| 摘要: |
| 【 目的】 明确一种侵染猕猴桃的新病毒基因组特征，构建其侵染性 cDNA 克隆，并分析其致病性。【 方法】 以采集的感染新病毒的猕猴桃叶片为材料，通过长链 RT-PCR 和 RACE 技术获得新病毒全基因组序列，暂命名为柑橘叶斑驳病毒 A（c citrus leaf blotch virus Ac，CLBV-Ac），与乙型线形病毒科（Betaflexiviridae）其他成员共同构建进化树，分析基因编码氨基酸序列和基因组核苷酸序列的一致性，并检测潜在的基因重组事件，明确 CLBV-Ac 的分类地位。利用同源重组方法构建 CLBV-Ac 侵染性 cDNA 克隆，通过农杆菌浸润法将其接种至健康心叶烟和本氏烟中，测定其致病性。【 结果】 CLBV-Ac 属于乙型线形病毒科柑橘病毒属（Citrivirus）成员，其基因组全长 8 678 nt，不包含Poly A，基因组为正义单链 RNA（+ssRNA），包含 3 个开放阅读框（open reading frame，ORF），分别编码病毒的复制酶蛋白（replicase polyprotein，RP）、运动蛋白（movement protein，MP）和外壳蛋白（coat protein，CP）。运动蛋白和外壳蛋白与柑橘病毒属其他成员的氨基酸序列一致性高于 90%，相比之下，复制酶蛋白的氨基酸序列相似性仅有 67. 03%。基因重组分析发现，CLBV-Ac 是柑橘叶斑驳病毒（citrus leaf blotch virus，CLBV）不同寄主分离物的重组体。成功构建了 CLBV-Ac 侵染性克隆载体 pCB301-CLBV-Ac，接种烟草 14 d 后，叶片出现严重扭曲，并伴随轻微的褪绿症状。RT-PCR 和 斑 点 ELISA（Dot-ELISA）试 验 证 实 ，构 建 的 侵 染 性 克 隆 pCB301-CLBV-Ac 具 有 生 物 活 性 。【 结 论】 CLBV-Ac 是柑橘病毒属新成员，为侵染猕猴桃的新病毒，成功构建了 CLBV-Ac 侵染性克隆，为后续深入研究其致病机制提供了基础工具。 |
| 关键词: 猕猴桃 柑橘叶斑驳病毒 Ac 基因组特征 侵染性克隆 |
| DOI：10. 13207/j. jnwafu. 2027. 01. 012 |
| 分类号: |
| 基金项目:国家自然科学基金项目（32000123）；陕西省创新人才推进计划项目（2021KJXX-54）；陕西省教育厅一般项目（24JK0286）；国 家级大学生创新训练项目（202411397014） |
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| Genome characteristics and infectious clone construction of a novel CLBV⁃Ac virus infecting kiwifruit |
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Liu Huan1, Wu Wei1, Liu Fei2, Zhang Wenhui3, Tang Xiaodong3
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1.College of Modern Agriculture and Biotechnology，Ankang University，Ankang，Shaanxi 725000，China;2.Xianyang Agricultural Research Academy，Xianyang，Shaanxi 712000，China;3.Ankang Agricultural Research Academy，Ankang，Shaanxi 725000，China
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| Abstract: |
| 【 Objective】 The study aims to clarify the genomic characteristics of a novel virus infecting kiwi?fruit，construct an infectious cDNA clone，and further analyze its pathogenicity.【Method】 Using collected kiwi?fruit leaves infected with the novel virus as materials，the complete genome sequence of the novel virus tenta?tively named citrus leaf blotch virus Ac（ CLBV-Ac） was obtained through long-chain RT-PCR and RACE techniques. To clarify the taxonomic position of CLBV-Ac，a phylogenetic tree was constructed with other members of the family Betaflexiviridae to analyze the consistency of the encoded amino acid sequences and genomic nucleotide sequences，and to detect potential gene recombination events. An infectious cDNA clone of CLBV-Ac was constructed using homologous recombination，and then inoculated into healthy Nicotiana benthamiana and N. glutinosa using the Agrobacterium-mediated infiltration method to determine its pathogenicity.【Result】 CLBV-Ac is a member of the genus Citrivirus in the family Betaflexiviridae. Its genome is a positive-sense single-stranded RNA（ +ssRNA） of 8 678 nt in length，excluding a Poly A tail，and contains three open reading frames（ ORFs） that encode the viral replicase polyprotein（ RP），movement protein（ MP） and coat protein （CP）. The sequences of the MP and CP share over 90% similarity with other members of the Citrivirus genus， while replicase protein shows only 67. 03% amino acid sequence similarity. Recombination analysis revealed that CLBV-Ac is a recombinant of CLBV isolates derived from different hosts. Furthermore，an infectious clone vector pCB301-CLBV-Ac was successfully constructed. Tobacco plants inoculated with this clone exhibited se?
vere leaf distortion accompanied by mild chlorosis at 14 days after inoculation. RT-PCR and Dot-ELISA tests confirmed the biological activity of the infectious clone pCB301-CLBV-Ac.【Conclusion】 CLBV-Ac is a new member of the genus Citrivirus and a novel virus that infects kiwifruit. The infectious clone of CLBV-Ac was successfully constructed，providing a basic tool for further in-depth research on its pathogenic mechanism. |
| Key words: kiwifruit citrus leaf blotch virus Ac genome characteristic infectious clone |
