番茄褐色皱纹果病毒分子检测与基因组部分序列分析

doi:10.7668/hbnxb.20194858

摘要/Abstract

摘要：

为了确定张家口及银川番茄产区是否发生番茄褐色皱纹果病毒病(ToBRFV),探究番茄ToBRFV的遗传信息和演化,为番茄褐色皱纹果病毒的诊断和防治及番茄抗病毒病基因工程提供重要的科学依据,采用张家口和银川两地区疑似病果进行分子检测,并利用相关分子生物学软件对该病毒基因进行序列分析及全基因组的系统进化分析。结果表明,张家口及银川果实病毒分离物与GenBank中大部分ToBRFV分离物的基因组结构相似性在99%以上,确定两地区番茄果实病毒为番茄褐色皱纹果病毒。ToBRFV分离物的地域性很强,中国不同地区的ToBRFV病毒,与来自欧洲和亚洲的多个国家地区都有关联,张家口分离物与中国分离物(MT018320.1)亲缘关系最近,银川分离物与秘鲁分离物聚类到一起,说明银川分离物可能来自南美洲。张家口分离物与中国分离物(MT018320.1)的4个ORF氨基酸相似性最高,而银川分离物与张家口分离物相似性较低。另外,研究结果首次发现,银川分离物CP蛋白的第444位碱基由A突变成G,从而导致CP蛋白的氨基酸第131位由V(缬氨酸)突变为A(丙氨酸),CP蛋白发生了有义突变。综上所述,张家口及银川番茄产区发生了番茄褐色皱纹果病毒病,且两地区的病毒来自不同的地方。

关键词: 番茄, 番茄褐色皱纹果病毒, 分子检测, 序列分析

Abstract:

In order to determine whether Tomato brown rugose fruit virus(ToBRFV)occurs in Zhangjiakou and Yinchuan tomato producing areas,and to explore the genetic information and evolution of ToBRFV,so as to provide an important scientific basis for the diagnosis and control of ToBRFV and the genetic engineering of tomato resistance to viral diseases.Molecular detection was carried out in Zhangjiakou and Yinchuan suspected diseased fruits,and sequence analysis and genome-wide phylogenetic analysis of the virus gene were carried out by relevant molecular biology software.The results showed that the genome structure similarity between the virus isolates from Zhangjiakou and Yinchuan fruit was more than 99% similar to most of the ToBRFV isolates in GenBank,and the tomato fruit virus in the two places was determined to be ToBRFV.ToBRFV isolates were highly regional,and ToBRFV viruses from different regions of China were associated with many countries and regions from Europe and Asia,and the Zhangjiakou isolate was most closely related to the Chinese isolate(MT018320.1),and the Yinchuan isolate was clustered with the Peruvian isolate,indicating that the Yinchuan isolate may have originated from South America.The similarity of the four ORF amino acids of the Zhangjiakou isolate from China(MT018320.1)was the highest,while the similarity between the Yinchuan isolate and the Zhangjiakou isolate was low.In addition,this study found for the first time that the 444th base of the Yinchuan isolate CP protein changed from A to G,resulting in a meaningful mutation of the 131st amino acid of the CP protein from V(valine)to A(alanine).In summary,ToBRFV disease occurred in Zhangjiakou and Yinchuan tomato producing areas,and the virus came from different places in the two regions.

Key words: Tomato, Tomato brown rugose fruit virus, Molecular detection, Sequence analysis

华明艳, 宋兰芳, 崔少杰, 孙海波, 金凤媚. 番茄褐色皱纹果病毒分子检测与基因组部分序列分析[J]. 华北农学报, 2024, 39(3): 179-186. doi: 10.7668/hbnxb.20194858.

HUA Mingyan, SONG Lanfang, CUI Shaojie, SUN Haibo, JIN Fengmei. Molecular Detection and Genome Sequence Analysis of Tomato brown rugose fruit virus[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 179-186. doi: 10.7668/hbnxb.20194858.

http://www.hbnxb.net/CN/Y2024/V39/I3/179

图/表 7

参考文献 24

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扩增片段 Amplified fragment	引物名称 Primer name	引物序列 (5'—3') Primer sequence (5'—3')	预期扩增片段大小/bp Expected product size
ToBRFV-A	ToBRFV-AF	CATATACCAACAACAACAAA	1 630
	ToBRFV-AR	TCAAGCACTGGCATATCCAC
ToBRFV-B	ToBRFV-BF	AGATTTCCCTGGCTTTTGGA	2 200
	ToBRFV-BR	AGATTTCCCTGGCTTTTGGA
ToBRFV-C	ToBRFV-CF	AAGTGTCTCCCAGGTAATAGC	1 460
	ToBRFV-CR	AACAGGTGTGAACAGACGG
ToBRFV-D	ToBRFV-DF	AGGTACGTGATACATCATGA	1 761
	ToBRFV-DR	GGCCCCTACCGGGGGTTCCGGG

扩增片段 Amplified fragment	引物名称 Primer name	引物序列 (5'—3') Primer sequence (5'—3')	预期扩增片段大小/bp Expected product size
ToBRFV-A	ToBRFV-AF	CATATACCAACAACAACAAA	1 630
	ToBRFV-AR	TCAAGCACTGGCATATCCAC
ToBRFV-B	ToBRFV-BF	AGATTTCCCTGGCTTTTGGA	2 200
	ToBRFV-BR	AGATTTCCCTGGCTTTTGGA
ToBRFV-C	ToBRFV-CF	AAGTGTCTCCCAGGTAATAGC	1 460
	ToBRFV-CR	AACAGGTGTGAACAGACGG
ToBRFV-D	ToBRFV-DF	AGGTACGTGATACATCATGA	1 761
	ToBRFV-DR	GGCCCCTACCGGGGGTTCCGGG

序号 Serial number	分离物代号 Isolate code	来源地 Source	登录号 Accession No.	基因组大小/bp Genome size
1	GBVC_ToBRFV_02	比利时	MZ945420.1	6 379
2	GBVC_ToBRFV_01	比利时	MZ945419.1	6 391
3	ToBRFV-Gr2	希腊	OQ190155.1	6 405
4	ToBRFV-CH	瑞士	OM305070.1	6 386
5	ToBRFV-SD	中国	MT018320.1	6 392
6	41903150	荷兰	OM515272.1	6 372
7	41106995	以色列	OM515266.1	6 364
8	4110842	秘鲁	OM515258.1	6 376
9	41108448	以色列	OM515257.1	6 367
10	40732089_3	秘鲁	OM515235.1	6 335
11	2020015323_B	英国	OM515232.1	6 373
12	36783860_1	中国	MW314114.1	6 390
13	36689794_1	约旦	MW314110.1	6 388
14	33314743	埃及	MW314094.1	6 390
15	33314313_B	埃及	MW314093.1	6 379
16	6189975_2	中国	MW314091.1	6 367
17	41106813	中国	OM515256.1	6 361
18	2020015323_A	英国	OM515231.1	6 373
19	6165965	比利时	OM515265.1	6 375
20	39474756	比利时	OM515261.1	6 376
21	36689655	约旦	MW314107.1	6 376
22	36783571_2	秘鲁	MW314111.1	6 376
23	38950951	荷兰韦斯特兰	OM515248.1	6 366
24	39986364	荷兰克鲁恩	OM515246.1	6 374
25	Zhangjiakou	中国张家口	OR500699	6 376
26	Yinchuan	中国银川	OR500698	6 376

序号 Serial number	分离物代号 Isolate code	来源地 Source	登录号 Accession No.	基因组大小/bp Genome size
1	GBVC_ToBRFV_02	比利时	MZ945420.1	6 379
2	GBVC_ToBRFV_01	比利时	MZ945419.1	6 391
3	ToBRFV-Gr2	希腊	OQ190155.1	6 405
4	ToBRFV-CH	瑞士	OM305070.1	6 386
5	ToBRFV-SD	中国	MT018320.1	6 392
6	41903150	荷兰	OM515272.1	6 372
7	41106995	以色列	OM515266.1	6 364
8	4110842	秘鲁	OM515258.1	6 376
9	41108448	以色列	OM515257.1	6 367
10	40732089_3	秘鲁	OM515235.1	6 335
11	2020015323_B	英国	OM515232.1	6 373
12	36783860_1	中国	MW314114.1	6 390
13	36689794_1	约旦	MW314110.1	6 388
14	33314743	埃及	MW314094.1	6 390
15	33314313_B	埃及	MW314093.1	6 379
16	6189975_2	中国	MW314091.1	6 367
17	41106813	中国	OM515256.1	6 361
18	2020015323_A	英国	OM515231.1	6 373
19	6165965	比利时	OM515265.1	6 375
20	39474756	比利时	OM515261.1	6 376
21	36689655	约旦	MW314107.1	6 376
22	36783571_2	秘鲁	MW314111.1	6 376
23	38950951	荷兰韦斯特兰	OM515248.1	6 366
24	39986364	荷兰克鲁恩	OM515246.1	6 374
25	Zhangjiakou	中国张家口	OR500699	6 376
26	Yinchuan	中国银川	OR500698	6 376

分离物代号 Isolate code	来源地 Source	登录号 Accession No.	Genome	RDRP		RDRPSRS		MP		CP
分离物代号 Isolate code	来源地 Source	登录号 Accession No.	NS	NS	AAS	NS	AAS	NS	AAS	NS	AAS
GBVC_ToBRFV_02	比利时	MZ945420.1	99.4	99.6	99.7	99.6	99.6	99.2	99.2	99.6	100.0
GBVC_ToBRFV_01	比利时	MZ945419.1	99.3	99.6	99.7	99.6	99.6	99.1	99.2	99.6	100.0
ToBRFV-Gr2	希腊	OQ190155.1	99.0	99.4	99.4	99.5	99.6	99.2	98.9	100.0	100.0
ToBRFV-CH	瑞士	OM305070.1	99.5	99.6	99.7	99.6	99.7	99.6	99.2	99.8	100.0
ToBRFV-SD	中国	MT018320.1	99.5	99.8	99.9	99.8	99.8	99.9	99.6	99.8	100.0
41903150	荷兰	OM515272.1	99.4	99.5	99.6	99.5	99.5	99.3	98.5	100.0	100.0
41106995	以色列	OM515266.1	99.5	99.7	99.8	99.7	99.7	99.6	99.6	100.0	100.0
4110842	秘鲁	OM515258.1	99.6	99.6	99.8	99.6	99.7	99.3	99.2	100.0	100.0
41108448	以色列	OM515257.1	99.5	99.7	99.8	99.7	99.7	99.6	99.6	100.0	100.0
40732089_3	秘鲁	OM515235.1	98.9	99.6	99.6	99.6	99.5	99.2	99.2	100.0	100.0
2020015323_B	英国	OM515232.1	99.6	99.6	99.8	99.6	99.6	99.6	99.2	100.0	100.0
36783860_1	中国	MW314114.1	99.4	99.7	99.8	99.7	99.6	99.4	98.9	100.0	100.0
36689794_1	约旦	MW314110.1	99.5	99.7	99.8	99.7	99.6	99.4	98.9	100.0	100.0
33314743	埃及	MW314094.1	99.4	99.5	99.6	99.5	99.6	99.7	99.6	100.0	100.0
33314313_B	埃及	MW314093.1	99.6	99.6	99.6	99.6	99.6	99.7	99.6	100.0	100.0
6189975_2	中国	MW314091.1	99.6	99.7	99.8	99.7	99.6	99.3	98.9	99.8	100.0
41106813	中国	OM515256.1	99.4	99.6	99.8	99.7	99.7	99.7	99.6	100.0	100.0
2020015323_A	英国	OM515231.1	99.6	99.7	99.8	99.7	99.6	99.6	99.2	100.0	100.0
6165965	比利时	OM515265.1	99.5	99.5	99.6	99.6	99.6	99.4	98.9	100.0	100.0
39474756	比利时	OM515261.1	99.6	99.6	99.7	99.6	99.6	99.3	99.6	99.8	100.0
36689655	约旦	MW314107.1	99.6	99.7	99.6	99.6	99.5	99.4	98.9	100.0	100.0
36783571_2	秘鲁	MW314111.1	99.5	99.5	99.7	99.5	99.6	99.3	99.2	100.0	100.0
38950951	荷兰韦斯特兰	OM515248.1	99.4	99.6	99.7	99.6	99.6	99.2	98.5	100.0	100.0
39986364	荷兰克鲁恩	OM515246.1	99.5	99.5	99.6	99.6	99.6	99.4	98.9	100.0	100.0
Yinchuan	中国银川	OR500698	99.4	99.4	99.6	99.5	99.6	99.3	99.2	99.4	99.4
平均相似性 Average similarity			99.45	99.60	99.70	99.62	99.62	99.43	99.17	99.91	99.98

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