Welcome to Acta Agronomica Sinica,

Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (12): 1822-1831.doi: 10.3724/SP.J.1006.2019.94054


Cloning and analysis of candidate gene resistant to SC3 in soybean

Wen-Yang XIANG,Yong-Qing YANG,Qiu-Yan REN,Tong-Tong JIN,Li-Qun WANG,Da-Gang WANG,Hai-Jian ZHI()   

  1. Institute of Soybean, Nanjing Agricultural University / Key Laboratory of Soybean Biology and Genetic Breeding, Ministry of Agriculture / National Center for Soybean Improvement / State Key Laboratory of Crop Genetics & Germplasm Innovation, Nanjing 210095, Jiangsu, China
  • Received:2019-04-02 Accepted:2019-06-22 Online:2019-12-12 Published:2019-07-15
  • Contact: Hai-Jian ZHI E-mail:zhj@njau.edu.cn
  • Supported by:
    This study was supported by the Fund of Transgenic Breeding for Soybean Resistance to Soybean mosaic virus(2016ZX08004-004);the National Natural Science Foundation of China(31571690);the National Natural Science Foundation of China(31571687);the Fundamental Research Funds for the Central Universities(KYT201801);the Program for Changjiang Scholars and Innovative Research Team in University(PCSIRT_17R55);the National Soybean Industrial Technology System of China(CARS-004);Jiangsu Collaborative Innovation Center for Modern Crop Production(JCIC-MCP);the National Key R&D Program of China(2017YFD0101501)


Soybean mosaic virus (SMV) is one of the prevalent pathogens of soybean, causing great reduction of soybean yield worldwide. Soybean disease resistance-breeding is currently the most cost-effective measure to control SMV, and identification of resistance genes is the basis of disease resistance breeding. According to the previous mapping result of resistance gene to SMV strain SC3, two genes (GmR47, GmR51) with TIR-NBS-LRR domain were cloned. Bioinformatics analysis showed that both GmR47 and GmR51 genes have SNP mutations in the susceptible varieties and resistant varieties, and the mutation sites are located in the conserved domain. These two proteins encoded by GmR47 and GmR51 genes are predicted to be Tobacco mosaic virus (TMV) resistant N proteins. The results of homologous alignment between species indicated that GmR47 and GmR51 genes were close to those of wild soybean. The expression of GmR47 and GmR51 was analyzed after inoculation with soybean mosaic virus in soybean, demonstrating that GmR47 and GmR51 could increase the expression level in response to SMV infection, with the higher level in resistant varieties than in susceptible varieties. Analysis of the alternative splicing of GmR47 and GmR51 revealed that the two genes have different splice variants IN1, IN2, and IN3. The response analysis of splices to SMV showed that all splices were able to increase the expression in response to virus induction, with the higher level in resistant varieties than in susceptible varieties. It indicated that these alternative splicing may be involved in the disease resistance process of soybean to SMV. The result of this study lay a foundation for the study of subsequent gene function.

Key words: Soybean mosaic virus (SMV), resistance gene, inducing expression, alternative splicing

Table 1

Primers in gene cloning and qPCR"

Primer name
Forward primer (5'-3')
Reverse primer (5'-3')

Table 2

Primer used in alternative splicing detection and qPCR"

Primer name
Primer (5'-3')

Fig. 1

Conserved domain prediction of protein encoded by GmR47 and GmR51 A: conservative domain prediction of GmR47-encoded proteins. B: conservative domain prediction of GmR51-encoded proteins."

Fig. 2

Alignment result of the Amino acid sequence encoded by GmR47 and GmR51 in resistant (PI96983) and susceptible (Nannong 1138-2) cultivar A: alignment result of amino acid sequence encoded by GmR47; B: alignment result of amino acid sequence encoded by GmR51."

Fig. 3

Phylogenetic trees of GmR47 and GmR51 in different species"

Fig. 4

Prediction of secondary structure of amino acid sequence encoded by GmR47 (A) and GmR51 (B)"

Fig. 5

Predicted three-dimensional structure of protein encoded by gene GmR47 and GmR 51 A: PI96983 GmR47; B: PI96983 GmR51; C: Nannong 1138-2 GmR47; D: Nannong 1138-2 GmR51."

Fig. 6

Total expression of GmR47 and GmR51 genes at different time in PI96983 and Nannong 1138-2 after SMV inoculation"

Fig. 7

Gel electrophoresis detection results of alternative splicing The upper bands are the result of amplification of theconventional primer, the lower bands are the amplification result of the intron-specific primer, adjacent four bands are amplification from four varieties. A: Qihuang 1; B: PI96983; C: Nannong 1138-2; D: Williams 82; M: marker."

Fig. 8

Alternative splicing sequencing results A: schematic diagram of alternative splicing of GmR47 gene; B: IN1 sequence; C: IN2 sequence; D: IN3 sequence."

Fig. 9

Total expression of GmR47 and GmR51 genes at different time in PI96983 and Nannong 1138-2 after SMV inoculation A: total expression of alternative splicing in Intron 1; B: total expression of alternative splicing in Intron 2; C: total expression of alternative splicing in Intron 3."

Fig. 10

Subcellular localization results of candidate genes and GFP fusion proteins in tobacco cells"

[1] Hill J H, Whitham S A . Control of virus diseases in soybeans. Adv Virus Res, 2014,90:355-390.
[2] Kendrick J B, Gardner M W . Soybean mosaic: seed transmission and effect on yield. J Agric Res, 1924,27:91-98.
[3] Heinze K, Köhler E . The mosaic disease of the soybean and its transmission by insects. Phytopathol Z, 1940,13:207-242.
[4] Yu Y G, Maroof M A S, Buss G R . Divergence and allelomorphic relationship of a soybean virus resistance gene based on tightly linked DNA microsatellite and RFLP markers. Theor Appl Genet, 1996,92:64-69.
doi: 10.1007/BF00222952
[5] Hayes A J, Ma G, Buss G R, Maroof S . Molecular marker mapping of RSV4, a gene conferring resistance to all known strains of Soybean mosaic virus. Crop Sci, 2000,40:1434-1437.
[6] Jeong S C, Hayes A J, Biyashev R M, Maroof S . Diversity and evolution of a non-TIR-NBS sequence family that clusters to a chromosomal “hotspot” for disease resistance genes in soybean. Theor Appl Genet, 2001,103:406-414.
doi: 10.1007/s001220100567
[7] Klepadlo M, Chen P, Shi A, Mason R E, Korth K L, Srivastava V . Single nucleotide polymorphism markers for rapid detection of the Rsv4 locus for Soybean mosaic virus resistance in diverse germplasm. Mol Breed, 2017,37:10.
doi: 10.1007/s11032-016-0595-3
[8] Bent A F, Kunkel B N, Dahlbeck D, Brown K L, Schmidt R L, Giraudat J, Leung J L, Staskawicz B J . RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes. Science, 1994,265:1856-1860.
doi: 10.1126/science.8091210
[9] Grant M R, Godiard L, Straube E, Ashfield T, Lewald J, Sattler A, Innes R W, Dangl J L . Structure of the Arabidopsis RPM1 gene enabling dual specificity disease resistance. Science, 1995,269:843-846.
doi: 10.1126/science.7638602
[10] Parker J E, Coleman M J, Szab V, Frost L N, Schmidt R, Biezen E A V D, Moores T, Dean C, Daniels M J, Jones J D . The arabidopsis downy mildew resistance gene RPP5 shares similarity to the toll and interleukin-1 receptors with N and L6. Plant Cell, 1997,9:879-894.
doi: 10.1105/tpc.9.6.879
[11] Yoshimura S, Yamanouchi U, Katayose Y, Toki S, Wang Z X, Kono I, Kurata N, Yano M, Iwata N, Sasaki T . Expression of Xa1, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation. Proc Natl Acad Sci USA, 1998,95:1663-1668.
doi: 10.1073/pnas.95.4.1663
[12] Davis C L . Identification, Validation, Mapping of Phytophthora sojae and Soybean mosaic virus Resistance Genes in Soybean. PhD Dissertation of Virginia Tech, Blacksburg, USA, 2017.
[13] Tran P T, Widyasari K, Seo J K, Kim K H . Isolation and validation of a candidate Rsv3 gene from a soybean genotype that confers strain-specific resistance to Soybean mosaic virus. Virology, 2018,513:153-159.
doi: 10.1016/j.virol.2017.10.014
[14] 郭小勤, 李德葆 . 植物前体mRNA的选择性剪接. 农业生物技术学报, 2006,14:809-815.
Guo X Q, Li D B . Pre-mRNA alternative splicing in plants. Chin J Agric Biotechol, 2006,14:809-815 (in Chinese with English abstract).
[15] Brack C, Hirama M, Lenhardschuller R, Tonegawa S . A complete immunoglobulin gene is created by somatic recombination. Cell, 1978,15:1-14.
doi: 10.1016/0092-8674(78)90078-8
[16] Ali G S, Reddy A S N . Regulation of Alternative Splicing of Pre-mRNAs by Stresses. Heidelberg: Springer, 2008. pp 257-275.
[17] Gassmann W . Alternative splicing in plant defense. Curr Top Microbiol, 2008,326:219-233.
[18] Jang Y H, Lee J H, Park H Y, Kim S K, Lee B Y, Suh M C, Kim J K . OsFCA transcripts show more complex alternative processing patterns than its Arabidopsis counterparts. J Plant Biol, 2009,52:161-166.
doi: 10.1007/s12374-009-9018-x
[19] Modrek B, Lee C . A genomic view of alternative splicing. Nat Genet, 2001,30:13-19.
doi: 10.1038/ng0102-13
[20] Lal S, Choi J H, Shaw J R, Hannah L C . A splice site mutant of maize activates cryptic splice sites, elicits intron inclusion and exon exclusion, and permits branch point elucidation. Plant Physiol, 1999,121:411-418.
doi: 10.1104/pp.121.2.411
[21] Yang Y, Zheng G, Han L, Wang D G, Yang X F, Yuan Y, Huang S H, Zhi H J . Genetic analysis and mapping of genes for resistance to multiple strains of Soybean mosaic virus in a single resistant soybean accession PI96983. Theor Appl Genet, 2013,126:1783-1791.
doi: 10.1007/s00122-013-2092-y
[22] 刘玉芝, 廖林, 孙大敏 . 对大豆花叶病毒(SMV)病抗源的筛选. 吉林农业科学, 1997,1:30-34.
Liu Y Z, Liao L, Sun D M . Screening for resistant sources of soybean germplasm to SMV. J Jilin Agric Sci. 1997,1:30-34 (in Chinese with English abstract).
[23] 纪冬, 辛绍杰 . 实时荧光定量PCR的发展和数据分析. 生物技术通讯, 2009,20:598-600.
Ji D, Xin S J . Development and data analysis of real-time fluorescent quantitative PCR. Lett Biotech, 2009,20:598-600 (in Chinese with English abstract).
[24] 李晓君, 王绍梅, 谢艳兰, 和敏 . 农杆菌渗透法转化烟草条件的优化. 江苏农业科学, 2014,42(9):45-47.
Li X J, Wang S M, Xie Y L, He M . Optimization of agrobacterium-infiltration method for transformation of tobacco. Jiangsu Agric Sci, 2014,42(9):45-47 (in Chinese).
[25] Hayes A J, Jeong S C, Gore M A, Yu Y G, Buss G R, Tolin S, Maroof S . Recombination within a Nucleotide-Binding-Site/ Leucine-Rich-Repeat gene vluster produces new variants conditioning resistance to Soybean mosaic virus in soybeans. Genetics, 2004,166:493-503.
doi: 10.1534/genetics.166.1.493
[26] 黄赛花, 郑桂杰, 杨永庆, 智海剑 . 利用VIGS技术对抗SMV候选基因GmZ15的功能分析. 大豆科学, 2015,34:582-587.
Huang S H, Zheng G J, Yang Y Q, Zhi H J . Analysis on the candidate resistance gene GmZ15 to soybean mosaic virus by VIGS. Soybean Sci, 2015,34:582-587 (in Chinese with English abstract).
[27] Cesari S, Thilliez G, Ribot C, Chalvon V, Michel C, Jauneau A, Rivas S, Alaux L, Kanzaki H, Okuyama Y, Morel J B, Fournier E, Tharreau D, Terauchi R, Kroj T . The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding. Plant Cell, 2013,25:1463-1481.
doi: 10.1105/tpc.112.107201
[1] DENG Zhao, JIANG Nan, FU Chen-Jian, YAN Tian-Zhe, FU Xing-Xue, HU Xiao-Chun, QIN Peng, LIU Shan-Shan, WANG Kai, YANG Yuan-Zhu. Analysis of blast resistance genes in Longliangyou and Jingliangyou hybrid rice varieties [J]. Acta Agronomica Sinica, 2022, 48(5): 1071-1080.
[2] ZHU Zheng, WANG Tian-Xing-Zi, CHEN Yue, LIU Yu-Qing, YAN Gao-Wei, XU Shan, MA Jin-Jiao, DOU Shi-Juan, LI Li-Yun, LIU Guo-Zhen. Rice transcription factor WRKY68 plays a positive role in Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae [J]. Acta Agronomica Sinica, 2022, 48(5): 1129-1140.
[3] WEI Yi-Hao, YU Mei-Qin, ZHANG Xiao-Jiao, WANG Lu-Lu, ZHANG Zhi-Yong, MA Xin-Ming, LI Hui-Qing, WANG Xiao-Chun. Alternative splicing analysis of wheat glutamine synthase genes [J]. Acta Agronomica Sinica, 2022, 48(1): 40-47.
[4] JIANG Wei, PAN Zhe-Chao, BAO Li-Xian, ZHOU Fu-Xian, LI Yan-Shan, SUI Qi-Jun, LI Xian-Ping. Genome-wide association analysis for late blight resistance of potato resources [J]. Acta Agronomica Sinica, 2021, 47(2): 245-261.
[5] ZHANG Rong-Yue, WANG Xiao-Yan, YANG Kun, SHAN Hong-Li, CANG Xiao-Yan, LI Jie, WANG Chang-Mi, YIN Jiong, LUO Zhi-Ming, LI Wen-Feng, HUANG Ying-Kun. Identification of brown rust resistance and molecular detection of Bru1 gene in new and main cultivated sugarcane varieties [J]. Acta Agronomica Sinica, 2021, 47(2): 376-382.
[6] ZHANG Huan, LUO Huai-Yong, LI Wei-Tao, GUO Jian-Bin, CHEN Wei-Gang, ZHOU Xiao-Jing, HUANG Li, LIU Nian, YAN Li-Ying, LEI Yong, LIAO Bo-Shou, JIANG Hui-Fang. Genome-wide identification of peanut resistance genes and their response to Ralstonia solanacearum infection [J]. Acta Agronomica Sinica, 2021, 47(12): 2314-2323.
[7] SONG Ni-Xi, LI Xia, WANG Jin, WU Bo-Han, CAO Yue, YANG Jie, XIE Yin-Feng. Effects on drought tolerance by pladienolide B and rice with high expression of C4-PEPC [J]. Acta Agronomica Sinica, 2021, 47(10): 1927-1940.
[8] WEN Jing, SHEN Yan-Qi, HAN Si-Ping, XING Yue-Xian, ZHANG Ye, WANG Zi-Yu, LI Shi-Jie, YANG Xiao-Hong, HAO Dong-Yun, ZHANG Yan. Exploration of specific gene(s) for ear rot resistance to Fusarium verticilloides in maize [J]. Acta Agronomica Sinica, 2020, 46(9): 1303-1311.
[9] FENG Tao,TAN Hui,GUAN Mei,GUAN Chun-Yun. Mechanism of BnaBZR1 and BnaPIF4 regulating photosynthetic efficiency in oilseed rape (Brassica napus L.) under poor light [J]. Acta Agronomica Sinica, 2020, 46(8): 1146-1156.
[10] ZHANG Xue-Cui,ZHONG Chao,DUAN Can-Xing,SUN Su-Li,ZHU Zhen-Dong. Fine mapping of Phytophthora resistance gene RpsZheng in soybean cultivar Zheng 97196 [J]. Acta Agronomica Sinica, 2020, 46(7): 997-1005.
[11] XIAO Yan, YAO Jun-Yue, LIU Dong, SONG Hai-Xing, ZHANG Zhen-Hua. Expression profile analysis of low nitrogen stress in Brassica napus [J]. Acta Agronomica Sinica, 2020, 46(10): 1526-1538.
[12] Hui-Min WANG,Xin-Guo LI,Shu-Bo WAN,Zhi-Meng ZHANG,Hong DING,Guo-Wei LI,Wen-Wei GAO,Zhen-Ying PENG. Structure and expression analysis of the members of peanut annexin gene family [J]. Acta Agronomica Sinica, 2019, 45(3): 390-400.
[13] ZHANG An-Ning,LIU Yi,WANG Fei-Ming,XIE Yue-Wen,KONG De-Yan,NIE Yuan-Yuan,ZHANG Fen-Yun,BI Jun-Guo,YU Xin-Qiao,LIU Guo-Lan,LUO Li-Jun. Pyramiding and evaluation of brown planthopper resistance genes in water-saving and drought-resistance restorer line [J]. Acta Agronomica Sinica, 2019, 45(11): 1764-1769.
[14] Xiang-Yi XIAO,Xue-Tao SHI,Hao-Wen SHENG,Jin-Ling LIU,Ying-Hui XIAO. Fine Mapping and Candidate Gene Analysis of Rice Blast Resistance Gene Pi47 [J]. Acta Agronomica Sinica, 2018, 44(7): 977-987.
[15] WU Qiu-Hong,CHEN Yong-Xing,LI Dan,WANG Zhen-Zhong,ZHANG Yan,YUAN Cheng-Guo,WANG Xi-Cheng,ZHAO Hong,CAO Ting-Jie,LIU Zhi-Yong. Large Scale Detection of Powdery Mildew Resistance Genes in Wheat via SNP and Bulked Segregate Analysis [J]. Acta Agron Sin, 2018, 44(01): 1-14.
Full text



No Suggested Reading articles found!