Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (18): 3445-3454.doi: 10.3864/j.issn.0578-1752.2018.18.002

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

Cloning and Functional Analysis of the GmWRKY148 in Soybean

ShaSha WANG(), XiaoXia CUI, YanZhong HUANG, HuiDong XUAN, Na GUO, Han XING()   

  1. College of Agriculture, Nanjing Agricultural University/National Center for Soybean Improvement/Key Laboratory for Biology and Genetic Improvement of Soybean (General), Ministry of Agriculture/National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing 210095
  • Received:2018-04-11 Accepted:2018-06-19 Online:2018-09-16 Published:2018-09-16

RichHTML

18

PDF

278

Abstract:

【Objective】WRKY transcription factor is closely related to the biological and abiotic stress responses of plants. In order to study the function of GmWRKY148, GmWRKY148 was cloned and its expression was analyzed, then the gene was over-expressed in soybean hairy roots for analyzing resistance to Phytophthora root rot.【Method】Based on the sequence of AtWRKY44, the homologous gene Glyma.14G199800 was obtained from the root tissue of soybean Williams 82 by method of homologous cloning, named GmWRKY148. Phylogenetic analysis was performed. The transcriptional level of GmWRKY148 under four tissues and P. sojae infection was analyzed by qRT-PCR. For subcellular localization analysis, the recombinant plasmid pBinGFP2-GmWRKY148 was constructed, and transformed into onion epidermal cells by gene gun method. Transgenic soybean hairy roots were obtained by using A. rhizogenes-mediated method. The transgenic over-expressing hairy roots and empty-vector hairy roots were obtained by GFP fluorescence screening and qRT-PCR. To confirm whether GmWRKY148 is involved in the interaction between soybean and P. sojae, the positive soybean hairy roots were inoculated by P6497, and measured the lesions length, accumulation of P. sojae and the germination of oospores.【Result】The CDS of GmWRKY148 is 999 bp, encoding 332 amino acids, and the isoelectric point is 7.61. Phylogenetic analysis indicated that the WRKYs from Phaseolus vulgaris and Medicago truncatula were highly similar to GmWRKY148. Subcellular localization results showed that GmWRKY148 was located in the nucleus. Tissues expression analysis showed that GmWRKY148 had the highest expression in roots, followed in stems and leaves, and lowest in cotyledons. qRT-PCR analysis showed that the expression level of GmWRKY148 was up-regulated upon infection by P. sojae both in resistant variety Williams 82 (containing the Rps1k) and susceptible variety Williams, which has the highest expression at 24 h post infection. The transcript of GmWRKY148 is higher in Williams 82 than that in Williams. Transgenic over-expressing hairy roots (OE-GmWRKY148) and empty-vector hairy roots (EV) were inoculated with P. sojae P6497 mycelium blocks, the lesions length and the accumulation of P. sojae were compared after 24 h inoculation. The lesion length in OE-GmWRKY148 soybean hairy roots was significantly shorter than that in the EV, and the accumulation of P. sojae was significantly reduced. The rate of mycelial infection and zoospores germination was observed by microscopy at 24, 36 and 48 h after inoculated with P. sojae zoospores. Compared to the control EV, the rate of mycelia infection and zoospores germination of over-expressing soybean hairy roots was significantly decreased.【Conclusion】GmWRKY148 is a positive regulator in the interaction between soybean and P. sojae.

Key words: Glycine max, GmWRKY148, Phytophthora root rot, resistance

Fig. S1

Formation of mycelium of P6497a: Choose fresh mycelium edges, then cut them into small pieces, to carry on the propagation. b-f: Growth situation of P6497, after 2 to 6 days"

Fig. S2

Formation of zoospores of P6497 a: After 4 days of propagation, the growth situation of P6497. b: Choose fresh mycelium edges, cut them into tiny pieces, then put them into liquid V8 medium. c: Thoroughly clean the debris with sterile water. d: Growth situation of zoospores of P6497, 10h later. e: Strain the liquid through a piece of gauze, in order to get transparent zoospores fluid. f: Put positive soybean hairy roots in to zoospores fluid"

Fig. S3

GFP fluorescence in enlarged positive transgenic soybean hairy roots a, b, c: GFP Williams hairy roots. d, e, f : OE-GmWRKY148 Williams hairy roots"

Table S1

Primers used in this study"

基因Gene 引物序列
Sequence of primer (5'-3')		 引物用途
Function of primer
GmWRKY148 F：GCAGCTTCTAACTCTGGCGA GmWRKY148克隆
Cloning of GmWRKY148
R：TTTATTATAAGTGATAAGATCTACAAGCAA
GmWRKY148-MQ F：CGGGGTACCATGCCTTTCCGAAATTCCAATATGA
R：CGCGGATCCTCATATTTCATTTGGATATTGAGGA
GmWRKY148-2 F：ATGCACACAACCCACTTGCT GmWRKY148实时荧光定量PCR
qRT-PCR of GmWRKY148
R：CAGATGAAGAGTTTCTCTTC
GmEF1β F：GTTGAAAAGCCAGGGGACA 疫霉积累量实时荧光定量内参
Reference gene in qRT-PCR
R：TCTTACCCCTTGAGCGTGG
GmActin F：GGTGGTTCTATCTTGGCATC 实时荧光定量PCR内参
Reference gene in qRT-PCR
R：CTTTCGCTTCAATAACCCTA
pBinGFP2 F：AAGACCCCAACGAGAAGC pBinGFP2载体引物
Primer of vector
R：GAACCCTAATTCCCTTATCTG

Fig. 1

Phylogenetic tree analysis of GmWRKY148"

Fig. 2

Tissue-specific expression of GmWRKY148 Asterisks indicate statistically significant differences at P < 0.01. The same as below"

Fig. 3

Expression pattern of GmWRKY148 response to P. sojae in Williams and Williams82 A: Expression pattern of GmWRKY148 response to P. sojae in Williams. B: Expression pattern of GmWRKY148 response to P. sojae in Williams 82"

Fig. 4

Subcellular localization analysis of GmWRKY148 protein in onion epidermal cells a, b, c, d: 35S-GFP alone location; e, f, g, h: 35S-WRKY148::GFP location"

Fig. 5

GFP fluorescence in transgenic hairy roots a: GFP Williams hairy roots. b: OE-GmWRKY148 Williams hairy roots"

Fig. 6

Analysis of over expression efficiency in Williams transgenic soybean hairy roots"

Fig. 7

GmWRKY148 could improve the resistance to P. sojae in soybean Williams A：Disease phenotypes of GFP Williams hairy roots after inoculated with P6497 at 24 hpi. B：Disease phenotypes of OE-GmWRKY148 Williams hairy roots after inoculated with P6497 at 24 hpi. C: P6497 infected hairy roots 24h later, the lesion length of GFP Williams hairy roots and OE-GmWRKY148 Williams hairy roots. D: P. sojae biomass was determined by qPCR in inoculated Williams hairy roots OE-GmWRKY148 or EV at 24 and 36 hpi"

Fig. 8

Microscopic analysis of P. sojae infection in soybean Williams hairy roots"

Table S2

Table S2 Summary of the numbers of roots that were successfully infected by P. sojae or oospores development"

24 hpi 36 hpi 48 hpi
GFP OE-GmWRKY148 GFP OE-GmWRKY148 GFP OE-GmWRKY148
被成功侵染的大豆发状根数
Number of roots infected		 7/20 3/20 13/20 5/20 16/20 9/20
萌发卵孢子的大豆发状根数
Number of roots developed oospores		 3/20 0/20 6/20 2/20 12/20 4/20
[1] 张宝强. 大豆对大豆疫霉根腐病的抗源筛选及抗性遗传分析[D]. 南京: 南京农业大学, 2010.
ZHANG B B Q.Studies on screening for resistance source and genetic analysis to P. sojae in soybean [D]. Nanjing: Nanjing Agricultural University, 2010. (Chinese)
[2] LI D, LIU P, YU J, DOSSA K, ZHANG Y, ZHOU R, WEI X, ZHANG X.Genome-wide analysis of WRKY gene family in the sesame genome and identification of the WRKY genes involved in responses to abiotic stresses.BMC Plant Biology, 2017, 17(1): 152.
doi: 10.1186/s12870-017-1099-y pmid: 5594535
[3] RUSHTON P J, TORRES J T, PARNISKE M, WERNERT P, HAHIBROCK K, SOMSSICH I E.Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes.The EMBO Journal, 1996, 15(20): 5690-5700.
doi: 10.1002/j.1460-2075.1996.tb00953.x pmid: 8896462
[4] ROCHER A, DUMAS C, COCK J M.A W-box is required for full expression of the SA-responsive gene SFR2.Gene, 2005, 344(1): 181-192.
doi: 10.1016/j.gene.2004.09.016 pmid: 15656984
[5] KIM C Y, ZHANG S.Activation of a mitogen-activated protein kinase cascade induces WRKY family of transcription factors and defense genes in tobacco.The Plant Journal, 2004, 38(1): 142-151.
doi: 10.1111/j.1365-313X.2004.02033.x pmid: 202020202020042020203820142151
[6] PANDEY S P, SOMSSICH I E.The role of WRKY transcription factors in plant immunity.Plant Physiology, 2009, 150(4): 1648-1655.
doi: 10.1104/pp.109.138990 pmid: 19420325
[7] BANERJEE A, ROYCHOUDHURY A.WRKY proteins: Signaling and regulation of expression during abiotic stress responses.The Scientific World Journal, 2015, 2015(807560): 807560.
doi: 10.1155/2015/807560 pmid: 4387944
[8] HAN Y, ZHANG X, WANG Y, MING F.Correction: The suppression of WRKY44 by GIGANTEA- miR172 pathway is involved in drought response ofArabidopsis thaliana. PLOS ONE, 2015, 10(4): e0124854.
doi: 10.1371/journal.pone.0124854 pmid: 25844872
[9] ZELICOURT A D, COLCOMBET J, HIRT H.The role of MAPK modules and ABA during abiotic stress signaling.Trends in Plant Science, 2016, 21(8): 677-685.
doi: 10.1016/j.tplants.2016.04.004 pmid: 27143288
[10] ASAI T, TENA G, PLOTNIKOVA J, WILLMANN M R, CHIU W L, LOURDES G G, BOLLER T, AUSUBEL F M, SHEEN J.MAP kinase signalling cascade inArabidopsis innate immunity. Nature, 2002, 415(6875): 977-983.
[11] LI Y, WILLIAMS B, DICKMAN M.Arabidopsis B-cell lymphoma 2 (Bcl-2)-associated athano gene 7(BAG7)-mediated heat tolerance requires translocation, sumoylation and binding to WRKY29. New Phytologist, 2016, 214(2): 695-705.
[12] DANQUAH A, DE A Z, COLCOMBET J, HIRT H.The role of ABA and MAPK signaling pathways in plant abiotic stress responses.Biotechnology Advances, 2014, 32(1): 40.
doi: 10.1016/j.biotechadv.2013.09.006
[13] JIANG Y, LIANG G, YANG S, YU D.Arabidopsis WRKY57 functions as a node of convergence for jasmonic-acid and auxin- mediated signaling in jasmonic acid-induced leaf senescence. The Plant Cell, 2014, 26(1): 230-245.
[14] ZHANG Y, YU H, YANG X, LI Q, LING J, WANG H, GU X, HUANG S, JIANG W.CsWRKY46, a WRKY transcription factor from cucumber, confers cold resistance in transgenic-plant by regulating a set of cold-stress responsive genes in an ABA-dependent manner.Plant Physiology & Biochemistry, 2016, 108: 478.
doi: 10.1016/j.plaphy.2016.08.013 pmid: 27592172
[15] JOSHI R, WANI S H, SINGH B, BOHRA A, DAR Z A, LONE A A, PAREE K A, SINGLA-PAREEK S L. Transcription factors and plants response to drought stress: Current understanding and future directions.Frontiers in Plant Science, 2016, 7(e102529): 1029.
doi: 10.3389/fpls.2016.01029 pmid: 27471513
[16] OHAMA N, SATO H, SHINOZAKI K, YAMAGUCHI-SHINOZAKI K.Transcriptional regulatory network of plant heat stress response.Trends in Plant Science, 2017, 22(1): 53-65.
doi: 10.1016/j.tplants.2016.08.015 pmid: 27666516
[17] GU L, LI L, WEI H, WANG H, SU J, GUO Y, YU S.Identification of the group IIa WRKY subfamily and the functional analysis of GhWRKY17 in upland cotton (Gossypium hirsutum L.). PLOS ONE, 2018, 13(1): e0191681.
doi: 10.1371/journal.pone.0191681 pmid: 5784973
[18] WU X, SHIROTO Y, KISHITANI S, ITO Y, TORIYAMA K.Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter.Plant Cell Reports, 2009, 28(1): 21-30.
doi: 10.1007/s00299-008-0614-x pmid: 2018818929
[19] HE G H, XU J Y, WANG Y X, LIU J M, LI P S, CHEN M, MA Y Z, XU Z S.Drought-responsive WRKY transcription factor genes TaWRKY1, and TaWRKY33, from wheat confer drought and/or heat resistance inArabidopsis. BMC Plant Biology, 2016, 16(1): 116.
doi: 10.1186/s12870-016-0806-4 pmid: 27215938
[20] JIA H, WANG C, WANG F, LIU S, LI G, GUO X.GhWRKY68 reduces resistance to salt and drought in transgenic Nicotiana benthamiana. PLOS ONE, 2015, 10(3): e0120646.
[21] CHANG C, YU D, JIAO J, JING S, SCHULZE-LEFERT P, SHEN Q H.Barley MLA immune receptors directly interfere with antagonistically acting transcription factors to initiate disease resistance signaling.The Plant Cell, 2013, 25(3): 1158-1173.
doi: 10.1105/tpc.113.109942 pmid: 23532068
[22] DESLANDES L, OLIVIER J, THEULIERES F, HIRSCH J, FENG D X, BITTNER-EDDY P, BEYON J, MARCO Y.Resistance toRalstonia solanacearum in Arabidopsis thaliana is conferred by the recessive RRS1-R gene, a member of a novel family of resistance genes. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99(4): 2404-2409.
[23] 成洪涛, 王石平. 水稻-病原互作中的重要角色: WRKY类转录因子. 中国科学: 生命科学, 2014(8): 784-793.
CHENG H T, WANG S P.WRKY- type transcription factors: A significant factor in rice- pathogen interactions.Scientia Sinica, 2014, 44(8): 784-793.(in Chinese)
[24] WANG J, TAO F, AN F, ZOU Y P, TIAN W, CHEN X M, XU X M, HU X P.Wheat transcription factor TaWRKY70 is positively involved in high-temperature seedling-plant resistance toPuccinia striiformis f. sp. tritici. Molecular Plant Pathology, 2017, 18(5): 649-661.
[25] SUGIMOTO T, WATANABE K, YOSHIDA S, BIGGS A R.Field application of calcium to reduce Phytophthora stem rot of soybean, and calcium distribution in plants.Plant Disease, 2010, 94(7): 812-819.
doi: 10.1094/PDIS-94-7-0812
[26] 杨晓贺, 张瑜, 顾鑫, 赵海红, 姚亮亮, 刘伟, 申宏波, 丁俊杰. 大豆疫霉根腐病的综合防治. 大豆科学, 2014, 33(4): 554-558.
YANG X H, ZHANG Y, GU X, ZHAO H H, YAO L L, LIU W, SHEN H B, DING J J.Integrated control of soybean phytophthora root rot.Soybean Science, 2014, 33(4): 554-558. (in Chinese)
[27] 陶恺. 大豆疫霉侵染过程的分子细胞学研究[D]. 南京: 南京农业大学, 2012.
TAO K.Molecular and cellular biology of interactions between Phytophthora sojae and host soybean[D]. Nanjing: Nanjing Agricultural University, 2012. (in Chinese)
[28] MOY P, QUTOB D, CHAPMAN B P, ATKINSON I, GIJZEN M.Patterns of gene expression upon infection of soybean plants byPhytophthora sojae. Molecular Plant Microbe Interactions, 2004, 17(10): 1051-1062.
[29] DUNIWAY J M.Water relations of water molds. Annual Review of Phytopathology, 1979, 17(17): 431-460.
doi: 10.1146/annurev.py.17.090179.002243
[30] NIKLAUS J G, WILBERT G F.The biology of phytophthora infestans at its center of origin.Annual Review of Phytopathology, 2005, 43(43): 171.
doi: 10.1146/annurev.phyto.43.040204.135906
[31] SINCLAIR J B.Compendium of Soybean Diseases. Urbana- Champaign:American Phytopathological Society and University of Illinois, 1982.
[32] HARDHAM A, GUBLER F.Polarity of attachment of zoospores of a root pathogen and pre- alignment of the emerging germ tube.Cell Biology International Reports, 1990, 14(11): 947-956.
doi: 10.1016/0309-1651(90)90107-A
[33] HARDHAM A R.The cell biology behind Phytophthora pathogenicity. Australasian Plant Pathology, 2001, 30(2): 91-98.
doi: 10.1071/AP01006
[34] 张鑫. 大豆疫霉G蛋白α亚基调控游动孢子趋化性的机制研究[D]. 南京: 南京农业大学, 2015.
ZHANG X.Molecular mechanisms of Phytophthora sojae PsGPA1 in regulation of zoospore chemotaxis to soybean[D]. Nanjing: Nanjing Agricultural University, 2015. (in Chinese)
[1] YAN Qiang,XUE Dong,HU YaQun,ZHOU YanYan,WEI YaWen,YUAN XingXing,CHEN Xin. Identification of the Root-Specific Soybean GmPR1-9 Promoter and Application in Phytophthora Root-Rot Resistance [J]. Scientia Agricultura Sinica, 2022, 55(20): 3885-3896.
[2] XiaoShuai HAO,MengMeng FU,ZaiDong LIU,JianBo HE,YanPing WANG,HaiXiang REN,DeLiang WANG,XingYong YANG,YanXi CHENG,WeiGuang DU,JunYi GAI. Genome-Wide QTL-Allele Dissection of 100-Seed Weight in the Northeast China Soybean Germplasm Population [J]. Scientia Agricultura Sinica, 2020, 53(9): 1717-1729.
[3] LiYuan PAN,JianBo HE,JinMing ZHAO,WuBin WANG,GuangNan XING,DeYue YU,XiaoYan ZHANG,ChunYan LI,ShouYi CHEN,JunYi GAI. Detection Power of RTM-GWAS Applied to 100-Seed Weight QTL Identification in a Recombinant Inbred Lines Population of Soybean [J]. Scientia Agricultura Sinica, 2020, 53(9): 1730-1742.
[4] ShuGuang LI,YongCe CAO,JianBo HE,WuBin WANG,GuangNan XING,JiaYin YANG,TuanJie ZHAO,JunYi GAI. Genetic Dissection of Protein Content in a Nested Association Mapping Population of Soybean [J]. Scientia Agricultura Sinica, 2020, 53(9): 1743-1755.
[5] ZaiDong LIU,Shan MENG,JianBo HE,GuangNan XING,WuBin WANG,TuanJie ZHAO,JunYi GAI. A Comparative Study on Linkage and Association QTL Mapping for Seed Isoflavone Contents in a Recombinant Inbred Line Population of Soybean [J]. Scientia Agricultura Sinica, 2020, 53(9): 1756-1772.
[6] HAO QingTing,ZHANG Fei,JI XiaJie,XUE JinAi,LI RunZhi. Phenotypic Analysis of Epoxygenase-Transgenic Soybeans [J]. Scientia Agricultura Sinica, 2019, 52(2): 191-200.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd