[9] Asano T, Tanaka N, Yang G, et al. Genome-wide identification of the rice calcium-dependent protein kinase and its closely related kinase gene families: comprehensive analysis of the CDPKs gene family in rice[J]. Plant and Cell Physiology, 2005, 46(2):356-366.

[10] Bai X, Dai L, Sun H, et al. Effects of moderate soil salinity on osmotic adjustment and energy strategy in soybean under drought stress[J]. Plant Physiology and Biochemistry, 2019, 139:307-313.

[11] Barbier-Brygoo H, De Angeli A, Filleur S, et al. Anion channels/transporters in plants: from molecular bases to regulatory networks[J]. Annual Review of Plant Biology, 2011, 62:25-51.

[12] Basu S, Kumar A, Benazir I, et al. Reassessing the role of ion homeostasis for improving salinity tolerance in crop plants[J]. Physiologia Plantarum, 2021, 171(4):502-519.

[13] Behera S, Long Y, Schmitz-Thom I, et al. Two spatially and temporally distinct Ca2+ signals convey Arabidopsis thaliana responses to K+ deficiency[J]. New Phytologist, 2017, 213(2):739-750.

[14] Borkiewicz L, Polkowska-Kowalczyk L, Cieśla J, et al. Expression of maize calcium-dependent protein kinase (ZmCPK11) improves salt tolerance in transgenic Arabidopsis plants by regulating sodium and potassium homeostasis and stabilizing photosystem II[J]. Plant Physiology, 2020, 168(1):38-57.

[15] Bredow M, Bender KW, Johnson DA, et al. Phosphorylation-dependent subfunctionalization of the calcium-dependent protein kinase CPK28[J]. Proceedings of the National Academy of Sciences of the United States of America, 2021, 118(19): e2024272118.

[16] Bulle M, Yarra R, Abbagani S. Enhanced salinity stress tolerance in transgenic chilli pepper (Capsicum annuum L.) plants overexpressing the wheat antiporter (TaNHX2) gene[J]. Molecular Breeding, 2016, 36:1-12.

[17] Chang C, Tian L, Ma L, et al. Differential responses of molecular mechanisms and physiochemical characters in wild and cultivated soybeans against invasion by the pathogenic Fusarium oxysporum Schltdl[J]. Plant Physiology, 2019, 166: 1008-1025.

[18] Chen KQ, Song MR, Guo YN, et al. MdMYB46 could enhance salt and osmotic stress tolerance in apple by directly activating stress-responsive signals[J]. Plant Biotechnology Journal, 2019, 17:2341-2355.

[19] Cheng C, Liu YM, Liu X, et al. Recretohalophyte Tamarix TrSOS1 confers higher salt tolerance to transgenic plants and yeast than glycophyte soybean GmSOS1[J]. Environmental and Experimental Botany, 2019, 165:196-207.

[20] Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J]. The Plant Journal, 1998, 16(6): 735-743.

[21] Cui Y, Lu S, Li Z, et al. Cyclic Nucleotide-Gated ion Channels 14 and 16 promote tolerance to heat and chilling in Rice[J]. Plant Physiology, 2020, 183: 1794-1808.

[22] Cui YN, Lin ZR, Cai MM. PcCLCg is involved in the accumulation of Cl- in shoots for osmotic adjustment and salinity resistance in the Cl-tolerant xerophyte Pugionium cornutum[J]. Plant Soil, 2023, 059267.

[23] Cui YX, Wang JX, Bai YX, et al. Identification of CNGCs in Glycine max and screening of related resistance genes after Fusarium solani infection[J]. Biology, 2023, 12(3):439.

[24] Dammann C, Ichida A, Hong B, et al. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis[J]. Plant Physiology, 2003, 132(4):1840-8.

[25] Davenport RJ, Muñoz-Mayor A, Jha D, et al. The Na+ transporter AtHKT1;1 controls retrieval of Na+ from the xylem in Arabidopsis[J]. Plant Cell and Environment, 2010, 30(4):497-507.

[26] De Angeli A, Monachello D, Ephritikhine G, et al. The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles[J]. Nature, 2006, 442: 939-942.

[27] De Angeli A, Zhang J, Meyer S, et al. AtALMT9 is a malate-activated vacuolar chloride channel required for stomatal opening in Arabidopsis[J]. Nature Communication, 2013, 4:1804.

[28] DeFalco TA, Bender KW, Snedden WA. Breaking the code: Ca2+ sensors in plant signaling [J]. Biochemical Journal, 2010, 425(1):27-40.

[29] DeFalco TA, Moeder W, Yoshioka K. Opening the Gates: Insights into Cyclic Nucleotide-Gated Channel-mediated signaling[J]. Trends in Plant Science, 2016, 21(11):903-906.

[30] Dong H, Wu C, Luo C, et al. Overexpression of MdCPK1a gene, a calcium dependent protein kinase in apple, increase tobacco cold tolerance via scavenging ROS accumulation[J]. PLoS One, 2020, 15(11): e0242139.

[31] Du H, Wang N, Cui F, et al. Characterization of the beta-carotene hydroxylase gene DSM2 conferring drought and oxidative stress resistance by increasing xanthophylls and abscisic acid synthesis in rice[J]. Plant Physiology, 2010, 154:1304-1318.

[32] Duszyn M, Świeżawska B, Szmidt-Jaworska A, et al. Cyclic Nucleotide Gated Channels (CNGCs) in plant signaling-current knowledge and perspectives[J]. Journal of Plant Physiology, 2019, 241:153035.

[33] Feng X, Feng P, Yu H, et al. GsSnRK1 interplays with transcription factor GsERF7 from wild soybean to regulate soybean stress resistance[J]. Plant Cell and Environment, 2020, 43(5):1192-1211.

[34] Fischer C, Kugler A, Hoth S, et al. An IQ domain mediates the interaction with calmodulin in a plant cyclic nucleotide-gated channel[J]. Plant Cell Physiology, 2013, 54: 573-584.

[35] Flowers TJ. Improving crop salt tolerance[J]. Journal of Experimental Botany, 2004, 55:307-319.

[36] Fu J, Zhu C, Wang C, et al. Maize transcription factor ZmEREB20 enhanced salt tolerance in transgenic Arabidopsis[J]. Plant Physiology Biochemistry, 2021, 159:257-267.

[37] Gao F, Han X, Wu J, et al. A heat-activated calcium-permeable channel-Arabidopsis cyclic nucleotide-gated ion channel 6-is involved in heat shock responses[J]. the Plant Jounal, 2012, 70(6):1056-1069.

[38] Gobert A, Park G, Amtmann A, et al. Arabidopsis thaliana cyclic nucleotide gated channel 3 forms a non-selective ion transporter involved in germination and cation transport[J]. Journal of Experimental Botany, 2006, 57(4):791-800.

[39] Gong Z, Xiong L, Shi H, et al. Plant abiotic stress response and nutrient use efficiency[J]. Science China Life Sciences, 2020, 63(5):635-674.

[40] Grossi CEM, Santin F, Quintana SA, et al. Calcium‑dependent protein kinase 2 plays a positive role in the salt stress response in potato[J]. Plant Cell Reports, 2022, 41(3):535-548.

[41] Guan R, Qu Y, Guo Y, et al. Salinity tolerance in soybean is modulated by natural variation in GmSALT3[J]. The Plant Journal, 2014, 80(6):937-950.

[42] Guo J, Islam MA, Lin H, et al. Genome-wide identification of Cyclic Nucleotide-Gated ion Channel gene family in wheat and functional analyses of TaCNGC14 and TaCNGC16[J]. Frontiers in Plant Science, 2018, 9:18.

[43] Guo KM, Babourina O, Christopher DA, et al. The cyclic nucleotide-gated channel, AtCNGC10, influences salt tolerance in Arabidopsis[J]. Plant Physiology, 2008, 134(3):499-507.

[44] Guo R, Yang Z, Li F, et al. Comparative metabolic responses and adaptive strategies of wheat (Triticum aestivum) to salt and alkali stress[J]. BMC Plant Biology, 2015, 15:170.

[45] Hadiarto T, Tran LS. Progress studies of drought-responsive genes in rice[J]. Plant Cell Reports, 2011, 30(3):297-310.

[46] Hao LD, Qiao XL. Genome-wide identification and analysis of the CNGC gene family in maize[J]. PeerJ, 2018, 6:e5816.

[47] Harmon AC, Gribskov M, Gubrium E, et al. The CDPK superfamily of protein kinases[J]. New Phytologist, 2001, 151(1):175-183.

[48] Harmon AC, Putnam-Evans C, Cormier MJ. A calcium-dependent but calmodulin-independent protein kinase from soybean[J]. Plant Physiology, 1987, 83(4):830-837.

[49] Hettenhausen C, Sun G, He Y, et al. Genome-wide identification of calcium-dependent protein kinases in soybean and analyses of their transcriptional responses to insect herbivory and drought stress[J]. Scientific Reports, 2016, 6:18973.

[50] Hirsch RE, Lewis BD, Spalding EP, et al. A role for the AKT1 potassium channel in plant nutrition[J]. Science, 1998, 280(5365):918-921.

[51] Hojin R, Yong-Gu C. Plant hormones in salt stress tolerance[J]. Journal of Plant Biology, 2015, 58:147-155.

[52] Hrabak EM, Chan CWM, Gribskov M, et al. The Arabidopsis CDPK-SnRK superfamily of protein kinases[J]. Plant Physiology, 2003, 132(2):666-680.

[53] Jha S, Sharma M, Pandey G. Role of cyclic nucleotide gated channels in stress management in plants[J]. Current Genomics, 2016, 17:315-329.

[54] Jiang SS, Zhang D, Wang L, et al. A maize calcium-dependent protein kinase gene, ZmCPK4, positively regulated abscisic acid signaling and enhanced drought stress tolerance in transgenic Arabidopsis[J]. Plant Physiology and Biochemistry, 2013, 71:112-120.

[55] Jiang Z, Zhou X, Tao M, et al. Plant cell-surface GIPC sphingolipids sense salt to trigger Ca2+ influx[J]. Nature, 2019, 572:341346.

[56] Jin T, Sun YY, Zhao RR, et al. Overexpression of peroxidase gene GsPRX9 confers salt tolerance in soybean[J]. International Journal of Molecular Science, 2019, 20(15):3745.

[57] Jing XQ, Li WQ, Zhou MR, et al. Correction to: rice carbohydrate‑binding malectin‑like protein, OsCBM1, contributes todrought‑stress tolerance by participating in NADPH oxidase‑mediated ROS production[J]. Rice, 2022, 15(1):3.

[58] Jossier M, Kroniewicz L, Dalmas F, et al. The Arabidopsis vacuolar anion transporter, AtCLCc, is involved in the regulation of stomatal movements and contributes to salt tolerance[J]. The Plant Journal, 2010, 64(4):563-576.

[59] Kakar KU, Nawaz Z, Kakar K, et al. Comprehensive genomic analysis of the CNGC gene family in Brassica oleracea: novel insights into synteny, structures, and transcript profiles[J]. BMC Genomics, 2017, 18(1):869.

[60] Kim Y, Wang M, Bai Y, et al. Bcl-2 suppresses activation of VPEs by inhibiting cytosolic Ca²⁺ level with elevated K⁺ efflux in NaCl-induced PCD in rice[J]. Plant Physiology and Biochemistry, 2014, 80:168-75.

[61] Köhler Neuhaus C, Neuhaus G. Characterisation of calmodulin binding to cyclic nucleotide-gated ion channels from Arabidopsis thaliana[J]. FEBS Letters, 2000, 471(2-3):133-136.

[62] Kong X, Lv W, Jiang S, et al. Genome-wide identification and expression analysis of calcium-dependent protein kinase in maize[J]. BMC Genomics, 2013, 14:433.

[63] Kovermann P, Meyer S, Hörtensteiner S, et al. The Arabidopsis vacuolar malate channel is a member of the ALMT family[J]. The Plant Journal, 2007, 52(6):1169-1180.

[64] Kudla J, Becker D, Grill E, et al. Advances and current challenges in calcium signaling[J]. New Phytologist, 2018, 218: 414-431.

[65] Kugler A, Köhler B, Palme K, et al. Salt-dependent regulation of a CNG channel subfamily in Arabidopsis[J]. BMC Plant Biology, 2009, 9:140.

[66] Latz A, Mehlmer N, Zapf S, et al. Salt stress triggers phosphorylation of the Arabidopsis vacuolar K+ channel TPK1 by calcium-dependent protein kinases (CDPKs)[J]. Molecular Plant, 2013, 6(4):1274-1289.

[67] Laurie S, Feeney KA, Maathuis FJ, et al. A role for HKT1 in sodium uptake by wheat roots[J]. The Plant Journal, 2002, 32(2):139-149.

[68] Lee HJ, Seo PJ. Ca2+ talyzing Initial responses to environmental stresses[J]. Trends in Plant Science, 2021, 26(8):849-870.

[69] Li QQ, Yang SQ, Ren J, et al. Genome-wide identification and functional analysis of the cyclic nucleotide-gated channel gene family in Chinese cabbage[J]. 3 Biotech, 2019, 9(3):114.

[70] Li WF, Wong FL, Tsai SN, et al. Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells[J]. Plant Cell and Environment, 2006, 29(6):1122-1137.

[71] Li X, Gao YQ, Wu WH, et al. Two calcium-dependent protein kinases enhance maize drought tolerance by activating anion channel ZmSLAC1 in guard cells[J]. Plant Biotechnology Journal, 2022b, 20(1):143-157.

[72] Li X, Hu D, Cai L, et al. Calcium-Dependent Protein Kinase 38 regulates flowering time and common cutworm resistance in soybean[J]. Plant Physiology, 2022c, 190(1):480-499.

[73] Li X, Liu X, Gu M, et al. Overexpression of GsCNGC20-f from Glycine soja confers submergence tolerance to hairy-root composite soybean plants and Arabidopsis seedlings by enhancing anaerobic respiration[J]. Environmental and Experimental Botany, 2022a, 199:104901.

[74] Li X, Wang Y, Liu F, et al. Transcriptomic analysis of Glycine soja and Glycine max seedlings and functional characterization of GsGSTU24 and GsGSTU42 genes under submergence stress[J]. Environmental and Experimental Botany, 2020, 171:103963.

[75] Liang W, Cui W, Ma X, et al. Function of wheat Ta-UnP gene in enhancing salt tolerance in transgenic Arabidopsis and rice[J]. Biochemical and Biophysical Research Communications, 2014, 450(1):794-801.

[76] Liang W, Ma X, Wan P, et al. Plant salt-tolerance mechanism: A review[J]. Biochemical and Biophysical Research Communications, 2018, 495(1):286-291.

[77] Liao Q, Zhou T, Yao JY, et al. Genome-scale characterization of the vacuole nitrate transporter Chloride Channel (CLC) genes and their transcriptional responses to diverse nutrient stresses in allotetraploid rapeseed[J]. PLoS One, 2018, 13(12):e0208648.

[78] Liu H, Che Z, Zeng X, et al. Genome-wide analysis of calcium-dependent protein kinases and their expression patterns in response to herbivore and wounding stresses in soybean[J]. Functional and Integrative Genomics, 2016, 16(5):481-493.

[79] Liu W, Feng J, Ma W, et al. GhCLCg-1, a vacuolar chloride channel, contributes to salt tolerance by regulating ion accumulation in upland cotton[J]. Frontiers in Plant Science, 2021b, 12: 765173.

[80] Liu X, Liu F, Zhang L, et al. GsCLC-c2 from wild soybean confers chloride/salt tolerance to transgenic Arabidopsis and soybean composite plants by regulating anion homeostasis[J]. Physiologia Plantarum, 2021a, 172:1867-1879.

[81] Liu X, Pi BY, Du ZY, et al. The transcription factor GmbHLH3 confers Cl-/salt tolerance to soybean by upregulating GmCLC1 expression for maintenance of anion homeostasis[J]. Environmental and Experimental Botany, 2022a, 194:104755.

[82] Liu X, Pi BY, Pu JW, et al. Genome-wide analysis of chloride channel-encoding gene family members and identification of CLC genes that respond to Cl-/salt stress in upland cotton[J]. Molecular Biology Reports, 2020, 47(12):9361-9371.

[83] Liu Y, Cheng H, Cheng P, et al. The BBX gene CmBBX22 negatively regulates drought stress tolerance in chrysanthemum[J]. Horticulture Research, 2022b, 25:9.

[84] Lu S, Dong L, Fang C, et al. Stepwise selection on homeologous PRR genes controlling flowering and maturity during soybean domestication[J]. Nature Genetics, 2020, 52(4):428-436.

[85] Lu W, Guo C, Li X, et al. Overexpression of TaNHX3, a vacuolar Na+/H+ antiporter gene in wheat, enhances salt stress tolerance in tobacco by improving related physiological processes[J]. Plant Physiolgy and Biochemistry, 2014, 76:17-28.

[86] Lu Y, Yu M, Jia Y, et al. Structural basis for the activity regulation of a potassium channel AKT1 from Arabidopsis[J]. Nature Communications, 2022a, 13(1):5682.

[87] Lu ZY, Yin G, Chai M, et al. Systematic analysis of CNGCs in cotton and the positive role of GhCNGC32 and GhCNGC35 in salt tolerance[J]. BMC Genomics, 2022b, 23(1):560.

[88] Luo QY, Yu BJ, Liu YL. Differential sensitivity to chloride and sodium ions in seedings of Glycine max and Glycine soja under NaCl stress[J]. Journal of Plant Physiology, 2005, 162:1003-1012.

[89] Ma W, Berkowitz GA. Ca2+ conduction by plant cyclic nucleotide gated channels and associated signaling components in pathogen defense signal transduction cascades[J]. New Phytologist, 2011, 190: 566-572.

[90] Ma XJ, Fu JD, Tang YM, et al. GmNFYA13 improves salt and drought tolerance in transgenic soybean plants[J]. Frontiers in Plant Science, 2020, 11:587244.

[91] Maach M, Rodríguez-Rosales MP, Venema K, et al. Improved yield, fruit quality, and salt resistance in tomato co-overexpressing LeNHX2 and SlSOS2 genes[J]. Physiology and Molecular Biology Plants, 2021, 27(4):703-712.

[92] Maathuis FJ. Vacuolar two-pore K+ channels act as vacuolar osmosensors[J]. New Phytologist, 2011, 191:84-91.

[93] Marmagne A, Vinauger-Douard M, Monachello D, et al. Two members of the Arabidopsis CLC (chloride channel) family, AtCLCe and AtCLCf, are associated with thylakoid and Golgi membranes, respectively[J]. Journal of Experimental Botany, 2007, 58(12):3385–3393.

[94] Meng L, Zhang Q, Yang J, et al. PtrCDPK10 of Poncirus trifoliata functions in dehydration and drought tolerance by reducing ROS accumulation via phosphorylating PtrAPX[J]. Plant Science, 2020a, 291:110320.

[95] Meng X, Liu S, Dong T, et al. Comparative transcriptome and proteome analysis of salt-tolerant and salt-sensitive sweet potato and overexpression of IbNAC7 confers salt tolerance in Arabidopsis[J]. Frontiers in Plant Science, 2020b, 11:572540.

[96] Mittler R. Abiotic stress, the field environment and stress combination[J]. Trends in plant science, 2006, 11:15-19.

[97] Moon JY, Belloeil C, Ianna ML, et al. Arabidopsis CNGC family members contribute to heavy metal ion uptake in plants[J]. International Journal of Molecular Sciences, 2019, 20(2):413.

[98] Nadarajah KK. ROS homeostasis in abiotic stress tolerance in plants[J]. International Journal of Molecular Sciences, 2020, 21:5208.

[99] Nawaz Z, Kakar KU, Saand MA, et al. Cyclic nucleotide-gated ion channel gene family in rice, identification, characterization and experimental analysis of expression response to plant hormones, biotic and abiotic stresses[J]. BMC Genomics, 2014, 15(1):853.

[100] Oh DH, Lee SY, Bressan RA, et al. Intracellular consequences of SOS1 deficiency during salt stress[J]. Journal of Experimental Botany, 2010, 61(4):1205-1213.

[101] Pan Y, Chai X, Gao Q, et al. Dynamic interactions of plant CNGC subunits and calmodulins drive oscillatory Ca2+ channel activities[J]. Development Cell, 2019, 48:710-725.

[102] Panchy N, Lehti-Shiu M, Shiu S H. Evolution of gene duplication in plants[J]. Plant Physiology, 2016, 171(4):2294-2316.

[103] Pantha P, Dassanayake M. Living with Salt[J]. The Innovation, 2020, 1(3):2.

[104] Piñeros MA, Cançado GM, Maron LG, et al. Not all ALMT1-type transporters mediate aluminumactivated organic acid responses: the case of ZmALMT1-an anion-selective transporter[J]. The Plant Journal, 2008, 53(2):352-367.

[105] Qi X, Li MW, Xie M, et al. Identification of a novel salt tolerance gene in wild soybean by whole-genome sequencing[J]. Nature Communication, 2014, 5:4340.

[106] Qiu QS, Guo Y, Dietrich MA, et al. Regulation of SOS1, a plasma membrane Na+/H+ exchanger in Arabidopsis thaliana, by SOS2 and SOS3[J]. Proceedings of the National Academy of Sciences of the USA, 2002, 99(12):8436-8441.

[107] Raghavendra AS, Gonugunta VK, Christmann A, et al. ABA perception and signalling[J]. Trends in Plant Science, 2010, 15(7):395-401.

[108] Rao KP, Richa T, Kumar K, et al. In silico analysis reveals 75 members of mitogen-activated protein kinase kinase kinase gene family in rice[J]. DNA Research, 2010, 17:139-153.

[109] Ren ZH, Gao JP, Li LG, et al. A rice quantitative trait locus for salt tolerance encodes a sodium transporter[J]. Nature Genetics, 2005, 37(10):1141-1146.

[110] Rubio F, Gassmann W, Schroeder JI. Sodium-driven potassium uptake by the plant potassium transporter HKT1 and mutations conferring salt tolerance[J]. Science, 1995, 270(5242):1660-1663.

[111] Saand MA, Xu YP, Li W, et al. Cyclic nucleotide gated channel gene family in tomato: genome-wide identification and functional analyses in disease resistance[J]. Frontiers in Plant Science, 2015, 6:303.

[112] Saeng-ngam S, Takpirom W, Buaboocha T, et al. The role of the OsCam1-1 salt stress sensor in ABA accumulation and salt tolerance in rice[J]. Journal of Plant Biology, 2012, 55(3):198-208.

[113] Saijo Y, Hata S, Kyozuka J, et al. Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants[J]. The Plant Journal, 2000, 23:319-327.

[114] Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method[J]. Nature Protocol, 2008, 3(6):1101-1108.

[115] Schulz P, Piepenburg K, Lintermann R, et al. Improvingplant drought tolerance and growth under water limitationthrough combinatorial engineering of signalling networks[J]. Plant Biotechnology Journal, 2021, 19:74-86.

[116] Shi MQ, Liao XL, Ye Q, et al. Linkage and association mapping of wild soybean (Glycine soja) seeds germinating under salt stress[J]. Journal of Integrative Agriculture, 2022, 21(10):2833-2847.

[117] Simeunovic A, Mair A, Wurzinger B, et al. Know where your clients are: subcellular localization and targets of calcium-dependent protein kinases[J]. Journal of Experimental Botany, 2016, 67(13):3855-3872.

[118] Song H, Yin Z, Chao M, et al. Functional properties and expression quantitative trait loci for phosphate transporter GmPT1 in soybean[J]. Plant Cell and Environment, 2014, 37(2):462-472.

[119] Stadtman ER. Protein oxidation and aging[J]. Science, 1992, 257(5074):1220-1224.

[120] Szekely G, Abraham E, Cseplo A, et al. Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis[J]. The Plant Journal, 2008, 53:11-28.

[121] Tan YQ, Yang Y, Zhang A, et al. Three CNGC family members, CNGC5, CNGC6, and CNGC9, are required for constitutive growth of Arabidopsis root hairs as Ca2+-permeable channels[J]. Plant Communication, 2020, 1(1):100001.

[122] Tunc-Ozdemir M, Rato C, Brown E, et al. Cyclic nucleotide gated channels 7 and 8 are essential for male reproductive fertility[J]. PLoS One, 2013, 8(2): e55277.

[123] Uozumi N, Kim EJ, Rubio F, et al. The Arabidopsis HKT1 gene homolog mediates inward currents in Xenopus laevis oocytes and Na+ uptake in Saccharomyces cerevisiae[J]. Plant Physiology, 2000, 122(4):1249-1259.

[124] Vaidyanathan H, Sivakumar P, Chakrabarty R, et al. Scavenging of reactive oxygen species in NaCl-stressed rice (Oryza sativa L.) - differential response in salt-tolerant and sensitive varieties[J]. Plant Science, 2003, 165(6):1411-1418.

[125] van Zelm E, Zhang Y, Testerink C. Salt Tolerance Mechanisms of Plants[J]. Annual Review of Plant Biology, 2020, 71:403-433.

[126] Von der Fecht-Bartenbach J, Bogner M, Krebs M, et al. Function of the anion transporter AtCLC-d in the trans-Golgi network[J]. The Plant Journal, 2007, 50:466-474.

[127] Wang CF, Han GL, Yang ZR, et al. Plant salinity sensors: current understanding and future directions[J]. Frontiers in Plant Science, 2022, 13:859224.

[128] Wang D, Liu YX, Yu Q, et al. Functional analysis of the soybean GmCDPK3 gene responding to drought and salt stresses[J]. International Journal of Molecular Sciences, 2019b, 20(23):5909.

[129] Wang F, Liu J, Zhou L, et al. Senescence-specific change in ROS scavenging enzyme activities and regulation of various SOD isozymes to ROS levels in psf mutant rice leaves[J]. Plant Physiology and Biochemistry, 2016a, 109:248-261.

[130] Wang J, An C, Guo H, et al. Physiological and transcriptomic analyses reveal the mechanisms underlying the salt tolerance of Zoysia japonica Steud[J]. BMC Plant Biology, 2020a, 20(1):114.

[131] Wang J, Liu X, Zhang A, et al. A Cyclic Nucleotide-Gated Channel mediates cytoplasmic calcium elevation and disease resistance in rice[J]. Cell Research, 2019a, 29(10):820-831.

[132] Wang J, Ren Y, Liu X, et al. Transcriptional activation and phosphorylation of OsCNGC9 confer enhanced chilling tolerance in rice[J]. Molecular Plant, 2021b, 14(2):315-329.

[133] Wang L, Jia G, Jiang X, et al. Altered chromatin architecture and gene expression during polyploidization and domestication of soybean[J]. Plant Cell, 2021c, 33(5):1430-1446.

[134] Wang LX, Li M, Liu ZG, et al. Genome-wide identification of CNGC genes in Chinese jujube (Ziziphus jujuba Mill.) and ZjCNGC2 mediated signalling cascades in response to cold stress[J]. BMC Genomics, 2020b, 21(1):191.

[135] Wang M, Zhao X, Xiao Z, et al. A wheat superoxide dismutase gene TaSOD2, enhances salt resistance through modulating redox homeostasis by promoting nadph oxidase activity[J]. Plant Molecular Biology, 2016b, 91(1-2):115-130.

[136] Wang N, Zhang WX, Qin MY, et al. Drought tolerance conferred in soybean (Glycine max. L) by GmMYB84, a novel R2R3-MYB transcription factor[J]. Plant and Cell Physiology, 2017, 58:1764-1776.

[137] Wang X, Feng C, Tian L, et al. A transceptor-channel complex couples nitrate sensing to calcium signaling in Arabidopsis[J]. Molecular Plant, 2021a, 14(5):774-786.

[138] Wang YY, Hsu PK, Tsay YF. Uptake, allocation and signaling of nitrate[J]. Trends in Plant Science, 2012, 17:458-467.

[139] Ward JM, Hirschi KD, Sze H. Plants pass the salt[J]. Trends in Plant Science, 2003, 8(5):200-201.

[140] Wei M, Wang S, Dong H, et al. Characterization and comparison of the CPK gene family in the Apple (Malus×domestica) and other rosaceae species and its response to Alternaria alternata infection[J]. PLoS One, 2016b, 11(5): e0155590.

[141] Wei PP, Che BN, Shen LK, et al. Identification and functional characterization of the chloride channel gene, GsCLC-c2 from wild soybean[J]. BMC Plant Biology, 2019, 19(1):121.

[142] Wei PP, Wang L, Liu A, et al. GmCLC1 confers enhanced salt tolerance through regulating chloride accumulation in soybean[J]. Frontiers in Plant Science, 2016a, 7:1082.

[143] Wei QJ, Liu YZ, Zhou GF, et al. Overexpression of CsCLCc, a chloride channel gene from Poncirus trifoliata, enhances salt tolerance in Arabidopsis[J]. Plant Molecular Biology Reporter, 2013, 31(6):1548-1557.

[144] Willekens H, Langebartels C, Tiré C, et al. Differential expression of catalase genes in Nicotiana plumbaginifolia (L.)[J]. Proceedings of the National Academy of Sciences of USA, 1994, 91(22):10450-10454.

[145] Xu LH, Wang WY, Guo JJ, et al. Zinc improves salt tolerance by increasing reactive oxygen species scavenging and reducing Na+ accumulation in wheat seedlings[J]. Biologia plantarum, 2014, 58:751-757.

[146] Yamaguchi T, Aharon GS, Sottosanto JB, et al. Vacuolar Na+/H+ antiporter cation selectivity is regulated by calmodulin from within the vacuole in a Ca2+- and pH-dependent manner[J]. Proceedings of the National Academy of Sciences of USA, 2005, 102(44):16107-16112.

[147] Yang S, Cai WW, Shen L, et al. A CaCDPK29-CaWRKY27b module promotes CaWRKY40-mediated thermotolerance and immunity to Ralstonia solanacearum in pepper[J]. New phytologist, 2022, 233(4):1843-1863.

[148] Yu L, Liu W, Guo Z, et al. Interaction between MdMYB63 and MdERF106 enhances salt tolerance in apple by mediating Na+/H+ transport[J]. Plant Physiology and Biochemistry, 2020, 155:464-471.

[149] Yue Y, Zhang M, Zhang J, et al. SOS1 gene overexpression increased salt tolerance in transgenic tobacco by maintaining a higher K+/Na+ ratio[J]. Journal of Plant Physiology, 2012, 169(3):255-261.

[150] Zhang H, Feng H, Zhang J, et al. Emerging crosstalk between two signaling pathways coordinates K+ and Na+ homeostasis in the halophyte Hordeum brevisubulatum[J]. Journal of Experimental Botany, 2020, 71(14):4345-4358.

[151] Zhang JL, Wang JX, Jiang W, et al. Identification and analysis of NaHCO3 stress responsive genes in wild soybean (Glycine soja) roots by RNA-seq[J]. Frontiers in Plant Science, 2016, 7:1842.

[152] Zhang M, Liu S, Wang Z, et al. Progress in soybean functional genomics over the past decade[J]. Plant Biotechnology Journal, 2022, 20(2):256-282.

[153] Zhang XK, Zhou QH, Cao JH, et al. Differential Cl-/salt tolerance and NaCl induced alternations of tissue and cellular ion fluxes in Glycine max, Glycine soja and their hybrid seedlings[J]. Journal Of Agronomy and Crop Science, 2011, 197:329-339.

[154] Zhang YJ, Li YB, Yang J, et al. Genome-wide analysis and expression of cyclic nucleotide-gated ion channel (CNGC) family genes under cold stress in Mango (Mangifera indica)[J]. Plants, 2023, 12(3):592.

[155] Zhao JH, Peng S, Cui HT, et al. Dynamic Expression, Differential regulation and functional diversity of the CNGC family genes in cotton[J]. International Journal of Molecular Science, 2022, 23(4):2041.

[156] Zhao JY, Lu ZW, Sun Y, et al. The ankyrin-repeat gene GmANK114 confers drought and salt tolerance in Arabidopsis and soybean[J]. Frontiers in Plant Science, 2020, 11:584167.

[157] Zhao XF, Wei PP, Liu Z, et al. Soybean Na+/H+ antiporter GmsSOS1 enhances antioxidant enzyme activity and reduces Na+ accumulation in Arabidopsis and yeast cells under salt stress[J]. Acta Physiologiae Plantarum, 2017, 39:19.

[158] Zhao Y, Du H, Wang Y, et al. The calcium-dependent protein kinase ZmCDPK7 functions in heat-stress tolerance in maize[J]. Journal of Integrative Plant Biology, 2021, 63(3):510-527.

[159] Zhou L, Lan W, Jiang Y, et al. A calcium-dependent protein kinase interacts with and activates a calcium channel to regulate pollen tube growth[J]. Molecular Plant, 2014, 7(2):369-376.

[160] Zhu JK. Plant salt tolerance[J]. Trends in Plant Science, 2001, 6(2):66-71.

[161] Zhu JK. Salt and drought stress signal transduction in plants[J]. Annual Review of Plant Biology, 2002, 53:247-273.

[162] Zhu JK. Abiotic stress signaling and responses in plants[J]. Cell, 2016, 167:313-324.

[163] Zhuang Y, Wang X, Li X, et al. Phylogenomics of the genus Glycine sheds light on polyploid evolution and life-strategy transition[J]. Nature Plants, 2022, 8(3):233-244.

[164] Zia-Ur-Rehman M, Anayatullah S, Irfan E, et al. Nanoparticles assisted regulation of oxidative stress and antioxidant enzyme system in plants under salt stress: A review[J]. Chemosphere, 2023, 314:137649. u

[165] Zuo R, Hu R, Chai G, et al. Genome-wide identification, classification, and expression analysis of CDPK and its closely related gene families in poplar (Populus trichocarpa)[J]. Molecular Biology Reports, 2013, 40(3):2645-2662.