Welcome to Acta Agronomica Sinica，

Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (7): 1025-1032.doi: 10.3724/SP.J.1006.2020.94152

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and functional analysis of GmNRT1.2a and GmNRT1.2b in soybean

LI Guo-Ji**,ZHU Lin**,CAO Jin-Shan,WANG You-Ning()   

  1. College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2019-10-09 Accepted:2020-03-24 Online:2020-07-12 Published:2020-04-27
  • Contact: You-Ning WANG E-mail:youningwang@mail.hzau.edu.cn
  • About author:** Contributed equally to this work
  • Supported by:
    National Transgenic Major Project of China(2018ZX0800919B);National Natural Science Foundation of China(31872873)

RichHTML370

26

PDF

612

Abstract:

Nitrate transporters (NRTs) have been found to be involved in nitrate uptake, transport and allocation in Arabidopsis. The role of GmNRT1.2s in soybean (Glycine max) symbiotic nitrogen fixation process has been speculated by bioinformatics analysis, however, its biological function has not been explored yet. In this study, we mainly focused on analyzing the expression pattern and biological function of GmNRT1.2a and GmNRT1.2b in soybean. The relative expression levels of GmNRT1.2a and GmNRT1.2b were higher in leaves, which induced by nitrate and up-regulated with increasing nitrate concentration. GmNRT1.2a and GmNRT1.2b expressions were also induced by rhizobial inoculation and nod factor (NF) treatment. Overexpression of GmNRT1.2a or GmNRT1.2b caused dramatic increment of nodule number. The results provide some data for further investigating the molecular mechanism of GmNRT1.2a and GmNRT1.2b in regulating the symbiotic nitrogen fixation process of soybean.

Key words: soybean, nodule, expression analysis, nodulation, symbiotic nitrogen fixation

Fig. 1

Construction of GmNRT1.2a and GmNRT1.2b overexpression vectors A: the coding sequence of GmNRT1.2a and GmNRT1.2b were obtained through PCR amplification. B: the partial vector map of pEGAD."

Fig. 2

Expression pattern of GmNRT1.2a and GmNRT1.2b in different developmental stages of soybean Mean values indexed with different letters are significantly different at P < 0.05. DAI: days after inoculation."

Fig. 3

Expression pattern of GmNRT1.2a and GmNRT1.2b under different concentrations of nitrate Soybeans were germinated in vermiculite at different nitrate concentrations (0N-0 mmol L-1, LN-0.25 mmol L-1, HN-15.75 mmol L-1). The roots were collected at 15 days after germination. The expression of GmNRT1.2a (A) and GmNRT1.2b (B) was analyzed by RT-qPCR. Bars with different lowercase letters in each figure are significantly different at P < 0.05."

Fig. 4

Expression patterns of GmNRT1.2a and GmNRT1.2b in response to rhizobium inoculation and nod factors treatment Values followed by different letters are significantly different at P < 0.05."

Fig. 5

Nodule number increased by 35S::GmNRT1.2a or 35S::GmNRT1.2b under low nitrate condition A, B: RT-qPCR analysis of GmNRT1.2a or GmNRT1.2b in single hairy root transformed with empty vector, 35S::GmNRT1.2a or 35S::GmNRT1.2b inoculated with Bradyrhizobium japonicum USDA110 at 10 and 28 days treatment. GmELF1b was used as an endogenous control for gene expression. Bars with different lowercase letters in each figure are significantly different at P < 0.05. C: the phenotype of nodule per hairy root transformed with empty vector (EV), 35S::GmNRT1.2a and 35S::GmNRT1.2b at 28 DAI. Bar = 5 mm. D: nodule number per hairy root transformed with empty vector (EV), 35S::GmNRT1.2a or 35S::GmNRT1.2b were counted at 28 DAI. The values followed by * and *** are significantly different at P < 0.05 and P < 0.001."

[1] 张合琼, 张汉马, 梁永书, 南文斌. 植物硝酸盐转运蛋白研究进展. 植物生理学报, 2016,52:141-149.
Zhang H Q, Zhang H M, Liang Y S, Nan W B. Research progress of nitrate in plant transport mechanism. Acta Phytophysiol Sin, 2016,52:141-149 (in Chinese with English abstract).
[2] 姜丽娜, 张凯, 宋飞, 张新敏, 蒿宝珍, 李春喜. 拔节期追氮对冬小麦产量、效益及氮素吸收和利用的影响. 麦类作物学报, 2013,33:716-721.
doi: 10.7606/j.issn.1009-1041.2013.04.016
Jiang L N, Zhang K, Song F, Zhang X M, Hao B Z, Li C X. Effects of nitrogen topdressing at jointing stage on grain yield, benefit, absorption and utilization of nitrogen in winter wheat. J Triticeae Crops, 2013,33:716-721 (in Chinese with English abstract).
doi: 10.7606/j.issn.1009-1041.2013.04.016
[3] Masclaux-Daubresse C, Daniel-Vedele F, Dechorgnat J, Chardon F, Gaufichon L, Suzuki A. Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Ann Bot, 2010,105:1141-1157.
pmid: 20299346
[4] Dong C X, Shen Q R, Wang G. Tomato growth and organic acid changes in response to partial replacement of NO3--N by NH4+-N. Pedosphere, 2004 , 14:159-164.
[5] Vojtíšková L, Munzarová E, Votrubová O, Řihová A, Juřicová B. Growth and biomass allocation of sweet flag (Acorus calamus L.) under different nutrient conditions. Hydrobiologia, 2004,518:9-22.
doi: 10.1023/B:HYDR.0000025052.81373.f3
[6] 张富仓, 严富来, 范兴科, 李国栋, 刘翔, 陆军胜, 王英, 麻玮青. 滴灌施肥水平对宁夏春玉米产量和水肥利用效率的影响. 农业工程学报, 2018,34(22):111-120.
Zhang F C, Yan F L, Fan X K, Li G D, Liu X, Lu J S, Wang Y, Ma W Q. Effects of irrigation and fertilization levels on grain yield and water-fertilizer use efficiency of drip-fertigation spring maize in Ningxia. Trans CSAE, 2018,34(22):111-120 (in Chinese with English abstract).
[7] Ren Y Z, Qian Y Y, Xu Y H, Zou C Q, Liu D C, Zhao X Q, Zhang A M, Tong Y P. Characterization of QTLs for root traits of wheat grown under different nitrogen and phosphorus supply levels. Front Plant Sci, 2017,8:2096.
doi: 10.3389/fpls.2017.02096 pmid: 29312372
[8] Walch-Liu P, Forde B G. Nitrate signalling mediated by the NRT1.1 nitrate transporter antagonizes L-glutamate-induced changes in root architecture. Plant J, 2008,54:820-828.
doi: 10.1111/j.1365-313X.2008.03443.x pmid: 18266918
[9] Wang R, Okamoto M, Xing X, Crawford N M. Microarray analysis of the nitrate response in Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes and new linkages to glucose, trehalose-6-phosphate, iron, and sulfate metabolism. Plant Physiol, 2003,132:556-567.
doi: 10.1104/pp.103.021253 pmid: 12805587
[10] Forde B G. Nitrogen signalling pathways shaping root system architecture: an update. Curr Opin Plant Biol, 2014,21:30-36.
doi: 10.1016/j.pbi.2014.06.004 pmid: 24997289
[11] Alboresi A, Gestin C, Leydecker M T, Bedu M, Meyer C, Truong H M. Nitrate, a signal relieving seed dormancy in Arabidopsis. Plant Cell Environ, 2005,28:500-512.
doi: 10.1111/j.1365-3040.2005.01292.x pmid: 16229082
[12] Castro Marín I, Loef I, Bartetzko L, Searle I, Coupland G, Stitt M, Osuna D. Nitrate regulates floral induction in Arabidopsis, acting independently of light, gibberellin and autonomous pathways. Planta, 2011,233:539-552.
pmid: 21113723
[13] Madsen E B, Madsen L H, Radutoiu S, Rakwalska M, Szczyglowski K, Sato S, Kaneko T, Tabata S, Sandal N, Stougaard J. A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature, 2003,425:637-640.
doi: 10.1038/nature02045 pmid: 14534591
[14] Radutoiu S, Madsen L H, Madsen E B, Felle H H, Umehara Y, Grønlund M, Sato S, Nakamura Y, Tabata S, Sandal N, Stougaard J. Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature, 2003,425:585-592.
pmid: 14534578
[15] Arrighi J F, Barre A, Ben Amor B, Bersoult A, Soriano L C, Mirabella R, de Carvalho-Niebel F, Journet E P, Ghérardi M, Huguet T, Geurts R, Dénarié J, Rougé P, Gough C. The Medicago truncatula lysin motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes. Plant Physiol, 2006,142:265-279.
pmid: 16844829
[16] Indrasumunar, A, Searle I, Lin M H, Kereszt A, Men A, Carroll B J, Gresshoff P M. Nodulation factor receptor kinase 1α controls nodule organ number in soybean (Glycine max L. Merr.). Plant J, 2011,65:39-50.
pmid: 21175888
[17] Searle I, Miyagi M, Li D X, Nguyen C D T, Men A, Carroll B J, Gresshoff P M. Inactivation of duplicated nod factor receptor 5 (NFR5) genes in recessive loss-of-function non-nodulation mutants of allotetraploid soybean (Glycine max L. Merr.). Plant Cell Physiol, 2010,51:201-214.
doi: 10.1093/pcp/pcp178 pmid: 20007291
[18] Röhrig H, Schmidt J, Miklashevichs E, Schell J, John M. Soybean ENOD40 encodes two peptides that bind to sucrose synthase. Proc Natl Acad Sci USA, 2002,99:1915-1920.
doi: 10.1073/pnas.022664799 pmid: 11842184
[19] Wang Y N, Wang L X, Zou Y M, Chen L, Cai Z M, Zhang S L, Zhao F, Tian Y P, Jiang Q, Ferguson B J, Gresshoff P M, Li X. Soybean miR172c targets the repressive AP2 transcription factor NNC1 to activate ENOD40 expression and regulate nodule initiation. Plant Cell, 2014,26:4728-4801.
[20] Li X, Zhao J, Tan Z, Zeng R, Liao H. GmEXPB2, a cell wall β-expansin, affects soybean nodulation through modifying root architecture and promoting nodule formation and development. Plant Physiol, 2015,169:2640-2653.
pmid: 26432877
[21] Chen L Y, Qin L, Zhou L, Li X, Chen Z, Sun L, Wang W, Lin Z, Zhao J, Yamaji N, Ma J F, Gu M, Xu J, Liao H. A nodule-localized phosphate transporter GmPT7 plays an important role in enhancing symbiotic N2 fixation and yield in soybean. New Phytol, 2018,221:2013-2025.
doi: 10.1111/nph.15541 pmid: 30317659
[22] Yan Q Q, Wang L X, Li X. GmBEHL1, a BES1/BZR1 family protein, negatively regulates soybean nodulation. Sci Rep, 2018,8:7614.
pmid: 29769571
[23] Choudhury S R, Pandey S. Specific subunits of heterotrimeric G proteins play important roles during nodulation in soybean. Plant Physiol, 2013,162:522-533.
doi: 10.1104/pp.113.215400 pmid: 23569109
[24] Choudhury S R, Pandey S. Phosphorylation-dependent regulation of G-protein cycle during nodule formation in soybean. Plant Cell, 2015,27:3260-3276.
doi: 10.1105/tpc.15.00517 pmid: 26498905
[25] Wang Y N, Yang W, Zuo Y Y, Zhu L, Hastwell A H, Chen L, Tian Y P, Su C, Ferguson B J, Li X. GmYUC2a mediates auxin biosynthesis during root development and nodulation in soybean. J Exp Bot, 2019,10:3165-3176.
[26] Cai Z M, Wang Y N, Zhu L, Tian Y P, Chen L, Sun Z X, Ullah I, Li X. GmTIR1/GmAFB3‐based auxin perception regulated by miR393 modulates soybean nodulation. New Phytol, 2017,215:672-686.
doi: 10.1111/nph.14632 pmid: 28598036
[27] Bustos-Sanmamed P, Mao G, Deng Y, Elouet M, Khan G A, Bazin J, Lelandais-Brière C. Overexpression of miR160 affects root growth and nitrogen-fixing nodule number in Medicago truncatula. Funct Plant Biol, 2013,40:1208-1220.
doi: 10.1071/FP13123 pmid: 32481189
[28] Wang Y N, Li K X, Chen L, Zou Y M, Liu H P, Tian Y P, Li D X, Wang R, Zhao F, Ferguson B J, Gresshoff P M, Li X. MicroRNA167-directed regulation of the auxin response factors, GmARF8a and GmARF8b, is required for soybean nodulation and lateral root development. Plant Physiol, 2015,168:984-999.
doi: 10.1104/pp.15.00265 pmid: 25941314
[29] Wang Y Y, Hsu P K, Tsay Y F. Uptake, allocation and signaling of nitrate. Trends Plant Sci, 2012,17:458-467.
doi: 10.1016/j.tplants.2012.04.006 pmid: 22658680
[30] Huang N C, Liu K H, Lo H J, Tsay Y F. Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake. Plant Cell, 1999,11:1381-1392.
doi: 10.1105/tpc.11.8.1381 pmid: 10449574
[31] 朱林, 左妍妍, 曹金山, 王小迪, 杨薇, 王幼宁. 大豆NRT1.2同源基因的生物信息学分析. 大豆科学, 2019,38:371-378.
Zhu L, Zuo Y Y, Cao J S, Wang X D, Yang W, Wang Y N. Bioinformatic analysis of NRT1.2 homologous gene in soybean. Soybean Sci, 2019,38:371-378 (in Chinese with English abstract).
[32] Wang Y W, Li P C, Cao X F, Wang X J, Zhang A M, Li X. Identification and expression analysis of miRNAs from nitrogen-fixing soybean nodules. Biochem Biophys Res Commun, 2009,378:799-803.
doi: 10.1016/j.bbrc.2008.11.140 pmid: 19084500
[33] Kereszt A, Li D X, Indrasumunar A, Nguyen C D T, Nontachaiyapoom S, Kinkema M, Gresshoff P M. Agrobacterium rhizogenes mediated transformation of soybean to study root biology. Nat Protoc, 2007,2:948-952.
pmid: 17446894
[34] Jian B, Hou W S, Wu C X, Liu B, Liu W, Song S K, Bi Y R, Han T F. Agrobacterium rhizogenes-mediated transformation of Superroot-derived Lotus corniculatus plants: a valuable tool for functional genomics. BMC Plant Biol, 2009,9:78.
doi: 10.1186/1471-2229-9-78 pmid: 19555486
[35] Okamoto M, Vidmar J J, Glass A D. Regulation of NRT1 and NRT2 gene families of Arabidopsis thaliana: responses to nitrate provision. Plant Cell Physiol, 2003,44:304-317.
pmid: 12668777
[36] Araki R, Hasegawa H. Expression of rice (Oryza sativa L.) genes involved in high-affinity nitrate transport during the period of nitrate induction. Breed Sci, 2006,56:295-302.
doi: 10.1270/jsbbs.56.295
[37] Hu B, Wang W, Ou S J, Li H2, 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 Y, Li L G, Chu C C. Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies. Nat Genet, 2015,47:834-838.
doi: 10.1038/ng.3337 pmid: 26053497
[38] Hu B, Jiang Z M, Wang W, Qiu Y H, Zhang Z H, Liu Y Q, Li A F, Gao X K, Liu L C, Qian Y W, Huang X H, Yu F F, Kang S, Wang Y Q, Xie J P, Cao S Y, Zhang L H, Wang Y C, Xie Q, Kopriva S, Chu C C. Nitrate-NRT1.1B-SPX4 cascade integrates nitrogen and phosphorus signalling networks in plants. Nat Plants, 2019,5:401-413.
doi: 10.1038/s41477-019-0384-1 pmid: 30911122
[39] Zhang J Y, Liu Y X, Zhang N, Hu B, Jin T, Xu H R, Qin R Y, Yan P X, Zhang X N, Guo X X, Hui J, Cao S Y, Wang X, Wang C, Wang H, Qu B Y, Fan G Y, Yuan L X, Garrido-Oter R, Chu C C, Bai Y. NRT1.1B is associated with root microbiota composition and nitrogen use in field-grown rice. Nat Biotechnol, 2019,37:676-684.
doi: 10.1038/s41587-019-0104-4 pmid: 31036930
[40] Lauter F R, Ninnemann O, Bucher M, Riesmeier J W, Frommer W B. Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato. Proc Natl Acad Sci USA, 1996,93:8139-8144.
pmid: 8755617
[41] Zhao X Q, Li Y J, Liu J, Li B, Liu Q Y, Tong Y P, Li J Y, Li Z S. Isolation and expression analysis of a high-affinity nitrate transporter TaNRT2.3 from roots of wheat. Acta Bot Sin, 2004,46:347-354.
[42] Zhou J J, Theodoulou F L, Muldin I, Ingemarsson B, Miller A J. Cloning and functional characterization of a Brassica napus transporter that is able to transport nitrate and histidine. J Biol Chem, 1998,278:12017-12023.
[1] CHEN Ling-Ling, LI Zhan, LIU Ting-Xuan, GU Yong-Zhe, SONG Jian, WANG Jun, QIU Li-Juan. Genome wide association analysis of petiole angle based on 783 soybean resources (Glycine max L.) [J]. Acta Agronomica Sinica, 2022, 48(6): 1333-1345.
[2] CHEN Song-Yu, DING Yi-Juan, SUN Jun-Ming, HUANG Deng-Wen, YANG Nan, DAI Yu-Han, WAN Hua-Fang, QIAN Wei. Genome-wide identification of BnCNGC and the gene expression analysis in Brassica napus challenged with Sclerotinia sclerotiorum and PEG-simulated drought [J]. Acta Agronomica Sinica, 2022, 48(6): 1357-1371.
[3] YANG Huan, ZHOU Ying, CHEN Ping, DU Qing, ZHENG Ben-Chuan, PU Tian, WEN Jing, YANG Wen-Yu, YONG Tai-Wen. Effects of nutrient uptake and utilization on yield of maize-legume strip intercropping system [J]. Acta Agronomica Sinica, 2022, 48(6): 1476-1487.
[4] YU Chun-Miao, ZHANG Yong, WANG Hao-Rang, YANG Xing-Yong, DONG Quan-Zhong, XUE Hong, ZHANG Ming-Ming, LI Wei-Wei, WANG Lei, HU Kai-Feng, GU Yong-Zhe, QIU Li-Juan. Construction of a high density genetic map between cultivated and semi-wild soybeans and identification of QTLs for plant height [J]. Acta Agronomica Sinica, 2022, 48(5): 1091-1102.
[5] LI A-Li, FENG Ya-Nan, LI Ping, ZHANG Dong-Sheng, ZONG Yu-Zheng, LIN Wen, HAO Xing-Yu. Transcriptome analysis of leaves responses to elevated CO2 concentration, drought and interaction conditions in soybean [Glycine max (Linn.) Merr.] [J]. Acta Agronomica Sinica, 2022, 48(5): 1103-1118.
[6] PENG Xi-Hong, CHEN Ping, DU Qing, YANG Xue-Li, REN Jun-Bo, ZHENG Ben-Chuan, LUO Kai, XIE Chen, LEI Lu, YONG Tai-Wen, YANG Wen-Yu. Effects of reduced nitrogen application on soil aeration and root nodule growth of relay strip intercropping soybean [J]. Acta Agronomica Sinica, 2022, 48(5): 1199-1209.
[7] WANG Hao-Rang, ZHANG Yong, YU Chun-Miao, DONG Quan-Zhong, LI Wei-Wei, HU Kai-Feng, ZHANG Ming-Ming, XUE Hong, YANG Meng-Ping, SONG Ji-Ling, WANG Lei, YANG Xing-Yong, QIU Li-Juan. Fine mapping of yellow-green leaf gene (ygl2) in soybean (Glycine max L.) [J]. Acta Agronomica Sinica, 2022, 48(4): 791-800.
[8] JIN Min-Shan, QU Rui-Fang, LI Hong-Ying, HAN Yan-Qing, MA Fang-Fang, HAN Yuan-Huai, XING Guo-Fang. Identification of sugar transporter gene family SiSTPs in foxtail millet and its participation in stress response [J]. Acta Agronomica Sinica, 2022, 48(4): 825-839.
[9] LI Rui-Dong, YIN Yang-Yang, SONG Wen-Wen, WU Ting-Ting, SUN Shi, HAN Tian-Fu, XU Cai-Long, WU Cun-Xiang, HU Shui-Xiu. Effects of close planting densities on assimilate accumulation and yield of soybean with different plant branching types [J]. Acta Agronomica Sinica, 2022, 48(4): 942-951.
[10] DU Hao, CHENG Yu-Han, LI Tai, HOU Zhi-Hong, LI Yong-Li, NAN Hai-Yang, DONG Li-Dong, LIU Bao-Hui, CHENG Qun. Improving seed number per pod of soybean by molecular breeding based on Ln locus [J]. Acta Agronomica Sinica, 2022, 48(3): 565-571.
[11] ZHOU Yue, ZHAO Zhi-Hua, ZHANG Hong-Ning, KONG You-Bin. Cloning and functional analysis of the promoter of purple acid phosphatase gene GmPAP14 in soybean [J]. Acta Agronomica Sinica, 2022, 48(3): 590-596.
[12] WANG Juan, ZHANG Yan-Wei, JIAO Zhu-Jin, LIU Pan-Pan, CHANG Wei. Identification of QTLs and candidate genes for 100-seed weight trait using PyBSASeq algorithm in soybean [J]. Acta Agronomica Sinica, 2022, 48(3): 635-643.
[13] DONG Yan-Kun, HUANG Ding-Quan, GAO Zhen, CHEN Xu. Identification, expression profile of soybean PIN-Like (PILS) gene family and its function in symbiotic nitrogen fixation in root nodules [J]. Acta Agronomica Sinica, 2022, 48(2): 353-366.
[14] ZHANG Guo-Wei, LI Kai, LI Si-Jia, WANG Xiao-Jing, YANG Chang-Qin, LIU Rui-Xian. Effects of sink-limiting treatments on leaf carbon metabolism in soybean [J]. Acta Agronomica Sinica, 2022, 48(2): 529-537.
[15] YU Tao-Bing, SHI Qi-Han, NIAN-Hai , LIAN Teng-Xiang. Effects of waterlogging on rhizosphere microorganisms communities of different soybean varieties [J]. Acta Agronomica Sinica, 2021, 47(9): 1690-1702.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Add: No. 80 Xueyuan South Rd, Beijing 100081, P. R. China E-mail: zwxb301@caas.cn
Supported by Beijing Magtech Co.Ltd