Ligustrum Virus A Isolates from Syringa lindl: Identification and Whole Genome Sequence Analysis

WANGYunxuan, LIULi, SUNChunhui, LUShuhao, LIANGYuqing, CUIWenli, GAOJiayi, LIYongqiang

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Chinese Agricultural Science Bulletin ›› 2025, Vol. 41 ›› Issue (3) : 107-113. DOI: 10.11924/j.issn.1000-6850.casb2024-0125

Ligustrum Virus A Isolates from Syringa lindl: Identification and Whole Genome Sequence Analysis

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Abstract

To identify and characterize the candidate viruses infecting Beijing lilac (Syringa lindl) associated with the symptoms of leaf chlorosis, mosaic and leaf curling, small RNA deep sequencing and RT-PCR were conducted with the diseased leaf samples. Ligustrum virus A (LVA) was detected and the complete genome sequences of two isolates were obtained. The Beijing lilac isolate LVA-NXY is 8488 nt in length, while the isolate LVA-HLG is 8478 nt. Sequence similarity analysis showed that LVA-NXY and LVA-HLG were most similar to the LVA-DBLQ isolate from Forsythia suspensa in Shenyang, China. No recombination events were detected among the seven reported LVA isolates. The phylogenetic tree based on the genome sequences indicated that LVA-NXY and LVA-HLG clustered together with LVA-DBLQ and the Syringa vulgaris isolate LVA-DX in the same branch, showing a close phylogenetic relationship. This study reports the complete genome sequence of ligustrum virus A infecting Beijing lilac and explores the molecular characteristics and evolutionary relationships of the virus which provides a theoretical basis for the study of genetic variation and the control of lilac virus disease.

Key words

Syringa lindl / Ligustrum virus A / small RNA / genome / sequence analysis / molecular characteristics / phylogenetic relationship

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WANG Yunxuan , LIU Li , SUN Chunhui , LU Shuhao , LIANG Yuqing , CUI Wenli , GAO Jiayi , LI Yongqiang. Ligustrum Virus A Isolates from Syringa lindl: Identification and Whole Genome Sequence Analysis. Chinese Agricultural Science Bulletin. 2025, 41(3): 107-113 https://doi.org/10.11924/j.issn.1000-6850.casb2024-0125

0 引言

丁香(Syringa lindl)是木犀科(Oleaceae)丁香属(Syringa)落叶灌木,耐寒耐旱,适应性强,是中国北方常见的园林绿化植物之一[1]。当前的国内外研究中已报道多种病毒可侵染丁香。丁香环斑驳病毒(lilac ring mottle virus,LRMV)侵染丁香后会导致叶片出现环斑[2];烟草花叶病毒(tobacco mosaic virus,TMV)侵染丁香后会导致叶片花叶[3];丁香叶褪绿病毒(lilac leaf chlorosis virus,LLCV)侵染丁香后会导致叶片畸形、褪绿和叶缘坏死[4];黄瓜花叶病毒(cucumber mosaic virus,CMV)侵染丁香后会导致叶片出现轻花叶、褪绿斑点和褪绿条纹症状[5];丁香类番茄丛矮病毒(syringa tombus-like virus,STIV)侵染丁香后会导致叶片斑驳褪绿[6]
水蜡A病毒(ligustrum virus A,LVA)最早于2016年在韩国的水蜡树(Ligustrum obtusifolium)上发现[7];2018年,中国首次在辽宁省的丁香上发现该病毒[1]。2022年,韩国在叶片表现坏死和萎蔫的丁香中首次发现该病毒[8]。LVA的病毒粒子为丝状颗粒[9],基因组为7.4 k~9 kb的正义单链RNA分子,具有3′-poly(a)尾巴和5′-cap结构。LVA基因组包含6个开放阅读框(open reading frame,ORF)[10],分别是复制蛋白(replicase,Rep)[11],三联体基因模块蛋白(triple gene block proteins 1-3,TGB1-3)[12],外壳蛋白(coat protein,CP)和核酸结合蛋白(nucleic acid-binding protein,NABP),其中核酸结合蛋白和外壳蛋白有重叠[13]
近年来,一种疑似病毒引起的叶片褪绿、花叶、卷叶症状在北京丁香上普遍发生。本研究采用小RNA高通量测序、逆转录聚合酶链反应(RT-PCR)技术明确造成该症状的病原物。本研究旨在明确造成北京丁香褪绿花叶卷叶病的病原物,克隆病原物基因组全长序列,明确病原物的基因组全序列及进化关系,为遗传变异提供理论依据。

1 材料与方法

1.1 试验材料

供试样品:对北京市海淀区丁香病情进行调查,采用随机取样法,对表现症状为褪绿、花叶、卷叶的疑似丁香病毒病的叶片进行采集。将同一植株的叶片取下放入干净密封袋中,储存在-80℃冰箱中,以备后续试验使用。试验在北京农学院生物与资源环境学院实验室,于2021年5月—2022年5月进行。
试验试剂:HiPure Plant RNA Mini Kit植物RNA小量提取试剂盒(美基生物科技有限公司);5×Reverse Transcriptase M-MLV buffer、dNTP Mixture、Random Primer、RRL、M-MLV(以上试剂均采购于TAKARA)、琼脂糖凝胶DNA回收试剂盒(天根生化科技有限公司)。
主要仪器:Retsch MM400型研磨仪、eppendorf minispin plus型低温离心机、eppendorf Centrifuge 5417R型低温离心机、BIO-RADS100TM Thermal Cycler PC仪、DYY-8C型电泳仪、BIO-RAD GelDosTM EZ IMAGER凝胶成像仪。

1.2 方法

1.2.1 小RNA高通量测序及分析

为对丁香样品的病毒种类进行鉴定,随机取具有明显症状的样品委托华大基因公司进行小RNA测序。利用Perl语言脚本对测序原始序列进行质控,剔除低质量数据,得到Clean数据,再从有效数据中筛选18~28 nt的小RNA序列。使用Velvet 1.2.10软件根据有效数据中的重叠区域将小RNA序列拼接为长片段[14]。将组装结果序列经NCBI Blast网站(https://www.ncbi.nlm.nih.gov/)同病毒核酸数据库比对,鉴定测序样品中病毒的种类。

1.2.2 RT-PCR验证

为验证小RNA高通量测序结果,根据测序结果设计目的片段为250~350 bp的特异性检测引物对LVA-F/R(表1),委托生工生物工程(上海)股份有限公司合成。
表1 本研究中所用引物
引物名称 序列(5′-3′) 引物位置 产物大小/bp
LVA-NXY-F ATGTCGAATCCACCGGAAGG 7232~7252 290
LVA-NXY-R GATGCCCCAAAGGATGTCAG 7502~7522
LVA-HLG-F GAGTAACCAGCCTCCGCCTC 7281~7300 310
LVA-HLG-R GACCCTCCAACTTGACCGC 7573~7591
LVA-NXY-F1 CCATTCACATCCGGCATG 282~300 3169
LVA-NXY-R1 GCTGCCGTCCAACAAACAC 3431~3451
LVA-NXY-F2 TTTGTTGGACGGCAGCAC 3434~3452 3081
LVA-NXY-R2 CACAGCAATTCCCCAACCTC 6495~6515
LVA-NXY-F3 TGGGGAACTGCTGTGCGCTC 6500~6520 1748
LVA-NXY-R3 CATACGAGGAAGTCCCGCCCC 8227~8248
LVA-HLG-F1 GCAATGCCGCACTCTCATCC 281~301 3498
LVA-HLG-R1 GCTGACCTCTGACCCCTCGAAG 3195~3217
LVA-HLG-F2 GAGGGGTCAGAGGTCAGCGTG 3199~3220 3067
LVA-HLG-R2 GCGCACAATGTGTACTCGTCGAC 6243~6266
LVA-HLG-F3 CATTGTGCGCTCGGGATTTAC 6251~6276 1979
LVA-HLG-R3 GATACACTCTATAGCAGCGATGGC 8278~8230
NXY-5RACE-out GGTGGATAGACAAGTGTAGCAAG 679~701 701
NXY-5RACE-in CAAGGTCTCTCAAAGTAGAATCTGC 541~565 565
NXY-3RACE-out GTCAAAGAACTTGCAGGTGTGAG 7741~7763 748
NXY-3RACE-in CCGCTATGGGTTTCAACTACAAC 7838~7860 651
HLG-5RACE-out GGATCTCATCATGCAAGAACACG 594~616 616
HLG-5RACE-in CCTGGATTTATCTGCACTCGTGAC 445~468 468
HLG-3RACE-out GTTCGTCTGTTATTGCGCTAGTGC 7632~7655 847
HLG-3RACE-in CAACTACAATACTCGGTTCGCAG 7848~7870 631
使用HiPure Plant RNA Mini Kit植物RNA小量提取试剂盒提取RNA,以此为模板合成cDNA。反应体系为:ddH2O 14.5 μL、5× Reverse Transcriptase M-MLV buffer 5 μL、dNTP Mixture 1 μL、Random Primer 1 μL、RRL 0.5 μL、M-MLV 1 μL、样品总RNA 2 μL。将上述反应体系轻轻摇晃均匀后放在42℃水浴锅中反应1 h后迅速转移至冰上。合成的cDNA保存于-20℃冰箱中。
以合成的cDNA为模板进行PCR扩增。PCR反应体系为25 μL:正向引物、反向引物各0.5 μL;2×Taq Mix 12.5 μL;反转录产物2.0 μL;ddH2O补足至25 μL。反应条件为:94℃预变性5 min;94℃变性30 s,退火温度参考引物Tm值,退火时间30 s,72℃延伸1 min/kb,完成35个扩增循环;于72℃条件下延伸10 min。PCR产物保存在4℃冰箱中。
取反应结束后的全部产物进行电泳检测,对目的条带进行回收纯化。委托生工生物工程(上海)股份有限公司测序,测序结果利用DNAMAN进行序列拼接与比对。

1.2.3 LVA北京丁香分离物的基因组全长测序

为对北京丁香分离物进行全序列克隆,根据小RNA高通量测序并组装的contigs分段设计基因组扩增引物对LVA-F1/R1、LVA-F2/R2、LVA-F3/R3(表1)。按照1.2.2方法,对丁香分离物的基因组进行RT-PCR扩增。根据候选序列设计5′和3′端RACE扩增引物(表1),对候选病毒基因组5′和3′末端序列进行扩增。扩增反应方法参照试剂盒说明书、体系和程序参照1.2.2。

1.2.4 LVA北京丁香分离物基因序列分析

为明确北京丁香分离物基因组的分子特征,使用DANMAN进行序列拼接后整理出北京丁香分离物完整序列信息。利用NCBI ORF Finder网站进行基因组结构分析,预测全序列的开放阅读框。从NCBI数据库中下载已报道的5个LVA分离物基因组序列,使用Sequence Demarcation Tool-version 1.3(SDT v1.3)软件计算核苷酸及氨基酸序列一致率[15]。使用Recombination Detection Program(RDP)v.4.101软件进行重组分析,分子类型选择线性,选用RDP、GENECONV、BootScan、MaxChi、Chimaera、SiScan和3Seq共7种程序进行重组检测,至少4个程序显示P值显著(P<1.0×10-6),判定该重组事件显著[16]

1.2.5 LVA北京丁香分离物系统发育分析

为明确北京丁香分离物的系统发育地位,使用MEGA7在NCBI数据库检索到的香石竹潜隐病毒属的病毒基因组序列、已报道的5个LVA分离物与本试验获得的基因序列一起进行多重比对[17]。采用邻近法(Neighbor Joining)基于基因组全长序列系统发育树,自展值设置为1000,分析该基因序列与相关基因之间的遗传关系。

2 结果与分析

2.1 小RNA高通量测序结果

通过对待测样品进行小RNA高通量测序,剔除低质量数据,将有效数据中的重叠区域将小RNA序列拼接为长片段,获得两段候选基因序列。初步鉴定两段候选基因序列均匹配为水蜡A病毒。

2.2 LVA北京丁香分离物的RT-PCR验证结果

为验证小RNA深度测序鉴定病毒种类的准确性,利用引物LVA-NXY-F/R、LVA-HLG-F/R对采集有明显症状的样品分别进行RT-PCR检测,获得大小约为290 bp、310 bp的条带。条带大小符合预期,且在健康植株中未检测到条带。结果表明,小RNA测序对病毒种类的鉴定正确。

2.3 LVA北京丁香分离物的基因序列分析

通过RT-PCR技术分别使用特异性引物LVA-NXY-F1/2/3、LVA-NXY-R1/2/3,扩增获得3个目标片段,再通过RACE技术获得末端序列。经拼接后得到北京丁香分离物基因组的全长序列,命名为LVA-NXY,提交至GenBank数据库,登录号为OM561079。
LVA-NXY基因组全长8488 nt,共编码6个ORF阅读框。ORF1位于nt 76~5982,编码1968 aa的RNA依赖性RNA聚合酶(RdRp);ORF2位于nt 6006~6698,编码230 aa的TGB1;ORF3位于nt 6673~6996,编码107 aa的TGB2;ORF4位于nt 6997~7179,编码60 aa的TGB3;ORF2-4共同组成三联体模块蛋白;ORF5位于nt 7222~8106,编码294 aa的外壳蛋白(CP);ORF6位于nt 8106~8465,编码119 aa的核酸结合蛋白(NABP)(图1)。
图1 水蜡A病毒北京丁香分离物基因组结构

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使用同样方法得到另外一条病毒分离物的全长序列,命名为LVA-HLG,将序列提交至GenBank数据库,登录号为OM671312。
LVA-HLG基因组全长8478 nt,与LVA-NXY分离物基因组结构相似。其中ORF1位于nt 73~5979;ORF2位于nt 6003~6692;ORF3位于nt 6670~6993;ORF4位于nt 6994~7176;ORF5位于nt 7219~8103;ORF6位于nt 8104~8462。
LVA-NXY基因组序列与LVA-DBLQ[中国:沈阳:东北连翘](MN786957.1)基因组序列一致率较高为95.75%,与LVA-SNU[韩国:暴马丁香](OL441191.1)基因组序列差异较大,一致率仅为73.12%(图2)LVA-NXY基因组序列与沈阳东北连翘分离物各OFR核苷酸序列一致率为93.95%~96.84%,氨基酸序列一致率为95.22%~98.98%(表2)。与韩国暴马丁香各OFR核苷酸序列一致率为64.51%~76.19%,氨基酸序列一致率为60.61%~86.39%(表2)。
图2 LVA相关病毒分离物全基因组序列对比热图

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表2 LVA-NXY与其他LVA分离物序列一致性对比(核苷酸/氨基酸)
分离物 LVA-DBLQ LVA-DX LVA-Sob LVA-SK LVA-SNU
开放阅读框1 ORF1 95.41/98.07 81.33/93.24 72.94/81.91 72.74/81.81 72.24/82.33
开放阅读框2 ORF2 93.95/95.22 82.28/89.57 70.19/70.74 69.03/69.87 64.51/60.61
开放阅读框3 ORF3 94.77/95.33 83.08/92.52 74.15/85.98 73.23/85.98 72.92/84.11
开放阅读框4 ORF4 95.11/96.67 90.76/93.33 75.54/73.33 74.46/73.33 75.00/76.67
开放阅读框5 ORF5 96.84/98.98 86.12/96.26 73.70/85.37 73.93/85.03 74.94/86.39
开放阅读框6 ORF6 97.51/98.32 91.41/95.80 79.50/83.19 78.67/83.19 76.19/77.97
LVA-HLG基因组序列同样与LVA-DBLQ基因组序列一致率较高为76.96%,与LVA-SNU基因组序列差异较大,一致率为73.61%(图2)。LVA-HLG与LVA-DBLQ各OFR阅读框核苷酸序列一致率为70.89%~85.60%,氨基酸序列一致率为73.91%~91.50%(表3)。与LVA-SNU各OFR核苷酸序列一致率为65.95%~77.03%,氨基酸序列一致率为63.20%~86.39%(表3)。
表3 LVA-HLG与其他LVA分离物序列一致性对比(核苷酸/氨基酸)
分离物 LVA- DBLQ LVA-DX LVA-Sob LVA-SK LVA-SNU
开放阅读框1 ORF1 75.68/86.74 74.73/86.33 71.85/81.05 71.60/81.00 71.53/80.95
开放阅读框2 ORF2 70.89/73.91 72.19/77.39 69.61/71.18 69.75/72.05 65.95/63.20
开放阅读框3 ORF3 76.62/89.72 76.62/89.72 71.38/85.05 70.77/85.05 71.08/84.11
开放阅读框4 ORF4 76.09/81.67 77.17/86.67 70.65/75.00 70.65/75.00 69.57/73.33
开放阅读框5 ORF5 77.65/91.50 77.54/91.84 71.11/85.71 72.23/85.37 73.48/86.39
开放阅读框6 ORF6 85.60/90.76 84.49/90.76 81.16/83.19 80.06/83.19 77.03/79.66
重组分析结果显示,本试验所获得的两个LVA分离物和已报道的5个LVA分离物中未检测到重组事件。

2.4 LVA北京丁香分离物系统发育分析

基于LVA-NXY分离物和LVA-HLG分离物的基因组序列构建系统发育树(图3),以亚洲李病毒1(Asian prunus virus 1,APV1)为外源病毒。根据发育树图发现,北京丁香分离物LVA-NXY、LVA-HLG与LVA-DBLQ、LVA-DX[中国:沈阳:丁香](MG865890.1)共聚同一分支中,LVA-HLG独立分支,LVA-NXY与LVA-DBLQ亲缘关系最近,说明该病毒可能来源于沈阳且有一定的变异。
图3 分离物及水蜡A病毒与其同属病毒的系统发育树

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3 讨论

丁香是木犀科丁香属的小乔木,不易受到病毒的侵染,因此侵染丁香的报道较少。目前用于检测和鉴定病毒种类的方法很多,主要包括生物学检测[18]、电镜观察检测[19]、血清学检测和分子生物学检测等[20-21]。新兴的小RNA深度测序技术可以发掘新的病毒种类,已被广泛应用于病毒的鉴定[22]。RNA干扰(RNA interference,RNAi)是指在生物体中由双链RNA诱导发生的基因沉默现象[23],小RNA深度测序则是应用了该现象。由于植物具有天然的抗病毒机制,当病毒侵染宿主植物时,病毒基因组会被释放到植物细胞内。宿主植物通过RNA干扰(RNAi)机制,靶向降解病毒核酸,形成大量长度为20~25 nt的病毒衍生小干扰RNA,从而使植物自身获得病毒免疫[24]。小RNA深度测序可以获得植物体内病毒衍生的小RNA序列,通过生物信息学软件进行重叠组装,获得侵染植物的病毒序列[25]
在对北京地区丁香进行病害调查时,发现多株丁香叶片出现褪绿、花叶、卷叶的症状,疑似感染病毒病。本试验采用小RNA深度测序技术对疑似感染病毒的丁香进行检测并明确病毒类型。结果表明,本试验获得了两条北京丁香分离物LVA-NXY、LVA-HLG的全基因序列,预测该序列的基因组结构特征与香石竹浅隐性病毒属的病毒相似。根据ICTV网站上乙型线状病毒科、香石竹浅隐性病毒属的物种分类标准,当cp基因的核苷酸序列同一性小于72%或氨基酸序列同一性小于80%时,可认定为香石竹浅隐性病毒属的新物种。LVA-NXY、LVA-HLG的氨基酸序列同一性均不符合分类标准,因此初步判断LVA-NXY、LVA-HLG是与LVA-DBLQ的相似度较高的分离物。经构建系统发育树后发现LVA-NXY、LVA-HLG可能来自沈阳。本研究是首次在北京丁香上分离出完整LVA序列。
本研究尚未进行科赫氏法则验证,后续需要通过人工接种健康丁香植株进行致病性检验,并通过构建LVA侵染性克隆进一步了解其致病机制。另外,LVA主要通过蚜虫以非持久性方式传播[7],目前尚不明确该病毒在丁香上的传播介体,因此,研究其传播方式将是下一步的重点工作。

4 结论

本研究发现引起北京丁香叶片褪绿花叶卷叶的病毒为LVA,并获得了两个北京丁香分离物的全长序列。经序列对比后发现LVA-NXY、LVA-HLG与中国沈阳东北连翘分离物LVA-DBLQ核苷酸同源性及氨基酸同源性最高。经构建系统发育树后发现,LVA-NXY、LVA-HLG与LVA-DBLQ、LVA-DX共聚同一分支中,且与LVA-DBLQ亲缘关系最近,与序列对比结果一致,表明LVA-NXY、LVA-HLG可能来自沈阳。综上所述,本研究证实了侵染北京丁香的病毒种类及其基因组分子特征,明确了病原物的基因组全序列及进化关系。本研究首次在北京丁香上发现了LVA,为丁香病毒病的研究和防控提供了新依据。

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