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作物学报 ›› 2022, Vol. 48 ›› Issue (3): 624-634.doi: 10.3724/SP.J.1006.2022.14017

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

黄麻双生病毒CoYVV的分子鉴定和抗性种质筛选

杨昕1,2,3(), 林文忠3, 陈思远1,2, 杜振国3, 林杰3, 祁建民1,2, 方平平1,2, 陶爱芬1,2, 张立武1,2,3,*()   

  1. 1福建农林大学农学院/作物遗传育种与综合利用教育部重点实验室/福建省作物设计育种重点实验室, 福建福州 350002
    2福建农林大学农业农村部东南黄红麻实验观测站/福建省麻类种质资源共享平台/福建省南方经济作物遗传育种与多用途开发国际科技合作基地, 福建福州 350002
    3闽台作物有害生物生态防控国家重点实验室, 福建福州 350002
  • 收稿日期:2021-01-28 接受日期:2021-06-16 出版日期:2022-03-12 网络出版日期:2021-07-22
  • 通讯作者: 张立武
  • 作者简介:杨昕, E-mail: 295548772@qq.com
  • 基金资助:
    国家自然科学基金项目(31771369);国家现代农业产业技术体系建设专项(CARS-19-E06)

Molecular identification of a geminivirus CoYVV and screening of resistant germplasms in jute

YANG Xin1,2,3(), LIN Wen-Zhong3, CHEN Si-Yuan1,2, DU Zhen-Guo3, LIN Jie3, QI Jian-Min1,2, FANG Ping-Ping1,2, TAO Ai-Fen1,2, ZHANG Li-Wu1,2,3,*()   

  1. 1Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops/Fujian Key Laboratory for Crop Breeding by Design/College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    2Experiment Station of Ministry of Agriculture and Rural Affairs for Jute and Kenaf in Southeast China/Fujian Public Platform for Germplasm Resources of Bast Fiber Crops/Fujian International Science and Technology Cooperation Base for Genetics, Breeding and Multiple Utilization Development of Southern Economic Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    3State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fuzhou 350002, Fujian, China
  • Received:2021-01-28 Accepted:2021-06-16 Published:2022-03-12 Published online:2021-07-22
  • Contact: ZHANG Li-Wu
  • Supported by:
    National Natural Science Foundation of China(31771369);China Agriculture Research System(CARS-19-E06)

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摘要：

越南黄麻黄脉病毒(Corchorus yellow vein Vietnam virus, CoYVV)是影响黄麻纤维品质和产量的双生病毒, 其基因组含2个环状单链DNA (DNA-A和DNA-B)。鉴定黄麻双生病毒CoYVV并筛选抗病毒种质, 不仅丰富对双生病毒分布和进化的认识, 而且为黄麻抗性基因克隆奠定了基础。2018年福建省三明市福建农林大学洋中科教基地发现黄麻种质资源圃长果种黄麻福农5号疑似感染黄色花叶病。利用CTAB法提取该品种感病叶片DNA, 通过PCR、滚环扩增及测序技术获得病毒基因组序列。通过同源序列比对和进化树构建明确该病毒与新旧世界双生病毒的关系。含有CoYVV DNA-A和CoYVV DNA-B侵染性克隆载体的农杆菌等比例混合, 子叶注射法侵染具有代表性的7份长果种和7份圆果种黄麻品种(系), 定期观察发病株数(病株率)并PCR检测接种黄麻的侵染率。PCR检测结果证实, 福农5号病样感染了CoYVV, 命名为CoYVV-FS。其DNA-A为2724 bp, DNA-B为2691 bp。进化分析表明, 相较于旧世界菜豆金黄花叶病毒(begomoviruses), CoYVV-FS与新世界begomoviruses更近。农杆菌接种14份黄麻品种43 d时, 统计病株率和侵染率。结果表明该病毒可以成功侵染13份黄麻品种, 病株率和侵染率分别在0~60%和0~100%之间, 说明该病毒在黄麻中具有较高侵染性。807元引马里的病株率和侵染率皆为0, 表现为抗性种质; 而巴长4号和印度墨绿子在接种30 d时产生黄脉、卷曲等典型的表型, 表现出对CoYVV具有高度感病性。黄麻种植资源圃发现的CoYVV-FS与前期已报道的CoYVV属于同一分离物, 它能侵染多数黄麻种质, 在长果种黄麻上引起严重症状。

关键词: 黄麻, 越南黄麻黄脉病毒, CoYVV, 侵染性克隆, 抗性鉴定

Abstract:

Corchorus yellow vein Vietnam virus (CoYVV) is a geminivirus that affects yield and quality in jute. Its genome is consisted of two circular single-stranded DNAs, named DNA-A and DNA-B, respectively. Detection of CoYVV and screening of resistant germplasm will not only increase our understanding of geminivirus distribution and evolution but also lay the foundation for resistance gene cloning. Funong 5 (C. olitorius.) was suspected to be infected with leaf mosaic symptoms in jute germplasm resource nursery of Yangzhong Science and Education Base of Fujian Agriculture and Forestry University (Sanming, Fujian, China) in 2018. The diseased samples were collected and subjected to total DNA extraction by CTAB. The virus in the sample was identified by PCR, RCA, and sequencing. Then the mixture of Agrobacterium tumefaciens containing two plasmids (infectious clone vector plasmids of CoYVV DNA-A and DNA-B) in 1:1 ratio was injected to 7 representative C. capsularis varieties and 7 representative C. olitorius ones. After inoculation, we investigated symptom development (disease incidence) and PCR was used to detect the rate of infection. The results showed that the symptomatic samples in the field were infected by an isolate of CoYVV (CoYVV-FS). The size of the DNA-A and DNA-B of CoYVV-FS were 2724 bp and 2691 bp, respectively. Phylogenetic analysis revealed that CoYVV-FS was more related to New World begomoviruses than to Old World begomoviruses. Moreover, the inoculation of 14 representative jute varieties showed that CoYVV was infectious to jute. The disease incidence was 0 in the 7 C. capsularis varieties and 0-60% in the 7 C. olitorius varieties. The infection rates for the 14 jute varieties was from 0 to 100%. Both disease incidence and infection rates of 807 yuanyinmali (C. olitorius) were 0, indicating that it was the resistant germplasm; while Bachang4 (C. olitorius) and Yindumolvzi (C. olitorius) produced yellow vein and curly-leaf phenotypes after 30 days of inoculation, showing that it was the sensitive germplasm. The CoYVV-FS in jute germplasm resource nursery belonged to the same isolate as the CoYVV previously reported, which could infect most jute varieties (lines) and cause serious symptoms in C. olitorius.

Key words: jute, Corchorus yellow vein Vietnam virus, CoYVV, infectious clone, resistance evaluation

表1

本研究检测与克隆CoYVV基因组全长所用引物"

引物Primer 序列Sequence (5°-3°)
DeP3 GANGSATGHGTRCADGCCATATA
AV494 GCCYATRTAYAGRAAGCCMAG
CoVV-A-CP-F-149 CCATGTATAGGAAGCCCAGGATC
CoVV-A-CP-R-542 ATGACCTGATAACGATCACGGAG
CoYVV-AF TTCGTGCATCTGTACTTCTTCTGG
CoYVV-AR CTGCAGATTGTTCGATCTATCCAA

图1

黄麻福农5号田间发病症状"

图2

黄麻CoYVV-FS的DNA-A和DNA-B基因组结构"

表2

黄麻CoYVV的DNA-A和DNA-B编码的开放阅读框"

开放阅读框
Open reading frame		 序列位置
Position
(nt)		 序列长度
Sequence length (bp)		 氨基酸数目
Amino acid residues (aa)		 蛋白分子量
Protein molecular weight (kD)		 预测功能
Function prediction		 参考文献
Reference
AC1 1449-2528 1080 359 40.6 复制相关蛋白
Replication-associated protein		 [26]
AC2 1139-1564 426 141 16.0 转录激活蛋白
Transcriptional activator protein		 [26]
AC3 1009-1401 393 130 15.8 增强病毒DNA复制蛋白
AC3 protein enhances viral DNA replication		 [27]
AC4 2087-2377 291 96 11.0 与病毒的移动相关蛋白
AC4 protein involved in virus movement		 [28]
AV1/V1 236-1012 777 258 29.9 外壳蛋白
Coat protein		 [26]
BC1 1246-2124 879 292 33.3 移动蛋白
Movement protein		 [29]
BV1 423-1184 762 253 29.1 核穿梭蛋白
Nuclear shuttle protein		 [30]

表3

本研究24个与CoYVV-FS进行同源性比较的双生病毒"

病毒名称
Name of viruses		 缩写名
Abbreviation		 GenBank登录号
GenBank accession No.		 属
Genus		 病毒类型
Type of viruses
DNA-A DNA-B
南瓜曲叶病毒
Squash leaf curl virus		 SMLCV DQ285016 DQ285017 菜豆金黄花叶病毒属Begomovirus 来自新世界的双组分双生病毒
It is a geminivirus from NW consist
of two DNA components
菜豆矮花叶病毒
Bean dwarf mosaic virus		 BDMV NC_001931 NC_001930 菜豆金黄花叶病毒属Begomovirus 来自新世界的双组分双生病毒
It is a geminivirus from NW consist
of two DNA components
鹿藿金花叶病毒
Rhynchosia golden mosaic virus		 RhGMV AF239671 — 菜豆金黄花叶病毒属Begomovirus 来自新世界的单组分双生病毒
It is a geminivirus from NW consist
of one DNA components
黄花捻斑点病毒
Sida mottle virus		 SiMoV AY090555 — 菜豆金黄花叶病毒属Begomovirus 来自新世界的单组分双生病毒
It is a geminivirus from NW consist
of one DNA components
胜红蓟耳突病毒
Ageratum enation virus		 AEV JX436472 — 菜豆金黄花叶病毒属Begomovirus 来自旧世界的单组分双生病毒
It is a geminivirus from OW consist
of one DNA components
泽兰黄脉病毒
Eupatorium yellow vein virus		 EpYVV AB007990 — 菜豆金黄花叶病毒属Begomovirus 来自旧世界的单组分双生病毒
It is a geminivirus from OW consist
of one DNA components
印度木薯花叶病毒
Indian cassava mosaic virus		 ICMV AJ314739 AJ314740 菜豆金黄花叶病毒属Begomovirus 来自旧世界的双组分双生病毒
It is a geminivirus from OW consist
of two DNA components
南非木薯花叶病毒
South African cassava mosaic virus		 SACMV AF155806 AF155807 菜豆金黄花叶病毒属Begomovirus 来自旧世界的双组分双生病毒
It is a geminivirus from OW consist
of two DNA components
病毒名称
Name of viruses		 缩写名
Abbreviation		 GenBank登录号
GenBank accession No.		 属
Genus		 病毒类型
Type of viruses
DNA-A DNA-B
中国南瓜曲叶病毒
Squash leaf curl China virus		 SLCCNV AF509743 AF509742 菜豆金黄花叶病毒属Begomovirus 来自中国(旧世界)的双组分双生病毒
It is a geminivirus from China (OW)
consist of two DNA components
苎麻花叶病毒
Ramie mosaic virus		 RaMoV KC171650 KC171651 菜豆金黄花叶病毒属Begomovirus 来自中国(旧世界)的双组分双生病毒
It is a geminivirus from China (OW)
consist of two DNA components
中国大青花叶病毒
Clerodendrum golden mosaic China virus		 ClGMCNV FJ011668 FJ011669 菜豆金黄花叶病毒属Begomovirus 来自中国(旧世界)的双组分双生病毒
It is a geminivirus from China (OW)
consist of two DNA components
番茄黄曲叶病毒
Tomato yellow leaf curl virus		 TYLCV AJ489258 — 菜豆金黄花叶病毒属Begomovirus 来自中国(旧世界)的单组分双生病毒
It is a geminivirus from China (OW)
consist of one DNA components
番茄曲叶病毒
Tomato leaf curl virus		 ToLCV S53251 — 菜豆金黄花叶病毒属Begomovirus 来自中国(旧世界)的单组分双生病毒
It is a geminivirus from China (OW)
consist of one DNA components
胜红蓟黄脉病毒
Ageratum yellow vein virus		 AYVV AM940137 — 菜豆金黄花叶病毒属Begomovirus 来自中国(旧世界)的单组分双生病毒
It is a geminivirus from China (OW)
consist of one DNA components
黄麻金色花叶病毒
Corchorus golden mosaic virus		 CoGMV AB971842 AB971843 菜豆金黄花叶病毒属Begomovirus 来自旧世界双组分双生病毒
It is a geminivirus from OW consist
of two DNA components
越南黄麻黄脉病毒
Corchorus yellow vein Vietnam virus		 CoYVV AY727903 AY727904 菜豆金黄花叶病毒属Begomovirus 越南(旧世界)报道的CoYVV分离物
The isolate of CoYVV reported in
Vietnam (OW)
玉米坏死线条病毒
Maize streak reunion virus		 MSRV JQ624879 — 玉米线条病毒属
Mastrevirus		 代表玉米线条病毒属的病毒
It represents Mastrevirus
甜菜曲顶病毒Beet curly top virus BCTV AY548948 — 曲顶病毒属
Curtovirus		 代表曲顶病毒属的病毒
It represents Curtovirus
番茄伪曲顶病毒
Tomato pseudo-curly top virus		 TPCTV X84735 — 番茄伪曲顶病毒属Topocuvirus 代表番茄伪曲顶病毒属的病毒
It represents Topocuvirus
亚利桑那州菠菜曲顶病毒
Spinach curly top Arizona virus		 SCTAV HQ443515 — 甜菜曲顶病毒属
Becurtovirus		 代表甜菜曲顶病毒属的病毒
It represents Becurtovirus
弯叶画眉草线条病毒
Eragrostis curvula streak virus		 ECSV FJ665629 — 画眉草病毒属
Eragrovirus		 代表画眉草病毒属的病毒
It represents Eragrovirus
芜菁曲顶病毒
Turnip curly top virus		 TCTV JQ742019 — 芜菁曲顶病毒属Turncurtovirus 代表芜菁曲顶病毒属的病毒
It represents Turncurtovirus
苜蓿曲叶病毒
Alfalfa leaf curl virus		 ALCV MH603810 — 大戟小刺潜伏病毒属Capulavirus 代表大戟小刺潜伏病毒属的病毒
It represents Capulavirus
葡萄藤红斑病毒
Grapevine red blotch virus		 GRBV JX559642 — 葡萄斑点病毒属
Grablovirus		 代表葡萄斑点病毒属的病毒
It represents Grablovirus

表4

CoYVV-FS与其他双生病毒相似性分析"

序号
No.		 缩写名
Abbreviation		 DNA-Aa DNA-Ba AC1b AC2 b AC3 b AC4 b AV1 b BC1 b BV1 b
1 CoYVV (the isolates in Vietnam) 100.00 99.93 100.00 100.00 100.00 100.00 100.00 100.00 99.60
2 CoGMV 79.80 71.73 78.93 65.96 65.38 51.06 83.72 86.30 63.60
3 SiMoV 77.09 — 49.72 63.72 48.55 26.67 84.50 — —
4 BDMV 83.09 77.74 55.03 53.44 50.38 — 83.72 75.00 51.56
5 RhGMV 82.45 — 66.29 56.34 47.06 — 84.50 — —
6 SMLCV 81.15 73.89 51.45 56.06 51.47 0 86.05 70.89 51.17
7 AYVV 77.88 — 68.17 31.88 46.84 37.23 74.90 — —
8 AEV 76.05 — 64.51 49.24 48.51 35.38 72.59 — —
9 RaMoV 76.02 0 65.92 51.16 50.00 32.14 73.36 0 0
10 ToLCV 73.93 — 67.64 45.80 46.67 38.10 71.54 — —
11 EpYVV 77.35 — 63.90 0 44.78 0 67.18 — —

图3

双生病毒DNA-A系统进化树 OW: 旧世界(东半球, 欧洲, 非洲, 亚洲)的菜豆金色花叶病毒; NW: 新世界(西半球, 美洲)的菜豆金色花叶病毒; China: 在中国(旧世界)报道的双生病毒; Vietnam: 越南报道的CoYVV分离物; Mastrevirus: 玉米线条病毒属的病毒; Curtovirus: 曲顶病毒属的病毒; Topocuvirus: 番茄伪曲顶病毒属的病毒; Becurtovirus: 甜菜曲顶病毒属的病毒; Eragrovirus: 属于画眉草病毒属的病毒; Turncurtovirus: 属于芜菁曲顶病毒属的病毒; Capulavirus: 代表大戟小刺潜伏病毒属的病毒; Grablovirus: 代表葡萄斑点病毒属的病毒。"

图4

双生病毒DNA-B系统进化树 OW: 旧世界(东半球, 欧洲, 非洲, 亚洲)的菜豆金色花叶病毒; NW: 新世界(西半球, 美洲)的菜豆金色花叶病毒; China: 在中国(旧世界)报道的双生病毒; Vietnam: 越南报道的CoYVV分离物。"

图5

含有CoYVV侵染性克隆的农杆菌接种黄麻后的表型 A: 阴性对照巴长4号; B: 接种后的黄麻巴长4号; C: 接种后的黄麻印度墨绿子; D: 阴性对照印度墨绿子。"

表5

黄麻14份代表性品种接种后的病情统计"

品种名称
Name of accessions		 种类
Species		 病株率
Disease incidence (%)		 侵染率
Infection rates (%)
爱店野生种Aidianyeshengzhong C. capsularis 0 100
粤圆5号Yueyuan 5 C. capsularis 0 60
黄麻179 Huangma 179 C. capsularis 0 100
新选1号Xinxuan 1 C. capsularis 0 90
梅峰2号Meifeng 2 C. capsularis 0 100
C46 C. capsularis 0 90
闽麻5号Minma 5 C. capsularis 0 90
奠边青麻Dianbianqingma C. olitorius 0 100
宽叶长果Kuanyechangguo C. olitorius 0 100
807元引马里807 Yuanyinmali C. olitorius 0 0
西贡长荚Xigongchangjia C. olitorius 0 20
福农5号Funong 5 C. olitorius 0 100
印度墨绿子Yindumolyuzi C. olitorius 40 80
巴长4号Bachang 4 C. olitorius 60 80
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