Complete Genomic Sequence Characteristics of Tomato chlorosis virus Shijiazhuang Isolate and Establishment of qPCR Detection Technique

doi:10.7668/hbnxb.20196175

Abstract

Abstract:

In order to identify the pathogenic source of Tomato chlorosis virus disease and clarify the taxonomic status of Tomato chlorosis virus (ToCV)in the Shijiazhuang area of Hebei Province,an analysis of the phylogenetic relationships of diseased samples collected from the Tomato Planting Base in Dahe Town,Shijiazhuang,was conducted.Additionally,an efficient method for ToCV identification was established.The published ToCV sequences in NCBI database were selected for similarity analysis,and the full-length RNA1 and RNA2 sequences of ToCV,Heat-shock protein 70 homologue(Hsp70h)and coat protein(CP)sequences were used to construct the phylogenetic tree.The results showed that the nucleotide(nt)sequences of RNA1 and RNA2 of the ToCV-SJZ shared 85.2% and 92.4% similarity,respectively,with the corresponding sequences of the ToCV-XS Taiwan strain,indicating the closest genetic relationship.The evolutionary analysis showed that ToCV could be divided into two clades,one represented by the Florida strain and the other by the Taiwan strain.According to the CP gene sequences of ToCV-SJZ,ToCV-XS and ToCV-Florida strains,a RT-qPCR assay system based on SYBR Green Ⅰ was established.In conclusion,this study clarifies the taxonomic status of the ToCV-SJZ isolate and provides technical support for the identification of disease-resistant germplasm resources and scientific prevention and control.

Key words: Tomato chlorosis virus(ToCV), Sequence analysis, Phylogenetic analysis, SYBR Green Ⅰ Real-time PCR

CLC Number:

KANG Chen, WANG Peng, GAO Kang, TIAN Zhejuan, HAO Lingyu, LI Yadong, WU Zhiming. Complete Genomic Sequence Characteristics of Tomato chlorosis virus Shijiazhuang Isolate and Establishment of qPCR Detection Technique[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(3): 185-197. doi: 10.7668/hbnxb.20196175.

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References 39

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引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	基因组位置 Location	产物大小/bp Product size	用途 Function
ToCV-CP-491-F	ACACTCCACTAGGGGTGATACTCTT	RNA2-4548	491	ToCV检测
ToCV-CP-491-R	AAGCGTTTCTTTTCATAAGCAGGTT	RNA2-5038
SJZ-RNA1-Ⅰ-F	GAAATAGTATTCGTGTGATTACACA	RNA1-1	2 078	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅰ-R	GTCCTTAAGTCCCCACATGGTATCG	RNA1-2078
SJZ-RNA1-Ⅱ-F	CTATGCTCGATACCATGTGGGGACT	RNA1-2046	2 088	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅱ-R	GTTTCATCCAATTCTGGCAACTTTT	RNA1-4133
SJZ-RNA1-Ⅲ-F	TTGGAGCGAAGATTTCTTGGTA	RNA1-3979	1 474	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅲ-R	CCATATCCGTGTCCATTTTACC	RNA1-5452
SJZ-RNA1-Ⅳ-F	GGAAAAACGACCACGATAGTAAAGA	RNA1-5254	1 848	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅳ-R	CCACTGATACCACACCTACCACG	RNA1-7101
SJZ-RNA1-Ⅴ-F	CAAGGTATTCTTTTCAAGGTTTATG	RNA1-6974	1 623	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅴ-R	CGACCTATTTATTTATATACTAGATCTACC	RNA1-8596
SJZ-RNA2-Ⅰ-F	GAAATACTAGTCCAGGTGTTTCCTG	RNA2-1	1 765	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅰ-R	TTATGAGTGACAGGCTATGATTGC	RNA2-1765
SJZ-RNA2-Ⅱ-F	TAACATAATAAGTGATGACGGAAG	RNA2-1536	1 910	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅱ-R	ACTCTCTTTATGAAACTCAACAACTC	RNA2-3445
SJZ-RNA2-Ⅲ-F	TGTGAATAGTAAAGGTTTCGGAGAT	RNA2-3365	1 829	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅲ-R	AAGTCACAGTCTGATACAAGAAGCC	RNA2-5193
SJZ-RNA2-Ⅳ-F	TAAGACAAATGGATGATAATGAGTTC	RNA2-5104	1 740	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅳ-R	ACGAGTTGAGAAATCTTGAGTGTG	RNA2-6843
SJZ-RNA2-Ⅴ-F	TTGCTGTAGTGACAACTCATCTTTC	RNA2-6746	1 497	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅴ-R	CGACCTATTTATTTATATACTAAATCTACCA	RNA2-8242
SJZ-CP-216-F	AGAGACGACGCAACTTCGCTTG	RNA2-4441	216	ToCV-SJZ CP基因特异性RT-qPCR
SJZ-CP-216-R	CTTATCAGTCAATGGAGTCTCACCG	RNA2-4656
Flo-CP-250-F	GAGGTAGATGGTGCGAGATTTAAG	RNA2-4753	250	ToCV-Florida CP基因特异性RT-qPCR
Flo-CP-250-R	GATCATCTGAGATATTCATCAACGA	RNA2-5002
ToCV-CP-154-F	GCACTCTACAAGGGGTGATAC	RNA2-4548	154	ToCV CP基因通用RT-qPCR
ToCV-CP-154-R	ATTTACAATCGCCTGACACAT	RNA2-4701

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	基因组位置 Location	产物大小/bp Product size	用途 Function
ToCV-CP-491-F	ACACTCCACTAGGGGTGATACTCTT	RNA2-4548	491	ToCV检测
ToCV-CP-491-R	AAGCGTTTCTTTTCATAAGCAGGTT	RNA2-5038
SJZ-RNA1-Ⅰ-F	GAAATAGTATTCGTGTGATTACACA	RNA1-1	2 078	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅰ-R	GTCCTTAAGTCCCCACATGGTATCG	RNA1-2078
SJZ-RNA1-Ⅱ-F	CTATGCTCGATACCATGTGGGGACT	RNA1-2046	2 088	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅱ-R	GTTTCATCCAATTCTGGCAACTTTT	RNA1-4133
SJZ-RNA1-Ⅲ-F	TTGGAGCGAAGATTTCTTGGTA	RNA1-3979	1 474	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅲ-R	CCATATCCGTGTCCATTTTACC	RNA1-5452
SJZ-RNA1-Ⅳ-F	GGAAAAACGACCACGATAGTAAAGA	RNA1-5254	1 848	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅳ-R	CCACTGATACCACACCTACCACG	RNA1-7101
SJZ-RNA1-Ⅴ-F	CAAGGTATTCTTTTCAAGGTTTATG	RNA1-6974	1 623	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅴ-R	CGACCTATTTATTTATATACTAGATCTACC	RNA1-8596
SJZ-RNA2-Ⅰ-F	GAAATACTAGTCCAGGTGTTTCCTG	RNA2-1	1 765	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅰ-R	TTATGAGTGACAGGCTATGATTGC	RNA2-1765
SJZ-RNA2-Ⅱ-F	TAACATAATAAGTGATGACGGAAG	RNA2-1536	1 910	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅱ-R	ACTCTCTTTATGAAACTCAACAACTC	RNA2-3445
SJZ-RNA2-Ⅲ-F	TGTGAATAGTAAAGGTTTCGGAGAT	RNA2-3365	1 829	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅲ-R	AAGTCACAGTCTGATACAAGAAGCC	RNA2-5193
SJZ-RNA2-Ⅳ-F	TAAGACAAATGGATGATAATGAGTTC	RNA2-5104	1 740	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅳ-R	ACGAGTTGAGAAATCTTGAGTGTG	RNA2-6843
SJZ-RNA2-Ⅴ-F	TTGCTGTAGTGACAACTCATCTTTC	RNA2-6746	1 497	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅴ-R	CGACCTATTTATTTATATACTAAATCTACCA	RNA2-8242
SJZ-CP-216-F	AGAGACGACGCAACTTCGCTTG	RNA2-4441	216	ToCV-SJZ CP基因特异性RT-qPCR
SJZ-CP-216-R	CTTATCAGTCAATGGAGTCTCACCG	RNA2-4656
Flo-CP-250-F	GAGGTAGATGGTGCGAGATTTAAG	RNA2-4753	250	ToCV-Florida CP基因特异性RT-qPCR
Flo-CP-250-R	GATCATCTGAGATATTCATCAACGA	RNA2-5002
ToCV-CP-154-F	GCACTCTACAAGGGGTGATAC	RNA2-4548	154	ToCV CP基因通用RT-qPCR
ToCV-CP-154-R	ATTTACAATCGCCTGACACAT	RNA2-4701

分离物 Isolate	年份 Year	来源 Source	RNA1 登录号 GenBank accession No.of RNA1	RNA2登录号 GenBank accession No.of RNA2
ToCV-PAK1	2019	巴基斯坦	MN869004.1	MN869006.1
ToCV-BR	2019	巴西	MT279194.1	MT279195.1
ToCV-PAP-JJ6	2023	韩国	OR865222.1	OR865223.1
ToCV-Florida	1998	美国	AY903447.1	AY903448.1
ToCR-186	2015	南非	KY471129.1	KY471130.1
ToCV-KGS	2020	日本	LC711108.1	LC711109.1
ToCV-Kas	2015	土耳其	KY419526.1	KY419528.1
ToCV-AT80/99-IC	2014	西班牙	KJ740256.1	KJ740257.1
ToCV-DSMZ PV-1242	2022	希腊	ON398512.1	ON398513.1
ToCV-FERA_160205	2016	英国	KY810786.1	KY810787.1
ToCV-BJ	2013	中国北京	KC887998.1	KC887999.1
ToCV-SDSG	2012	中国山东	KC709509.1	KC709510.1
ToCV-SDTA	2019	中国山东	OR246919.1	OR246920.1
ToCV-SH-SIA	2019	中国上海	MW490611.1	MW490612.1
ToCV-XS	2011	中国台湾	KY618796.1	KY618797.1
ToCV-CQ630	2019	中国重庆	PQ213439.1	PQ213440.1
BYVaV	2005	美国	NC_006962.1	NC_006963.1
CCYV	2009	中国台湾	KY618798.1	KY618799.1
CYSDV	2009	美国	EF547827.1	FJ492808.1
DVCV	2009	美国	GQ225585.1	GQ376201.1
LCV	2008	美国	NC_012909.1	NC_012910.1
LIYV	1994	美国	U15440.1	U15441.1
SPaV	2009	美国	AY488137.1	AY488138.1
SPCSV	2002	乌干达	AJ428554.1	AJ428555.1
TVCV	2011	韩国	KR002686.1	KR002687.1
TICV	2009	美国	FJ815440.1	FJ815441.1

分离物 Isolate	年份 Year	来源 Source	RNA1 登录号 GenBank accession No.of RNA1	RNA2登录号 GenBank accession No.of RNA2
ToCV-PAK1	2019	巴基斯坦	MN869004.1	MN869006.1
ToCV-BR	2019	巴西	MT279194.1	MT279195.1
ToCV-PAP-JJ6	2023	韩国	OR865222.1	OR865223.1
ToCV-Florida	1998	美国	AY903447.1	AY903448.1
ToCR-186	2015	南非	KY471129.1	KY471130.1
ToCV-KGS	2020	日本	LC711108.1	LC711109.1
ToCV-Kas	2015	土耳其	KY419526.1	KY419528.1
ToCV-AT80/99-IC	2014	西班牙	KJ740256.1	KJ740257.1
ToCV-DSMZ PV-1242	2022	希腊	ON398512.1	ON398513.1
ToCV-FERA_160205	2016	英国	KY810786.1	KY810787.1
ToCV-BJ	2013	中国北京	KC887998.1	KC887999.1
ToCV-SDSG	2012	中国山东	KC709509.1	KC709510.1
ToCV-SDTA	2019	中国山东	OR246919.1	OR246920.1
ToCV-SH-SIA	2019	中国上海	MW490611.1	MW490612.1
ToCV-XS	2011	中国台湾	KY618796.1	KY618797.1
ToCV-CQ630	2019	中国重庆	PQ213439.1	PQ213440.1
BYVaV	2005	美国	NC_006962.1	NC_006963.1
CCYV	2009	中国台湾	KY618798.1	KY618799.1
CYSDV	2009	美国	EF547827.1	FJ492808.1
DVCV	2009	美国	GQ225585.1	GQ376201.1
LCV	2008	美国	NC_012909.1	NC_012910.1
LIYV	1994	美国	U15440.1	U15441.1
SPaV	2009	美国	AY488137.1	AY488138.1
SPCSV	2002	乌干达	AJ428554.1	AJ428555.1
TVCV	2011	韩国	KR002686.1	KR002687.1
TICV	2009	美国	FJ815440.1	FJ815441.1

序列 Sequences	开放阅读框 ORFs	位置/nt Location	氨基酸序列长度/aa Amino acid sequence length	编码的蛋白 Protein
ToCV-SJZ-RNA1	ORF 1a	304~6 141	1 945	木瓜蛋白酶、甲基转移酶、斛旋酶
	ORF 1b	6 056~7 657	533	RdRp
	ORF 2	7 664~8 242	192	P22
	ORF 3	8 265~8 420	51	P5
ToCV-SJZ-RNA2	ORF 1	733~2 397	534	Hsp70h
	ORF 2	2 407~2 610	67	P8
	ORF 3	2 562~4 115	517	P59
	ORF 4	4 097~4 333	78	P9
	ORF 5	4 333~5 106	257	CP
	ORF 6	5 112~7 121	669	mCP
	ORF 7	7 125~7 823	232	P27
	ORF 8	7 832~8 029	65	P7

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