1、第 46 卷第 1 期2023 年 1 月河 北 农 业 大 学 学 报JOURNAL OF HEBEI AGRICULTURAL UNIVERSITYVol.46 No.1Jan.2 0 2 3河北苹果坏死花叶病毒的发生及近全基因组 序列的测定与分析董震杨,李紫腾,胡同乐,王树桐,王亚南,曹克强(河北农业大学 植物保护学院,河北 保定 071001)摘要:为明确苹果坏死花叶病毒(Apple necrotic mosaic virus,ApNMV)在河北的发生及其基因组特性,本试验利用 RT-PCR 技术对河北保定两地 ApNMV 发病情况进行检测,然后通过高通量测序技术对带毒样本进行序列测定
2、和分析。结果表明,河北农大果园红珍珠锦锈红信侬红3 个品种及保定唐县苹果实验基地富士品种 ApNMV 检出率分别为 9.09%、18.18%、100%和 65%。ApNMV 阳性果树高通量测序结果显示,果树为 ApNMV、苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus,ACLSV)、苹果茎沟病毒(Apple stem grooving virus,ASGV)和苹果茎痘病毒(Apple stem pitting virus,ASPV)复合侵染,ApNMV 河北分离物(ApNMV-HB)的近全基因组序列包含 RNA1 RNA3 序列,GenBank 登录号分别为
3、OP019626、OP019627和 OP019628。RNA1 编码 120 kD 的甲基转移酶/NTP-binding 解旋酶(MET/HEL),RNA2 编码 97 kD 的RNA 聚合酶(POL),RNA3 编码运动蛋白(Movement protein,MP)和外壳蛋白(Coat protein,CP),4 个基因核苷酸和氨基酸序列与代表性分离物相似性分别为 89.44%97.02%、92.14%98.67%。系统发育关系分析表明,以 ApNMV RNA1、RNA2、RNA3(CP 和 MP)编码的氨基酸为基础构建系统发育树,ApNMV-HB分别与 AM95、AM125、XJ、ZZ
4、亲缘关系最近。本研究首次揭示了 ApNMV 河北分离物的近全基因组序列,丰富了该病毒的基因组序列信息,明确了该分离物与其它分离物的系统发育关系,为进一步了解该病毒的系统发育及遗传进化提供参考。关 键 词:苹果坏死花叶病毒;高通量测序;基因组特性;系统进化中图分类号:S432.1 开放科学(资源服务)标识码(OSID):文献标志码:AOccurrence of apple necrotic mosaic virus in Hebei province and sequence analysis of its whole genome DONGZhenyang,LIZiteng,HUTongle,
5、WANGShutong,WANGYanan,CAOKeqiang(College of Plant Protection,Hebei Agricultural University,Baoding 071001,China)Abstract:In order to clarify the occurrence and genomic characteristics of apple necrotic mosaic virus(ApNMV)in Hebei,the incidence of ApNMV in two regions of Baoding was detected by RT-PC
6、R,and then the infected sample was sequenced by high-throughput sequencing technology.The results showed that the positive rates of ApNMV were 9.09%,18.18%,100%and 65%in the cultivars of Hongzhenzhu,Jinxiuhong and Xinnonghong in the 收稿日期:2022-09-13基金项目:河北省自然科学基金(C2022204196);河北省重点研发计划(21326506D);国家现
7、代农业(苹果)产业技术体系(CARS-27).第一作者:董震杨(1997),男,河北邢台人,硕士研究生,主要从事植物病毒学相关研究.E-mail:通信作者:王亚南(1979),女,河北三河人,博士,教授,主要从事植物病毒学相关研究.E-mail:本刊网址:http:/文章编号:1000-1573(2023)01-0072-08DOI:10.13320/ki.jauh.2023.001173第 1 期Apple Experimental Garden of Agricultural University of Hebei,and Fuji in the the Apple Experimenta
8、l Site of Tangxian County,Baoding.High-throughput sequencing revealed that the apple trees infected by ApNMV were also infected by apple chlorotic leaf spot virus(ACLSV),apple stem grooving virus(ASGV)and apple stem pitting virus(ASPV).The near-complete genome sequence of ApNMV was obtained from the
9、 sequencing results.The RNA1RNA3 GenBank accession numbers were OP019626,OP019627 and OP019628,respectively.The isolate was named ApNMV Hebei isolate(ApNMV-HB).RNA1 encoded a 120 kD methyltransferase/NTP-binding helicase(MET/HEL).RNA2 encoded a 97 kD RNA polymerase(POL).And RNA3 encoded movement pro
10、tein(MP)and coat protein(CP).The four genes shared sequence similarity with the representative isolates in 89.44%97.02%of the nucleotide and 92.14%98.67%of the amino acid.The phylogenetic analysis showed that all representative isolates were clustered in one branch based on the amino acids of ApNMV
11、RNA1,and ApNMV-HB was closest to AM95.All representative isolates were clustered in one branch,except for AM72,based on the amino acids encoded by ApNMV RNA2.ApNMV-HB was closest related to AM125.In the phylogenetic tree based on ApNMV CP,the representative isolates clustered in one branch rooted wi
12、th AM72,and ApNMV-HB and XJ were relatively close.In the phylogenetic tree based on ApNMV MP,all isolates were divided into 4 branches.ApNMV-HB was clustered with AM75,ApNMV-Qu,AM125 and ZZ in one branch,and was closest to ZZ.We revealed the near-complete genome sequence of the ApNMV Hebei isolate f
13、or the first time,which enriches the genome sequence information of the virus.Clarification of the phylogenetic relationship between the isolate and other isolates provides reference for further understanding of the phylogeny and genetic evolution of the virus.Keywords:apple necrotic mosaic virus;hi
14、gh-throughput sequencing;genomic characteristics;phylogenesis起,ApNMV 侵染果树后叶片会产生淡黄色不规则斑点、沿着叶脉发展后形成网状外观和叶脉之间的大型无定形失绿区域等症状,严重时会导致果树衰弱,甚至死亡11。据报道,ApNMV 也可从具有花叶症状的海棠(Malus spp.)叶片中分离出来12。ApNMV 属于雀麦花叶病毒科(Bromoviridae)、等轴不稳环斑病毒属(Ilarvirus)13,基因组由RNA 1(3 378 nt)、RNA 2(2 767 nt)和 RNA 3(1 956 nt)组成,其中的 RNA1 编
15、码甲基转移酶/NTP-binding 解旋酶(MET/HEL),蛋白质中存在分子内和分子间的互作,而 N 端部分在互作中起着关键作用。RNA2 可以编码 RNA 聚合酶(POL),包括高度保守的甘氨酸-天冬氨酸-天冬氨酸(GDD)序列,编码的 POL 与病毒的复制能力及致病性密切相关。RNA3 是双顺反子,编码 5 端的运动蛋白(Movement protein,MP)和 3 端的外壳蛋白(Coat protein,CP),后者由源于 RNA3 的亚基因组 RNA4编码10,14-16。邢飞17利用高通量测序技术对典型花叶症状的叶片进行了病毒种类的鉴定,并对其中 6株 ApNMV 分离物的全基
16、因组序列进行了测定和分析,结果表明,所有的样品中均未检出 ApMV,而ApNMV 检出率则高达 92%,ApNMV 与李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)的系统发育关系较近,但与 ApMV 有明显区别14。李正南等18用 RT-PCR 的方法检测 29 份内蒙古我国是苹果种植大国,苹果产业对农民收入的增加具有重要作用1。苹果病毒病在世界各苹果种植地广泛发生,严重影响苹果生产。在我国苹果产区,张力支等人2报道渤海湾产区、西北高原产区、西南高地产区、黄河故道产区栽培的果树带毒率为 100%,且苹果树受到多种病毒复合侵染的情况比较严重。郝璐等人3调查发现,陕西地区苹果病毒复合侵染率是 92.1%,山东、辽宁、山西分别为67.1%、88.2%、75%。果树感染病毒后终身带毒,严重影响果树的正常生长,降低果树产量和果实品质4-6,病毒病是限制苹果生产的最重要因素7。在已发现的 39 种苹果病毒中 25 种是非潜隐性病毒,其余为潜隐性病毒8。我国发现且鉴定的主要病毒有苹果褪绿叶斑病毒(Apple chlorotic leaf spot vir