大豆单锌指蛋白基因GmSZFP的克隆及其在SMV与寄主互作中的功能

【目的】由大豆花叶病毒（soybean mosaic virus,SMV）引起的大豆花叶病是世界性大豆病害之一,利用病毒诱导的基因沉默（virus induced gene silencing,VIGS）技术研究GmSZFP在大豆与SMV互作过程中发挥的功能,为深入探讨其分子机制奠定基础。【方法】以大豆品种冀豆7号与SMV毒株SC-8、N3组成的亲和、不亲和组合为试验材料,运用生物信息学预测GmSZFP的分子结构特征;以酵母转录激活试验检测其转录因子活性;借助实时定量PCR（RT-qPCR）验证GmSZFP在大豆与SMV互作中转录水平的表达特征;结合VIGS技术探究GmSZFP在大豆与SMV互作过程中的功能。【结果】通过PCR克隆GmSZFP的CDS区,序列全长为1 071 bp;氨基酸序列分析和酵母转录激活试验发现GmSZFP为C₂H₂型锌指蛋白转录因子,且具有转录激活活性;RT-qPCR结果显示,GmSZFP受SMV的强烈诱导,在亲和组合与不亲和组合中的表达模式不同,在不亲和组合中,GmSZFP呈先上升后下降的表达趋势,其表达水平明显高于亲和组合,若在接种病毒前给叶片预注射咪唑,GmSZFP的表达水平降低,并与亲和组合相近,说明GmSZFP在转录水平响应SMV的侵染并受胞内H₂O₂信号调控;利用VIGS技术沉默GmSZFP后,发现接种部位胼胝质的积累水平较对照大大降低,RT-qPCR检测胼胝质合酶基因GmGSL7c和GmGSL12b的表达量较对照降低,胼胝质水解酶基因BG的表达量较对照增加;在基因沉默植株叶片上点接种SMV后72 h,病毒向外扩散至2 mm,在96 h时病毒扩散至3 mm,而对照组叶片在接种点外始终未能检测到SMV外壳蛋白CP的表达;摩擦接种SMV后10 d,在基因沉默植株的上位叶表现出花叶、失绿和卷曲等感病症状,并检测到CP的表达,说明沉默GmSZFP后,SMV在胞间扩散和长距离运输的能力均增强。【结论】大豆GmSZFP是典型的C₂H₂型单锌指蛋白,GmSZFP在大豆抵抗SMV侵染过程中发挥正调控作用。

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

【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.