NbJAZ3在苜蓿花叶病毒侵染本氏烟过程中的作用

doi:10.13560/j.cnki.biotech.bull.1985.2025-0084

摘要/Abstract

摘要：

目的探究本氏烟（Nicotiana benthamiana）中编码茉莉酸ZIM结构域蛋白3（jasmonate ZIM-domain protein 3, JAZ3）基因NbJAZ3对苜蓿花叶病毒（alfalfa mosaic virus, AMV）侵染本氏烟的影响，为进一步解析AMV侵染机制提供理论依据。方法以本氏烟和AMV为材料，采用摩擦接种法将AMV接种于本氏烟，取接种后1、7、15、21 d的本氏烟叶片进行转录组测序分析，对茉莉酸（jasmonic acid, JA）信号转导途径中差异基因进行筛选，利用实时荧光定量PCR（RT-qPCR）检测NbJAZ3在AMV侵染中的表达变化；克隆NbJAZ3，进行生物学信息分析，构建系统进化树并进行多序列比对；构建pCAMBIA1300-NbJAZ3瞬时过表达载体，利用激光共聚焦显微镜观察NbJAZ3蛋白亚细胞定位，利用Western blot检测蛋白表达量；当NbJAZ3在本氏烟叶片中瞬时过表达后，接种AMV，采用RT-qPCR法检测接种后第5天的AMV CP相对表达量；利用烟草脆裂病毒（tobacco rattle virus, TRV）诱导的基因沉默（virus-induced gene silencing, VIGS）技术构建pTRV2-NbJAZ3沉默载体，沉默NbJAZ3后摩擦接种AMV，利用RT-qPCR检测AMV CP相对表达量。结果转录组测序和RT-qPCR进一步验证表明，AMV接种后的不同天数NbJAZ3均下调表达，且第15天NbJAZ3相对表达量最低，与对照相比下降了84.43%；NbJAZ3 CDS序列长度为1 143 bp，编码380个氨基酸，包含ZIM和Jas-motif 2个保守结构域；NbJAZ3与林烟草（Nicotiana sylvestris）的NsJAZ3同源性最高，相似度为96.58%；显微镜观察到NbJAZ3蛋白定位于本氏烟叶片的细胞核、细胞质与细胞膜中；与对照组相比，NbJAZ3瞬时过表达和瞬时沉默的本氏烟接种AMV第5天时，AMV CP相对表达量分别降低了83.82%和增加了78.58%。结论 AMV侵染会引起本氏烟中NbJAZ3下调，瞬时过表达NbJAZ3可抑制AMV对本氏烟的侵染，而沉默该基因可促进AMV的侵染，进一步表明NbJAZ3可作为本氏烟抗AMV侵染的正调控因子，在抵御病毒侵染中具有重要作用。

关键词: 苜蓿花叶病毒, 侵染, 本氏烟, NbJAZ3, 亚细胞定位, 瞬时过表达, 基因沉默, 生物信息学分析

Abstract:

Objective To explore the effect of NbJAZ3, a gene encoding jasmonate ZIM-domain protein 3 (JAZ3), on the infection of Nicotiana benthamiana by alfalfa mosaic virus (AMV), and to provide a theoretical basis for further analyzing the mechanism of AMV infection. Method Using N. benthamiana and AMV as materials, AMV was inoculated into N. benthamiana by friction inoculation method. The leaves of N. benthamiana at 1, 7, 15 and 21 d after inoculation were used for transcriptome sequencing analysis. The differential genes in jasmonic acid (JA) signal transduction pathway were screened, and the expression of NbJAZ3 in AMV infection was detected by real-time fluorescence quantitative PCR (RT-qPCR). NbJAZ3 was cloned, biological information was analyzed, phylogenetic tree was constructed, and multiple sequence alignment was performed.The transient overexpression vector of pCAMBIA1300-NbJAZ3 was constructed. The subcellular localization of NbJAZ3 protein was observed by laser confocal microscopy, and the protein expression was detected by Western blot. NbJAZ3 was transiently overexpressed in N. benthamiana leaves and inoculated with AMV, and the relative expression of AMV CP at 5 d after inoculation was detected by RT-qPCR. The pTRV2-NbJAZ3 silencing vector was constructed by gene silencing technology induced by tobacco brittle fracture virus. After silencing NbJAZ3, AMV was inoculated by friction. The relative expression of AMV CP was detected by RT-qPCR. Result Transcriptome sequencing and RT-qPCR further verified that NbJAZ3 was down-regulated on different days after AMV inoculation, and the relative expression of NbJAZ3 was the lowest on the day15, which was 84.43% lower than that of CK. The NbJAZ3 CDS sequence was 1 143 bp in length, encoding 380 amino acids, containing two conserved domains of ZIM and Jas-motif. NbJAZ3 had the highest homology with NsJAZ3 of Nicotiana sylvestris, and the similarity was 96.58%. Microscopic observation showed that NbJAZ3 was localized in the nucleus, cytoplasm and cell membrane of N. benthamiana leaves. Compared with the control group, the relative expression of AMV CP decreased by 83.82% and increased by 78.58% in N. benthamiana with transient overexpression and transient silencing of NbJAZ3 on the day 5 of AMV inoculation, respectively. Conclusion AMV infection can cause the down-regulation of NbJAZ3 in N. benthamiana. Transient overexpression of NbJAZ3 can inhibit the infection of AMV, while silencing this gene can promote the infection of AMV, further indicating that NbJAZ3 can be used as a positive regulator of N. benthamiana resistance to AMV infection and play an important role in resisting virus infection.

Key words: alfalfa mosaic virus (AMV), infection, Nicotiana benthamiana, NbJAZ3, subcellular localization, transient overexpression, virus-induced gene silencing (VIGS), bioinformatics analysis

赖诗雨, 梁巧兰, 魏列新, 牛二波, 陈应娥, 周鑫, 杨思正, 王博. NbJAZ3在苜蓿花叶病毒侵染本氏烟过程中的作用[J]. 生物技术通报, 2025, 41(8): 186-196.

LAI Shi-yu, LIANG Qiao-lan, WEI Lie-xin, NIU Er-bo, CHEN Ying-e, ZHOU Xin, YANG Si-zheng, WANG Bo. The Role of NbJAZ3 in the Infection of Nicotiana benthamiana by Alfalfa Mosaic Virus[J]. Biotechnology Bulletin, 2025, 41(8): 186-196.

图/表 8

表1 本试验采用引物及序列

Table 1 Primers and sequences were used in this experiment

引物名称	上游引物序列	下游引物序列
Primer name	Forward primer sequence （5′-3′）	Reverse primer sequence （5′-3′）
NbJAZ3	ATGGAGAGAGATTTTATGGG	CGTCTCCTTGACCAAATT
Kpn Ⅰ/Sac I-NbJAZ3	GGTACCATGGAGAGAGATTTTATGGG	GAGCTCCGTCTCCTTGACCAAATT
si-NbJAZ3	GGTACCCTATCTCCAGTTCAGGC	GAGCTCCCGAGCCAATGAT
T-vector-M13	CGCCAGGGTTTTCCCAGTCAC	AGCGGATAACAATTTCACACAGGA
pCAMBIA1300	CTATCCTTCGCAAGACCCTTC	TGACGAACGTTGTCGAAACC
pTRV2-PCR	ACATTGTTACTCAAGGAAGCACGA	AACTTCAGACACGGATCTACTT
25S	AAGGCCGAAGAGGAGAAAGGT	CGTCCCTTAGGATCGGCTTAC
AMV-CP	GCATCCCTAGGGGCATTCATGCA	ATCATTGATCGGTAATGGGCCGTT
qPCR-NbJAZ3	CTGGTGTCGGGCAGAAAA	TGGGTTGGAAACTGGGAG
Actin	CTTGAAACAGCAAAGACCAGC	CATCCTATCAGCAATGCCCG

图1 AMV侵染本氏烟不同时间差异基因热图（A-C）及AMV CP（D）和NbJAZ3（E）基因相对表达量A：接种AMV后1 d；B：接种AMV后15 d；C：接种AMV后21 d；D：AMV CP基因相对表达量；E：NbJAZ3基因相对表达量。统计分析采用Student’s t检验（**：0.001<P<0.01，***：P<0.001），每个处理进行3次生物学重复，每次生物学重复3株植株，数值代表3次生物学重复的平均值±标准误，下同

Fig. 1 Heat map of differential genes (A-C) and the relative expressions of AMV CP (D) and NbJAZ3 (E) of AMV-infected N. benthamiana at different timesA: 1 d after AMV infection. B:15 d after AMV infection. C:21d after AMV infection. D: Relative expressions of AMV CP. E: Relative expressions of NbJAZ3. Student's t test was used for statistical analysis (**: 0.001<P<0.01, ***: P<0.001). Each treatment was repeated three times, and three plants were used for repeated each time. The value indicates the mean ± standard error of the three biological replicates, the same below

图2 NbJAZ3与其同源基因系统发育分析（A）及氨基酸序列比对（B）

Fig. 2 Phylogenetic development analysis (A) and amino acid sequence alignment (B) of NbJAZ3 and homologous genes

图3 NbJAZ3蛋白理化性质分析A：蛋白亲疏水性结构预测；B：蛋白二级结构预测；C：蛋白三级结构预测

Fig. 3 Analysis for physicochemical properties ofNbJAZ3 proteinA: Prediction of protein hydrophilic structure. B: Prediction of protein secondary structure. C: Prediction of protein tertiary structure

图4 T-Vetor-NbJAZ3（A）和pCAMBIA1300-NbJAZ3（B）瞬时过表达载体双酶切验证M1：DNA marker（2 000 bp）；M2：DNA marker（15 000 bp）；T-vector载体、pCAMBIA1300载体及NbJAZ3长度分别为2 693、11 189和1 143 bp

Fig. 4 Double enzyme digestion of T-Vetor-NbJAZ3 (A) and pCAMBIA1300-NbJAZ3 (B) transient overexpressionM1: DNA marker (2 000 bp). M2: DNA marker (15 000 bp). The lengths of T-vector, pCAMBIA1300 and NbJAZ3 are 2 693, 11 189 and 1 143 bp, respectively

图5 NbJAZ3在本氏烟叶片中的亚细胞定位A：pCAMBIA1300-NbJAZ3在本氏烟中的表达；B：pCAMBIA1300-35S-eGFP在本氏烟中的表达

Fig. 5 Subcellular localization of NbJAZ3 in N. benthamiana leavesA: Expression of pCAMBIA1300-NbJAZ3 in N. benthamiana. B: Expression of pCAMBIA1300-35S-eGFP in N. benthamiana

图6 本氏烟中瞬时过表达NbJAZ3的表达量检测及对AMV复制的影响A：pCAMBIA1300-NbJAZ3瞬时过表达后第5天NbJAZ3相对表达量；B：Western blot检测pCAMBIA1300-NbJAZ3的表达；C：瞬时过表达NbJAZ3并接种AMV后第5天的本氏烟表型；D：NbJAZ3瞬时过表达对AMV CP相对表达量的影响

Fig. 6 Detection of transient overexpression of NbJAZ3 in N. benthamiana and its effect on AMV replicationA: The relative expression of NbJAZ3 on the day 5 after transient overexpression of pCAMBIA1300-NbJAZ3.B: The expression of pCAMBIA1300-NbJAZ3 was detected by Western blot. C: The phenotype of N. benthamiana on the day 5 after transient overexpression of NbJAZ3 and inoculation with AMV. D: Effect of transient overexpression of NbJAZ3 on the relative expression of AMV CP

图7 NbJAZ3沉默效率检测及对AMV复制的影响A： NbJAZ3第9天沉默效率；B：NbJAZ3沉默植株接种AMV后第5天叶片表型；C：NbJAZ3沉默植株接种AMV后第5天RT-qPCR检测AMV CP相对表达量

Fig. 7 Detection of silencing efficiency of NbJAZ3 and its effect on AMV replicationA: Silencing efficiency of NbJAZ3 on the day 9. B: The leaf phenotype of NbJAZ3 silenced plants on the day 5 after inoculation with AMV; C: The relative expression of AMV CP in NbJAZ3 silenced plants was detected by RT-qPCR on the day 5 after inoculation with AMV

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