小西葫芦黄花叶病毒弱毒疫苗的构建及其交叉保护防治作用

doi:10.13560/j.cnki.biotech.bull.1985.2025-0944

生物技术通报 ›› 2026, Vol. 42 ›› Issue (1): 271-278.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0944

小西葫芦黄花叶病毒弱毒疫苗的构建及其交叉保护防治作用

张悦¹^,²(), 任倩¹^,²(), 栾雅梦³, 李翌琛¹, 孙无瑕¹, 任姝颖¹, 许小洁¹^,²(), 孙晓辉³()

^1.鲁东大学农林工程研究院植物病毒学研究室，烟台 264025
^2.烟台市作物高产抗逆分子育种及高效栽培重点实验室，烟台 264025
^3.山东省烟台市农业科学研究院，烟台 265500

收稿日期:2025-09-03 出版日期:2026-01-26 发布日期:2026-02-04
通讯作者: 许小洁，讲师，研究方向：植物病毒学；E-mail: xiaojiexua105@163.com；
孙晓辉，高级农艺师，研究方向：作物育种学；E-mail: 123615969@qq.com
作者简介:张悦，硕士研究生，研究方向：植物病毒学；E-mail: z17763388238@163.com
张悦，硕士研究生，研究方向：植物病毒学；E-mail: z17763388238@163.com
基金资助:
山东省重点研发计划项目(2023LZGC002);国家自然科学基金项目(32302310);山东省自然科学基金项目(ZR2023QC094);山东省烟台市高校与地方企业合作项目(2021XDRHXMPT09)

Construction of a Mild Vaccine of Zucchini Yellow Mosaic Virus and Its Cross-protective Efficacy

ZHANG Yue¹^,²(), REN Qian¹^,²(), LUAN Ya-meng³, LI Yi-chen¹, SUN Wu-xia¹, REN Shu-ying¹, XU Xiao-jie¹^,²(), SUN Xiao-hui³()

^1.Laboratory of plant Virology, The Engineering Research Institute of Agriculture and Forestry, Ludong University, Yantai 264025
^2.Yantai Key Laboratory of Molecular Breeding for High-Yield and Stress-Resistant Crops and Efficient Cultivation, Yantai 264025
^3.Shandong Yantai Academy of Agricultural Sciences, Yantai 265500

Received:2025-09-03 Published:2026-01-26 Online:2026-02-04

摘要/Abstract

摘要：

目的通过研究辅助成分-蛋白酶（helper component-proteinase, HC-Pro）中第364位保守的赖氨酸（lysine, K³⁶⁴）突变对小西葫芦黄花叶病毒（zucchini yellow mosaic virus, ZYMV）致病力的影响，构建ZYMV弱毒突变体，并评估其遗传稳定性和交叉保护防治效果，为利用弱毒株开发针对ZYMV的绿色防治策略提供理论依据。方法利用定点突变的方法，将HC-Pro中K³⁶⁴位点替换为天冬氨酸（aspartic acid, D），研究K364D突变对ZYMV致病力和HC-Pro RNA沉默抑制活性的影响，构建弱毒突变体ZYMV-HC-Pro_K364D。通过继代接种实验，测试弱毒突变体ZYMV-HC-Pro_K364D的遗传稳定性；通过交叉保护实验，评估弱毒突变体ZYMV-HC-Pro_K364D的交叉保护防治作用。结果 HC-Pro中K364D突变影响了ZYMV致病力，减轻了病毒在甜瓜（Cucumis melo）植株上引起的典型黄化症状，显著降低了病毒积累水平，同时，该突变削弱了ZYMV HC-Pro的RNA沉默抑制活性。ZYMV-HC-Pro_K364D-GFP突变体在甜瓜植株中连续3次继代接种后未发生回复突变，保持遗传稳定性。在交叉保护实验中，ZYMV-HC-Pro_K364D突变体在保护间隔期11 d时，可诱导甜瓜植株产生对野生型ZYMV的显著抗性。结论 HC-Pro中保守的K³⁶⁴残基是影响ZYMV致病力和HC-Pro RNA沉默抑制活性的关键位点，ZYMV-HC-Pro_K364D突变体在甜瓜中具有良好的交叉保护作用，可作为弱毒疫苗防治甜瓜ZYMV侵染。

关键词: 小西葫芦黄花叶病毒, 辅助成分-蛋白酶, 弱毒疫苗, 遗传稳定性, 交叉保护

Abstract:

Objective By investigating the effects of the mutation at the 364th conserved lysine (K³⁶⁴) in helper component-proteinase (HC-Pro) on the virulence of zucchini yellow mosaic virus (ZYMV), an attenuated ZYMV mutant were developed, and its genetic stability and cross-protection efficacy were assessed. Our study provided the theoretical basis for developing sustainable control strategies against ZYMV using attenuated strains. Method Using site-directed mutagenesis, the K³⁶⁴ residue in HC-Pro was substituted with aspartic acid (D) to investigate the effect of the K364D mutation on the virulence of ZYMV and the RNA silencing suppression activity of HC-Pro, thereby constructing the attenuated ZYMV-HC-Pro_K364D mutant. Furthermore, the genetic stability of ZYMV-HC-Pro_K364D mutant was assessed through serial passage of progeny viruses. The cross-protection efficacy of ZYMV-HC-Pro_K364D mutant was evaluated through cross-protection assays. Result The K364D mutation in HC-Pro attenuated ZYMV virulence, alleviated typical yellowing symptoms and reduced ZYMV accumulation in Cucumis melo plants. Simultaneously, K364D mutation suppressed the RNA silencing suppression activity of ZYMV HC-Pro. The attenuated mutant ZYMV-HC-Pro_K364D-GFP maintained genetic stability without reversion mutation after three successive passages in C. melo plants. In cross-protection assays, ZYMV-HC-Pro_K364D mutant induced significant resistance in C. melo plants against the wild-type ZYMV infection when the protection interval was 11 d. Conclusion The conserved K³⁶⁴residue in HC-Pro was crucial in determining the RNA silencing suppression activity and the virulence of ZYMV. The ZYMV-HC-Pro_K436D mutant present excellent cross-protection efficacy and is a potential mild vaccine against the wild-type ZYMV infection in C. melo plants.

Key words: Zucchini yellow mosaic virus, helper component-proteinase, mild vaccine, genetic stability, cross-protection

张悦, 任倩, 栾雅梦, 李翌琛, 孙无瑕, 任姝颖, 许小洁, 孙晓辉. 小西葫芦黄花叶病毒弱毒疫苗的构建及其交叉保护防治作用[J]. 生物技术通报, 2026, 42(1): 271-278.

ZHANG Yue, REN Qian, LUAN Ya-meng, LI Yi-chen, SUN Wu-xia, REN Shu-ying, XU Xiao-jie, SUN Xiao-hui. Construction of a Mild Vaccine of Zucchini Yellow Mosaic Virus and Its Cross-protective Efficacy[J]. Biotechnology Bulletin, 2026, 42(1): 271-278.

图/表 5

表1 本实验中用到的引物名称和序列

Table 1 Primer names and sequences used in this study

引物名称 Primer name	引物序列 Primer sequence （5'-3'）
ZYMV-HC-Pro_K364D-F	ATTTCACCGATATGATTCGTGATGTTTTGATCCCTATGCTTG
ZYMV-HC-Pro_K364D-R	CGAATCATATCGGTGAAATCCTTCGCCTCGTTCTCATTAAC
ZYMV-HC-Pro-F	GCGCTCTAGCAAGGCTATG
ZYMV-HC-Pro-R	CACCTAGTATGTATGCTGCAGT
GFP-F	ATGAGTAAAGGAGAAGAAC
GFP-R	TTTGTAGAGCTCATCCATG
GFP-qRT-F	GTGGAGAGGGTGAAGGTGAT
GFP-qRT-R	CGGATAACGGGAAAAGCATTGA
actin-qRT-F	CTGATGAAGATACTCACAGAAAGAG
actin-qRT-R	CAGGATACGGGGAGCTAATG
EF1α-qRT-F	CCACGAGTCTCTCCCAGAAG
EF1α-qRT-R	CACGCTTGAGATCCTTGACA

图1 K364D突变降低了ZYMV在甜瓜植株中的致病力A：比对马铃薯病毒属6种病毒部分HC-Pro的氨基酸序列。ZYMV HC-Pro中K364残基由红色虚线框标注。B：甜瓜植株在野生型ZYMV及ZYMV-HC-ProK364D-GFP突变体侵染后第11天出现的症状。C：Western blotting检测甜瓜上部叶片中ZYMV CP积累量。内参为丽春红染色条带（Ponceau S staining, PSS），条带灰度值使用ImageJ软件测定。ZYMV CP积累量以PSS归一化后的数据表示

Fig. 1 Mutation of K364D reduced ZYMV virulence in C. melo plantsA: Alignment of the partial amino acid sequences of 6 potyvirus HC-Pros. The K364 residue in ZYMV HC-Pro were pointed in the red box. B: The symptoms observed on C. melo plants at day 11 after infection with the wild-type ZYMV and ZYMV-HC-ProK364D-GFP mutant. C: Western blotting was used to detect the accumulation of ZYMV CP in the upper leaves of C. melo plants. The loading controls were the Ponceau S staining (PSS) bands, and the band gray values were measured using ImageJ software. The accumulation of ZYMV CP is represented by the data normalized to PSS

图2 ZYMV HC-Pro中K364D突变影响其抑制RNA沉默的活性A：野生型ZYMV HC-Pro及HC-ProK364D突变体抑制RNA沉默的活性；B：GFP mRNA在16c叶片不同区域的积累量。Tukey多重检验进行组间统计学分析，显著性差异用不同字母来表示（P<0.05）；C：GFP蛋白在16c叶片不同区域的积累量

Fig. 2 Mutation of K364D affected the activity of RNA silencing of ZYMV HC-ProA: Suppression activity of RNA silencing of wild-type ZYMV HC-Pro and HC-ProK364D mutant. B: The accumulation of GFP mRNA in different regions of 16c leaves. Tukey multiple range test was used for statistical analysis between groups. Significant differences are indicated by different letters (P<0.05). C: Accumulation of GFP protein in different regions of 16c leaves

图3 ZYMV-HC-ProK364D-GFP突变体的遗传稳定性A：在连续3次继代中，ZYMV-HC-ProK364D-GFP突变体在甜瓜植株上引起的症状；B： ZYMV-HC-ProK364D-GFP突变体F3代在甜瓜植株中的HC-ProRNA积累量；C： ZYMV-HC-ProK364D-GFP突变体F3代在甜瓜植株中HC-Pro序列分析。ZYMV HC-Pro中编码第364位氨基酸残基的密码子用下划线标注

Fig. 3 Genetic stability of ZYMV-HC-ProK364D-GFP mutantA: During three consecutive passages, the symptoms caused by the ZYMV-HC-ProK364D-GFP mutant on C. melo plants. B: The HC-Pro RNA accumulations of the ZYMV-HC-ProK364D-GFP mutant F3 generation in C. melo plants. C: Analysis of HC-Pro sequence in the F3 generation of ZYMV-HC-ProK364D-GFP mutant in C. melo plants. The codon encoding the 364th amino acid residues in ZYMV HC-Pro was underlined

图4 ZYMV-HC-ProK364D突变体介导的交叉保护作用A：间隔期为7或11 d的甜瓜植株在ZYMV-GFP挑战后第11天的症状表现。括号里列出的为有GFP荧光的甜瓜植株数/接种的总甜瓜植株数；B： GFP mRNA在ZYMV-GFP挑战后第11天的甜瓜植株中的积累量；C： GFP蛋白在ZYMV-GFP挑战后第11天的甜瓜植株中的积累量；D：在ZYMV-GFP挑战后第11天的甜瓜植株中病毒HC-Pro的序列分析。ZYMV HC-Pro中编码第364位氨基酸残基的密码子用下划线标注。每株甜瓜中ZYMV后代HC-Pro编码序列单独测序3次

Fig. 4 Cross protection efficacy mediated by ZYMV-HC-ProK364D mutantA: Symptoms of C. melo plants with an interval period of 7 or 11 d at the 11th day post ZYMV-GFP challenge. The numbers in parentheses indicate the number of C. melo plants with GFP fluorescence/the total number of inoculated C. melo plants. B: The accumulation of GFP mRNA in C. melo plants on the 11th day after ZYMV-GFP challenge. C: The accumulation of GFP protein in C. melo plants on the 11th day after ZYMV-GFP challenge. D: Analysis of HC-Pro sequence in C. melo plants on the 11th day after ZYMV-GFP challenge. The codon encoding the 364th amino acid residue in ZYMV HC-Pro was underlined. The HC-Pro coding sequence from the ZYMV progeny in each C. melo plant was individually sequenced three times

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小西葫芦黄花叶病毒弱毒疫苗的构建及其交叉保护防治作用

Construction of a Mild Vaccine of Zucchini Yellow Mosaic Virus and Its Cross-protective Efficacy

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