稻曲病菌Uv263菌株中一种新型真菌病毒的基因组结构与功能分析

doi:10.7668/hbnxb.20195221

摘要/Abstract

摘要：

为了深入挖掘稻曲病菌真菌病毒的多样性,以从海南省患病水稻样本中分离到的1株稻曲病菌异常菌株Uv263为试验材料,鉴定该菌株中潜在的真菌病毒,解析真菌病毒基因组结构与功能的关系。结果表明,Uv263菌株被1种新型真菌病毒侵染,命名为Ustilaginoidea virens RNA virus 7(UvRv7)。UvRV7为双链RNA病毒,全长5 082 bp,GC含量为60.29%。UvRV7编码的开放阅读框1编码外壳蛋白(CP),开放阅读框2编码RNA依赖的RNA聚合酶(RdRP)。BlastP比对表明,UvRV7的RdRP氨基酸序列与病毒Thelebolus microsporus totivirus 1的RdRP氨基酸序列的相似性最高,为48.49%。基于UvRV7的RdRP氨基酸序列的多重比对结果表明,UvRV7的RdRP序列共包含8个保守基序,其中在第Ⅵ个基序中鉴定到1个RdRP保守结构域中最典型的GDD基序。系统发育分析表明,UvRV7与Thelebolus microsporus totivirus 1的亲缘关系最近,并且与单分体病毒科中维多利亚病毒属的典型成员形成了1个进化分支。基因组特征和进化分析结果均表明,UvRV7是维多利亚病毒属中一个新发现的真菌病毒种类。透射电镜观察表明,UvRV7形成1个约45 nm的球状病毒粒子。水平传播和垂直传播试验也表明,UvRV7可以通过分生孢子高效垂直传播,并且在营养体亲和的菌株间高效水平传播。综上所述,阐明了稻曲病菌中新报道的单分体病毒UvRV7的全基因组结构特征及其进化关系。

关键词: 稻曲病, 真菌病毒, 生物防治, 单分体病毒科, 维多利亚病毒属

Abstract:

In order to deeply explore the diversity of mycoviruses in Ustilaginoidea virens,this study used an abnormal strain Uv263 of U.virens isolated from diseased rice samples collected from Hainan Province as experimental material to identify potential mycoviruses in this strain,and analyze the relationship between the genome organization and function of mycoviruses.The results showed that strain Uv263 was infected by a novel mycovirus named Ustilaginoidea virens RNA virus 7 (UvRV7).UvRV7 was a double stranded RNA virus with 5 082 bp in total length and 60.29% GC content.UvRV7 encoded two large open reading frames (ORF1 and ORF2),which encoded the coat protein (CP) and RNA-dependent RNA polymerase (RdRP),respectively.The BlastP comparison showed that the RdRP amino acid sequence of UvRV7 shared the highest similarity with that of Thelebolus microsporus totivirus 1,at 48.49%.The results of multiple alignment based on the amino acid sequence of UvRV7 RdRP showed that the RdRP sequence contained a total of eight conserved motifs,among which the most typical GDD motif in the RdRP conserved domain was identified in the Ⅵ motif.The phylogenetic analysis showed that UvRV7 was the most closely related to Thelebolus microsporus totivirus 1 and clustered with representative viruses of the genus Victorivirus in the family Totiviridae.The results of genome organization and evolutionary analyses both indicated that UvRV7 was a novel mycovirus in the genus Victorivirus.Transmission electron microscopy observations showed that UvRV7 formed a spherical viral particle of about 45 nm.Horizontal and vertical transmission experiments showed that UvRV7 could be efficiently transmitted vertically by conidia and efficiently transmitted horizontally between vegetatively compatible strains.Taken together,this study elucidated the genome organization and evolutionary relationships of the novel mycovirus UvRV7 in U.virens,and provided a potential biocontrol agent and theoretical basis for the biological control of rice false smut.

Key words: Rice false smut, Mycovirus, Biocontrol, Totiviridae, Victorivirus

中图分类号:

S435.111.46

何桢锐, 赵文华, 杨迎青, 杨媚, 周而勋. 稻曲病菌Uv263菌株中一种新型真菌病毒的基因组结构与功能分析[J]. 华北农学报, 2025, 40(3): 141-148. doi: 10.7668/hbnxb.20195221.

HE Zhenrui, ZHAO Wenhua, YANG Yingqing, YANG Mei, ZHOU Erxun. Genome Organization and Functional Analysis of a Novel Mycovirus in Ustilaginoidea virens Strain Uv263[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 141-148. doi: 10.7668/hbnxb.20195221.

http://www.hbnxb.net/CN/Y2025/V40/I3/141

图/表 6

图1 稻曲病菌Uv263菌株中真菌病毒的特征 A.Uv263菌株中真菌病毒的RT-PCR验证:M.2 000 bp Marker,1.酶解前,2.酶解后;B.DNase Ⅰ (酶解ssDNA和dsDNA) 和 S1 核酸酶(酶解ssDNA和dsDNA)处理前后的dsRNA进行RT-PCR检测。

Fig.1 Characterization of the mycovirus in Ustilaginoidea virens strain Uv263 A.RT-PCR verification of mycovirus in strain Uv263:M.2 000 bp Marker,1.Before digestion,2.After digestion;B.RT-PCR of dsRNA with mycovirus-specific primers before and after deoxyribonuclease Ⅰ(DNase Ⅰ) (digestion of ssDNA and dsDNA) and S1 nuclease (digestion of ssDNA or ssRNA) treatments.

图2 稻曲病菌Uv263菌株中真菌病毒UvRV7的末端序列克隆

Fig.2 Cloning of the terminal sequences of UvRV7 in Ustilaginoidea virens strain Uv263 by RACE

图3 稻曲病菌Uv263菌株中真菌病毒UvRV7的基因组结构模式图

Fig.3 Schematic representation of the genomic organization of mycovirus UvRV7 in Ustilaginoidea virens strain Uv263

图4 稻曲病菌Uv263菌株中真菌病毒UvRV7的多重比对和系统发育分析 A.真菌病毒UvRV7与来自单分体病毒科的代表性病毒的RdRP氨基酸基序的多序列比对:黑色.所选病毒的重叠保守序列,灰色.各病毒间部分重叠的保守序列;B.真菌病毒UvRV7的系统发育分析:红色星星.真菌病毒UvRV7。

Fig.4 Multiple alignment and phylogenetic analysis of mycovirus UvRV7 in Ustilaginoidea virens strain Uv263 A.Multiple alignment of the conserved RdRP amino acid motifs of UvRV7 and the representative viruses from the family Totiviridae:Black.The overlapped conserved sequences of selected viruses,Gray.The partially overlapped conserved sequences among viruses;B.Phylogenetic analysis of mycovirus UvRV7:Red stars.The mycovirus UvRV7.

图5 稻曲病菌Uv263菌株中真菌病毒UvRV7病毒粒子的验证和观察 A.不同蔗糖浓度混合液中UvRV7 PCR验证:1—6的蔗糖浓度分别为10%,20%,30%,40%,50%,60%; B.UvRV7 病毒粒子电镜图,红色箭头指示的为较明显的等轴二十面体对称性的病毒粒子。

Fig.5 Validation and observation of mycovirus UvRV7 viral particles in Ustilaginoidea virens strain Uv263 A.UvRV7 PCR validation in a mixture of different sucrose concentrations:Sucrose concentrations of 1—6 are 10%,20%,30%,40%,50%, and 60%,respectively;B.Electron micrograph of UvRV7 virus particles,red arrows indicate viral particles with icosahedral symmetry.

图6 稻曲病菌Uv263菌株中真菌病毒UvRV7的水平传播和垂直传播能力验证 A.真菌病毒UvRV7的水平传播能力验证;B.真菌病毒UvRV7的垂直传播能力验证。

Fig.6 Validation of horizontal and vertical transmission efficiencies of mycovirus UvRV7 in Ustilaginoidea virens strain Uv263 A.Validation of UvRV7 horizontal transmission efficiency;B.Validation of UvRV7 vertical transmission efficiency.

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