Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (14): 2685-2695.doi: 10.3864/j.issn.0578-1752.2022.14.001

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

Cloning of the Soybean Single Zinc Finger Protein Gene GmSZFP and Its Functional Analysis in SMV-Host Interactions

ZHAO DingLing1(),WANG MengXuan1,SUN TianJie1,SU WeiHua1,ZHAO ZhiHua1,XIAO FuMing2,ZHAO QingSong3,YAN Long3,ZHANG Jie1(),WANG DongMei1()   

  1. 1. School of Life Science, Hebei Agricultural University/National Key Laboratory of Crop Improvement and Regulation in North China/Hebei Provincial Key Laboratory of Plant Physiology and Molecular Pathology, Baoding 071001, Hebei
    2. Handan Municipal Academy of Agricultural Sciences, Hebei Province, Handan 056001, Hebei
    3. Hebei Academy of Agriculture and Forestry Institute of Cereals and Oils Crops, Shijiazhuang 050035
  • Received:2022-03-04 Accepted:2022-04-15 Online:2022-07-16 Published:2022-07-26
  • Contact: Jie ZHANG,DongMei WANG E-mail:491659835@qq.com;zhangjiezhaolh@163.com;dongmeiwang63@126.com

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Abstract:

【Objective】The molecular mechanism underlying the resistance to soybean mosaic virus (SMV) infection in soybean is of great importance as soybean mosaic caused by SMV has become one of the major soybean disease worldwide. We have previously performed transcriptome analysis of SMV-inoculated soybean after inhibition of H2O2 production and have identified a differentially expressed C2H2-type single zinc finger protein gene, Glyma.18G003600.1, named GmSZFP. In this study, we use virus induced gene silencing (VIGS) technique to investigate the function of GmSZFP in soybean-SMV interaction, providing a foundation for further investigation of the molecular mechanism of GmSZFP in soybean-SMV interaction. 【Method】Soybean cultivar Jidou 7 and SMV strains SC-8 (susceptive) and N3 (resistance) were used as the materials in this study. Bioinformatic analysis was conducted to predict the protein domains of GmSZFP; its transcription factor activity was measured by transcriptional activation assay in yeast; the expression characteristics of GmSZFP at the transcriptional level in soybean-SMV interaction were verified by real-time quantitative PCR (qPCR); and the function of GmSZFP in soybean-SMV interaction was investigated by VIGS technique. 【Result】The CDS region of GmSZFP gene was cloned; amino acid sequence analysis and transcriptional activation assay in yeast revealed that GmSZFP is a C2H2-type zinc finger protein transcription factor with transcriptional activation activity; qPCR results showed that GmSZFP was strongly induced by SMV inoculation, and the expression pattern was different between the compatible and the incompatible combinations. The expression of GmSZFP was elevated after SMV inoculation, and then decreased in the incompatible combination, and the expression level of GmSZFP was significantly lower in the compatible combination than that in the former. Moreover, the expression level of GmSZFP was found to be reduced to the level that is similar to the level in the compatible combination that was pre-inoculated with imidazole, indicating that GmSZFP responds to SMV infestation at the transcriptional level and is regulated by H2O2; After silencing GmSZFP, we found that callose at the SMV inoculation site was greatly reduced compared to the control, and the expression of callose synthase genes GmGSL7c and GmGSL12b was reduced compared to the control, and the expression of callose hydrolase gene BG was elevated compared to the control; In addition, after GmSZFP was silenced, the virus spread outward to a distance of 2 mm at 72 h and to a distance of 3 mm at 96 h from the central source after SMV was inoculated in a small area, while the expression of SMV capsid protein (CP) gene was not detectable outside the inoculation site in control leaves; 10 d after SMV inoculation, the upper leaves (of the SMV inoculated leaves) in the GmSZFP-silenced plants showed mosaic, greening and curling symptoms, and CP gene was expressed, indicating that silencing of GmSZFP enabled SMV to transport in an unrestricted manner. 【Conclusion】GmSZFP is a canonical C2H2-type mono-zinc finger protein, and the GmSZFP gene plays a positive regulatory role in soybean resistance to SMV infection.

Key words: Soybean mosaic virus, single zinc finger protein, virus-mediated gene silencing (VIGS), functional analysis

Table 1

Primer sequence information"

引物名称 Primer name 引物序列 Primer sequence（5′-3′） 引物用途 Primer application
GmSZFP CDS-F AATACCAAAACCACCACCACCAGATAC 扩增GmSZFP引物
Amplify GmSZFP primers
GmSZFP CDS-R TACACTGCCAAAACTTCTCTTCATATTCGA
qRT-GmSZFP-F CTAAAAAGCAGGCTGTTTCAGT 用于检测GmSZFP的相对转录水平
Used to detect the relative transcription level of GmSZFP
qRT-GmSZFP-R CTTCAGATGCTTCAGTAATGGC
M13-F GTAAAACGACGGCCAGT T载体通用测序引物
T vector universal sequencing primer
M13-R CAGGAAACAGCTATGAC
EF1b-F CCACTGCTGAAGAAGATGATGATG 用于检测EF1b的相对转录水平
For detecting the relative transcription level of EF1b
EF1b-R AAGGACAGAAGACTTGCCACTC
SMV-CP PCR-F GAGTGGGACAGGAGCAAAGAGCTTA 用于检测SMV-CP的表达水平
For detecting the expression level of SMV-CP
SMV-CP PCR-R TCAAGCAAGTGGTCCAAGCTAAGAA
pGBDT7-GmSZFP-F GAATTCATGGTTTCTTTTACAAGTTATTTC pGBDT7-GmSZFP载体构建引物
pGBDT7-GmSZFP vector construction primer
pGBDT7-GmSZFP-R GGATCCTCAATCAATATTATCATCTTCAGA

Fig. 1

Cloning of GmSZFP gene and its amino acid sequence analysis A: GmSZFP clone electrophoresis. M: DL 2000 Marker; 1: GmSZFP; B: Amino acid sequence analysis of GmSZFP"

Fig. 2

GmSZFP transcriptional activation activity verification A: Construction of pGBDT7-GmSZFP vector. M: DL 2000 Marker; 1: Before enzyme digestion; 2: After enzyme cutting; B: GmSZFP transcriptional activation activity Validation"

Fig. 3

Relative expression of GmSZFP in the soybean-SMV interaction The values in the figures represented the mean standard error of the three biological replicates, and the small letters represented the significant difference (P<0.05 as determined by one-way analysis of variance and Duncan's multiple comparisons). The same as below"

Fig. 4

The silencing efficiency of GmSZFP gene"

Fig. 5

Observation of callose fluorescence in GmSZFP silenced plants Bar = 50 μm"

Fig. 6

Statistics of callose area on GmSZFP silent plants"

Fig. 7

Expression analysis of callose synthase GmGSL7c, GmGSL12b and hydrolase BG genes A: GmGSL7c in transcriptome database data; B: GmGSL12b in transcriptome database data; C: BG in transcriptome database data; D: Changes in the expression of GmGSL7c in GmSZFP silent plants; E: Changes in the expression of GmGSL12b in GmSZFP silent plants; F: changes of BG expression in GmSZFP silent plants"

Fig. 8

Detection of SMV CP gene expression in soybean leaves by silencing GmSZFP A: RT-qPCR detection of SMV CP gene; M: DL 2000 Marker; B: Western blotting detection of SMV CP; M: PageRuler Prestained Protein Ladder"

Fig. 9

Phenotype observation and virus detection in the upper leaves of GmSZFP silenced plants inoculated with SMV A: Phenotype of the upper leaves at 10 d following SMV inoculation; B: Detection of SMV CP gene; M: DL 2000 Marker; +: SMV virus; -: H2O"

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