云南大理栽培春兰中ORSV和CymMV的分子检测及序列分析

doi:10.16420/j.issn.0513-353x.2020-1053

园艺学报 ›› 2022, Vol. 49 ›› Issue (3): 655-662.doi: 10.16420/j.issn.0513-353x.2020-1053

云南大理栽培春兰中ORSV和CymMV的分子检测及序列分析

陈健鑫¹, 魏玉倩¹, 唐婕¹, 竺永金¹, 马焕成², 伍建榕¹^,²^,^**()

¹西南林业大学生物多样性保护学院,云南省高校森林灾害预警控制重点实验室,昆明 650224
²西南林业大学林学院,西南地区生物多样性保育国家林业局重点实验室,昆明 650224

收稿日期:2021-07-07 修回日期:2021-11-25 出版日期:2022-03-25 发布日期:2022-03-25
通讯作者: 伍建榕 E-mail:1176279044@qq.com
基金资助:
国家自然科学基金项目(31860208);国家自然科学基金项目(31560207);国家重点研发计划项目(2019YFD100200X);西南林业大学木棉纤维人工林产业化培育省级创新团队项目(2018HC014)

Molecular Detection and Sequence Analysis of ORSV and CymMV in Cultivated Cymbidium goeringii in Dali,Yunnan

CHEN Jianxin¹, WEI Yuqian¹, TANG Jie¹, ZHU Yongjin¹, MA Huancheng², WU Jianrong¹^,²^,^**()

¹Key Laboratory of Forest Disaster Warning and Control in Universities of Yunnan Province,College of Biodiversity Conservation,Southwest Forestry University,Kunming 650224,China
²Key Laboratory of Biodiversity Conservation in Southwest China,State Forestry Administration,College of Biodiversity Forestry,Southwest Forestry University,Kunming 650224,China

Received:2021-07-07 Revised:2021-11-25 Online:2022-03-25 Published:2022-03-25
Contact: WU Jianrong E-mail:1176279044@qq.com

摘要/Abstract

摘要：

2018—2019年,在云南大理部分地区开展了春兰（Cymbidium goeringii）病毒病害的调查和典型植株收集,通过逆转录—聚合酶链式反应（Reverse Transcription-Polymerase Chain Reaction,RT-PCR）检测春兰中的病毒。70株春兰中均检测到齿兰环斑病毒（odontoglossum ringspot virus,ORSV）,未检出建兰花叶病毒（cymbidium mosaic virus,CymMV）,说明该地区潜在的病毒病害主要为ORSV。基于外壳蛋白（Coat Protein）的系统发育树分析表明,所有ORSV分离物位于一个分支。ORSV外壳蛋白属于烟草花叶病毒外壳蛋白家族,理论等电点为5.01,属于亲水性稳定蛋白质。本研究证实了ORSV已经开始感染大理地区栽培的春兰,建议尽快开展兰花病毒病的早期诊断、药剂筛选及防治工作。

关键词: 春兰, 建兰花叶病毒, 齿兰环斑病毒, 分子检测, 外壳蛋白基因, 生物信息

Abstract:

The survey on virus diseases was carried out and the samples of infected seedlings were collected in Dali,Yunnan,China during 2018—2019. The Reverse Transcription-Polymerase Chain Reaction （RT-PCR）was used to detect the virus. The results showed that total 70 sample were infected by ORSV （odontoglossum ringspot virus）. However,CymMV（cymbidium mosaic virus）was not detected,indicating the major potential infection thread may be from ORSV in Dali. Phylogenetic analysis based on Coat Protein gene（cp）showed that all ORSV clustered into a single clade. The results of protein analysis showed that ORSV coat protein belonged to TMV coat protein family,and the theoretical isoelectric point was 5.01,which was a hydrophilic stable protein. Thus,we confirmed that ORSV had infected in cultivated C. goeringii in Dali. The follow up study should be carried out soon on early diagnosing and screening of anti-virus medicine for orchid viral disease.

Key words: Cymbidium goeringii, CymMV, ORSV, molecular detection, cp gene, bioinformatics

中图分类号:

S682.31

陈健鑫, 魏玉倩, 唐婕, 竺永金, 马焕成, 伍建榕. 云南大理栽培春兰中ORSV和CymMV的分子检测及序列分析[J]. 园艺学报, 2022, 49(3): 655-662.

CHEN Jianxin, WEI Yuqian, TANG Jie, ZHU Yongjin, MA Huancheng, WU Jianrong. Molecular Detection and Sequence Analysis of ORSV and CymMV in Cultivated Cymbidium goeringii in Dali,Yunnan[J]. Acta Horticulturae Sinica, 2022, 49(3): 655-662.

图/表 5

表1 RT-PCR检测CymMV和ORSV cp基因的引物

Table 1 Primers for RT-PCR to detect cp gene of CymMV and ORSV

引物 Primer	序列（5′-3′） Sequence	预期片段/bp Expected size	参考文献 Reference
Cy-CPF1/Cy-CPR1	ATGGGAGAGYCCACTCCARCYCCAGC/ TTCAGTAGGGGGTGCAGGCA	669	Lee and Chang,2006
OR-CPF1/OR-CPR1	ATGTCTTACACTATTACAGACCCG/ GGAAGAGGTCCAAGTAAGTCC	474	Lee and Chang,2006

图1 病毒侵染春兰的症状 a、b：花叶和褪绿;c、d：褪绿和坏死;e：条纹;f：坏死和环斑;g、h：整株染病症状。

Fig. 1 Symptoms of orchids infected by virus a,b：Mosaic and chlorosis;c,d：Chlorosis and necrosis;e：Streak;f：Necrosis and ringspot;g,h：Symptoms of intact plant.

图2 RT-PCR检测ORSV和CymMV cp基因 M：100 ~ 2 000 bp Marker;N：无症植物;P：阳性对照;01 ~ 03：春兰样本。

Fig. 2 The RT-PCR detection of CymMV and ORSV with specific primers of cp gene M：Marker molecular weight 100-2 000 bp DNA ladder;N：Plant of symptom-free;P：Positive control;01-03：Samples of Cymbidium.

图3 邻接法构建的大理地区ORSV分离株与其他株系的系统发育树 a：基于cp基因核苷酸序列;b：基于cp基因编码的氨基酸序列。

Fig. 3 Phylogenetic trees of ORSV isolates from Dali and other isolates by Neighbor-joining method a：Based on cp gene nucleotide sequence;b：Based on amino acid sequence encoded by cp gene.

图4 蛋白质亲（疏）水性分析纵轴 > 0代表疏水性区域,< 0代表亲水性区域。

Fig. 4 Analysis of protein hydrophilicity and hydrophobicity Longitudinal axis > 0 represents hydrophobic region and < 0 represents hydrophilic region.

参考文献 33

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云南大理栽培春兰中ORSV和CymMV的分子检测及序列分析

Molecular Detection and Sequence Analysis of ORSV and CymMV in Cultivated Cymbidium goeringii in Dali,Yunnan

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云南大理栽培春兰中ORSV和CymMV的分子检测及序列分析

Molecular Detection and Sequence Analysis of ORSV and CymMV in Cultivated Cymbidium goeringii in Dali,Yunnan

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