云南大花蕙兰复合感染的ORSV和CyCMV病毒基因组序列特征分析

doi:10.16420/j.issn.0513-353x.2023-0555

园艺学报 ›› 2024, Vol. 51 ›› Issue (8): 1844-1852.doi: 10.16420/j.issn.0513-353x.2023-0555

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

云南大花蕙兰复合感染的ORSV和CyCMV病毒基因组序列特征分析

金晓香¹^,^*, 姚俊汐¹^,^*, 尹跃艳³, 李婷婷², 郭淼³, 钟静², 和寿星³, 陈越², 和家卫³, 鄢波¹^,^**(), 丁铭²^,^**()

¹ 西南林业大学园林园艺学院，昆明 650224
² 云南省农业科学院生物技术与种质资源研究所，昆明 6502233
³ 云南省农业科学院高山经济植物研究所，云南丽江 674100

收稿日期:2023-09-14 修回日期:2024-05-17 出版日期:2024-08-25 发布日期:2024-08-21
通讯作者:
^**E-mail：yanbodr@aliyun.com，
^**E-mail：yanbodr@aliyun.com，
作者简介:
^* 共同第一作者
基金资助:
国家自然科学基金地区基金项目(32360644); 云南省种子种业联合实验室项目(202205AR070001-02); 云南省乡村振兴科技专项(200202A); 云南省重大科技专项计划项目(202302AE090005); 云南省农业科学院生物技术与种质资源研究所开放项目(2023005)

Molecular Characterization of the Odontoglossum Ringspot Virus and Cymbidium Chlorotic Mosaic Virus Mix-Infecting Cymbidium hybridum in Yunnan

JIN Xiaoxiang¹, YAO Junxi¹, YIN Yueyan³, LI Tingting², GUO Miao³, ZHONG Jing², HE Shouxing³, CHEN Yue², HE Jiawei³, YAN Bo¹^,^**(), DING Ming²^,^**()

¹ Department of Landscape and Horticulture，Southwest Forestry University，Kunming 650224，China
² Institute of Biotechnology and Germplasm Resources，Yunnan Academy of Agricultural Sciences，Kunming 650223，China
³ Institute of Alpine Economic Plants，Yunnan Academy of Agricultural Sciences，Lijiang，Yunnan 674100，China

Received:2023-09-14 Revised:2024-05-17 Published:2024-08-25 Online:2024-08-21

摘要/Abstract

摘要：

从云南丽江采集了40个大花蕙兰（Cymbidium hybridum）感病样品，利用高通量转录组测序对其中的病毒种类进行分析，Blastn N比对发现样品中存在建兰花叶病毒（cymbidium mosaic virus，CymMV）、齿兰环斑病毒（odontoglossum ringspot virus，ORSV）和建兰褪绿花叶病毒（cymbidium chlorotic mosaic virus，CyCMV）。利用RT-PCR对样品中病毒分离物的CP序列进行扩增，10份样品为ORSV和CyCMV复合侵染。为进一步明确ORSV和CyCMV云南分离物（ORSV-DH和CyCMV-DH）的序列特征，分别对其全基因组序列进行了扩增及克隆，结果表明：ORSV-DH基因组全长序列为6 611 nt（Accession No. OP644547），CyCMV-DH基因组全长序列为4 083 nt（Accession NO. OP629812），将ORSV-DH和CyCMV-DH分别与已报道的分离物的全基因组序列进行核苷酸序列分析，发现ORSV-DH与其他分离物的核苷酸相似性为97.69% ~ 99.41%，CyCMV-DH为89.84% ~ 98.19%。根据病毒全基因组核苷酸序列分别构建了ORSV-DH和CyCMV-DH与本属其他分离物的系统进化树，明确了其分别与日本分离物ORSV-Cy-1（S83257）、CyCMV-Japan（NC_027123）亲缘关系最近。

关键词: 大花蕙兰, 齿兰环斑病毒, 建兰褪绿花叶病毒, 基因组序列特征, 复合侵染

Abstract:

Forty greenhouse-grown orchid samples were collected in Lijiang City，Yunnan Province，China，to assess the presence of viruses. Total RNAs from two samples were extracted and subjected them to high-throughput transcriptome sequencing（HTS）using the Illumina HiSeq platform. BLASTN searches revealed the presence of contigs with high similarities to cymbidium mosaic virus（CymMV），odontoglossum ringspot virus（ORSV），and cymbidium chlorotic mosaic virus（CyCMV）. RT-PCR assays were conducted to confirm the presence of these viruses in the individual collected samples，and 10 samples were found to be mix-infected by ORSV and CyCMV. To further ascertain their molecular characteristics，the complete genomic sequences of the ORSV-DH and CyCMV-DH isolates were cloned. The full-length sequences of the ORSV-DH and CyCMV-DH genomes were determined to be 6 611 nt （GeneBank accession No. OP644547）and 4 083 nt（GeneBank accession No. OP629812），respectively. Sequence alignments revealed that ORSV-DH displayed nucleotide sequence identities of 97.69%- 99.41% with previously reported ORSV isolates，whereas CyCMV-DH showed identities of 89.84%- 98.19% with other CyCMV isolates. Phylogenetic trees of ORSV-DH and CyCMV-DH based on their complete genomic sequences were constructed and showed closely related to the Japanese isolate ORSV-Cy-1（S83257）and the Japanese isolate CyCMV-Japan（NC_027123），respectively.

Key words: Cymbidium hybridum, ORSV, CyCMV, characterization of genomic sequence, mix-infection

金晓香, 姚俊汐, 尹跃艳, 李婷婷, 郭淼, 钟静, 和寿星, 陈越, 和家卫, 鄢波, 丁铭. 云南大花蕙兰复合感染的ORSV和CyCMV病毒基因组序列特征分析[J]. 园艺学报, 2024, 51(8): 1844-1852.

JIN Xiaoxiang, YAO Junxi, YIN Yueyan, LI Tingting, GUO Miao, ZHONG Jing, HE Shouxing, CHEN Yue, HE Jiawei, YAN Bo, DING Ming. Molecular Characterization of the Odontoglossum Ringspot Virus and Cymbidium Chlorotic Mosaic Virus Mix-Infecting Cymbidium hybridum in Yunnan[J]. Acta Horticulturae Sinica, 2024, 51(8): 1844-1852.

图/表 6

参考文献 19

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片段 Fragment	引物序列（5′-3′） Primer sequence
ORSV-1	F：GTATTGTTCGATTACTACAA；R：AAAGGCAGTGTGAGTTCGTTCTTC
ORSV-2	F：ATATCACCCCCGCAGATAAACTGG；R：CTATAAGATCCCGGCATCGA
ORSV-3	F：AGCAAACCGCTTAAAGGGAAA；R：GCCGTTAGAGCTGACGGTAGTA
ORSV-4	F：ATGGGTCGTTTGCGTTTTGT；R：TGGGCCTCTACCCGAGGTAA
ORSV CP	F：ATGTCTTACACTATTACAGACCC；R：GGAAGAGGTCCAAGTAAGTCC
ORSV 3′ RACE-F	GGGCTGACCCCAATTCACTAA
ORSV 5′ RACE-R	CCTTTTATCGCAGCACACAGC
CyCMV-1	F：ACAAAATATATGAGCGAGGG；R：TTAGGTTGACGTTGCTTTCGC
CyCMV-2	F：GCTCTCAGATCTCAGTTGGCC；R：TCATCGGTTCAACGAGCTAG
CyCMV CP	F：ATGATTTGCAAGCTGAATTGT；R：TCATCGGTTCAACGAGCTAG
CyCMV 3′ RACE-F	ATGATTTGCAAGCTGAATTGTGGAG
CyCMV 5′ RACE-R	ATGATTTGCAAGCTGAATTGTGGAG
M4	GTTTTCCCAGTCACGACAC
M4T	GTTTTCCCAGTCACGACACTTTTTTTTTTTTTTTT
CymMV CP	F：ATGGGAGAGCCCACTCCAAC；R：TTCAGTAGGGGGTGCAGGCA

片段 Fragment	引物序列（5′-3′） Primer sequence
ORSV-1	F：GTATTGTTCGATTACTACAA；R：AAAGGCAGTGTGAGTTCGTTCTTC
ORSV-2	F：ATATCACCCCCGCAGATAAACTGG；R：CTATAAGATCCCGGCATCGA
ORSV-3	F：AGCAAACCGCTTAAAGGGAAA；R：GCCGTTAGAGCTGACGGTAGTA
ORSV-4	F：ATGGGTCGTTTGCGTTTTGT；R：TGGGCCTCTACCCGAGGTAA
ORSV CP	F：ATGTCTTACACTATTACAGACCC；R：GGAAGAGGTCCAAGTAAGTCC
ORSV 3′ RACE-F	GGGCTGACCCCAATTCACTAA
ORSV 5′ RACE-R	CCTTTTATCGCAGCACACAGC
CyCMV-1	F：ACAAAATATATGAGCGAGGG；R：TTAGGTTGACGTTGCTTTCGC
CyCMV-2	F：GCTCTCAGATCTCAGTTGGCC；R：TCATCGGTTCAACGAGCTAG
CyCMV CP	F：ATGATTTGCAAGCTGAATTGT；R：TCATCGGTTCAACGAGCTAG
CyCMV 3′ RACE-F	ATGATTTGCAAGCTGAATTGTGGAG
CyCMV 5′ RACE-R	ATGATTTGCAAGCTGAATTGTGGAG
M4	GTTTTCCCAGTCACGACAC
M4T	GTTTTCCCAGTCACGACACTTTTTTTTTTTTTTTT
CymMV CP	F：ATGGGAGAGCCCACTCCAAC；R：TTCAGTAGGGGGTGCAGGCA

分离物 Isolate	登录号 Accession No.	国家 Country	相似性/% Identity
分离物 Isolate	登录号 Accession No.	国家 Country	全基因组 Genome	RdRP蛋白 P183	RdRp蛋白 P126	运动蛋白 MP	外壳蛋白 CP
ORSV-Cy-1	S83257	日本Japan	99.41	99.01	98.57	99.64	98.95
ORSV-DSMZ PV-0625	MW248355	德国Germany	99.38	99.05	99.10	99.23	100.00
ORSV-OS4	MW316660	中国台湾Taiwan，China	99.29	98.38	93.91	99.12	100.00
ORSV-CymK	LC381916	韩国Korea	99.27	99.30	98.93	99.12	100.00
ORSV-Ph	LC217742	朝鲜South Korea	99.02	99.11	98.57	99.12	100.00
ORSV	NC_001728	韩国Korea	97.69	98.68	94.00	98.14	98.32
ORSV-SW8	KF855954	澳大利亚Australia	99.17	99.30	97.67	99.45	99.58
ORSV-Singapore	ORU34586	新加坡Singapore	98.81	98.93	97.14	98.79	99.79

分离物 Isolate	登录号 Accession No.	国家 Country	相似性/% Identity
分离物 Isolate	登录号 Accession No.	国家 Country	全基因组 Genome	RdRP蛋白 P183	RdRp蛋白 P126	运动蛋白 MP	外壳蛋白 CP
ORSV-Cy-1	S83257	日本Japan	99.41	99.01	98.57	99.64	98.95
ORSV-DSMZ PV-0625	MW248355	德国Germany	99.38	99.05	99.10	99.23	100.00
ORSV-OS4	MW316660	中国台湾Taiwan，China	99.29	98.38	93.91	99.12	100.00
ORSV-CymK	LC381916	韩国Korea	99.27	99.30	98.93	99.12	100.00
ORSV-Ph	LC217742	朝鲜South Korea	99.02	99.11	98.57	99.12	100.00
ORSV	NC_001728	韩国Korea	97.69	98.68	94.00	98.14	98.32
ORSV-SW8	KF855954	澳大利亚Australia	99.17	99.30	97.67	99.45	99.58
ORSV-Singapore	ORU34586	新加坡Singapore	98.81	98.93	97.14	98.79	99.79

分离物 Isolate	登录号 Accession No.	国家 Country	相似性/% Identity
分离物 Isolate	登录号 Accession No.	国家 Country	全基因组 Genome	运动蛋白 P1	复制酶蛋白 P2a-P2b	外壳蛋白 CP
CyCMV-Japan	NC_027123	日本Japan	98.19	96.97	98.62	99.24
CyCMV-DL5	ON286969	中国China	96.08	96.21	94.47	93.13
CyCMV-Korea	LC381945	韩国Korea	90.65	84.09	92.34	95.04
CyCMV-TA-Br1	OL999300	韩国Korea	90.79	84.60	92.34	95.42
CyCMV-China	KR996515	中国China	89.84	83.33	90.74	95.42

云南大花蕙兰复合感染的ORSV和CyCMV病毒基因组序列特征分析

Molecular Characterization of the Odontoglossum Ringspot Virus and Cymbidium Chlorotic Mosaic Virus Mix-Infecting Cymbidium hybridum in Yunnan

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云南大花蕙兰复合感染的ORSV和CyCMV病毒基因组序列特征分析

Molecular Characterization of the Odontoglossum Ringspot Virus and Cymbidium Chlorotic Mosaic Virus Mix-Infecting Cymbidium hybridum in Yunnan

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