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

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

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)

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Abstract:

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"

引物名称
Primer name		 上游引物
Forward primer (5'-3')		 下游引物
Reverse primer (5'-3')
GmR47-1 TTTCAGAATCTATTGATATTAGGG TCTGTGAAACTATGCCTTGC
GmR51-1 GGATTGGAGTGGGAGGA GGGTTGAAGCAACGAAA
GmR47-2 TGGGATTGGAGTGGGAGGAGT CCATCAAAGTTGAATCCCAATTCAC
GmR51-2 GGGAGGAGTTGAATTAGCG CCAGTCTCCACCACTTTGTT
GmR47/51-qPCR GTAGGGGTCTTTGTTGATGTTGATT TCTGAGAAACTCCTATGTCCGCTA
Tubulin-qPCR AGTATGAGGACGAGGAGGACGAT TACGCATCACATAGCATAAGTAAGACAC

Table 2

Primer used in alternative splicing detection and qPCR"

引物名称
Primer name		 引物
Primer (5'-3')
Intron1-Uni F: TTCGGAGGCGATTGAACAT
R: TGCTCAATTTGATCAATTAATTAAACTCA
Intron2-Uni F: AAATTGTTCGACAGGAATCACC
R: TGGCAGAAACTCGACGTAATG
Intron3-Uni F: CTCTTGAATCCACCAAAGCAGG
R: AGTTCATATGATTAGAAGAACATGTAGAG
Intron1-Spe F: ATCTACTAGTCATATGGATTTGCTCAATTTGATCAATTAATTAAACTCA
R: CGGTACCCGGGGATCCGATTTTCGGAGGCGATTGAACAT
Intron2-Spe F: CGGTACCCGGGGATCCGATTAAATTGTTCGACAGGAATCACC
R: ATCTACTAGTCATATGGATTTGGCAGAAACTCGACGTAATG
Intron3-Spe F: CGGTACCCGGGGATCCGATTCTCTTGAATCCACCAAAGCAGG
R: ATCTACTAGTCATATGGATTAGTTCATATGATTAGAAGAACATGTAGAG
Intron1-qPCR F: GAAATGGAGGAACGCACTGC
R: AAGATCGACAAGAAGATATTACCTGAA
Intron2-qPCR F: GGCAAACGCAGTAGGTTATGG
R: ATACCATCAATAGAATGGCAGAAACT
Intron3-qPCR F: ATGTAGGAGAATGCTACTGAAACAGG
R: AGAATATCTCTTATTTCCAGCCTCAT

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"

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