小RNA深度测序技术分析西瓜花叶病毒蜀葵分离物
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摘要
蜀葵病毒病害的发生对其生长造成严重影响,明确蜀葵病毒病害的种类及变异进化对蜀葵病毒病害的防治具有重要意义。利用小RNA深度测序技术对具有明显脉明、花叶症状的蜀葵叶片进行鉴定。结果发现,感病蜀葵被西瓜花叶病毒(Watermelon mosaic virus, WMV)、锦葵脉明病毒(Mala vein cleaning virus, MVCV)和一种新的RNA病毒[暂命名为蜀葵病毒1号(Althaea rosea virus1, ArV1)]所侵染。为进一步明确WMV蜀葵分离物(WMV-Tg)的进化关系,对病毒WMV-Tg全基因组进行扩增,获得全长为10 046个核苷酸序列(nt)。序列分析结果显示,WMV-Tg与已报道的WMV分离物基因组核苷酸序列的同源性为83.3%～90.2%。系统进化关系表明,WMV-Tg与WMV-Pg聚为一簇,亲缘关系最近。对蜀葵WMV-Tg来源的小RNA(WMV-derived small interfering RNAs, WMV-vsiRNAs)的长度分布、5′碱基偏好性、极性分布以及热点区分布的分析,有助于加深对WMV-vsiRNAs的了解,并为进一步研究病毒来源的小RNA(virus-derived small interfering RNAs, vsiRNAs)在抗病毒防御中的功能,以及为蜀葵病毒病的防治奠定理论基础。
The occurrence of Althaea rosea(Linn.) virus disease has serious influence on its growth. It is of great significance to clarify the species and mutation evolution of Althaea rosea virus to prevent and control the disease.The small RNA deep sequencing technique was used to identify the leaves of althaea with obvious symptoms of vein clearing and mosaic. It was found that Althaea rosea leaves with typical mosaic symptoms were infected by Watermelon mosaic virus, Mala vein cleaning virus and a new RNA virus, tentatively named Althaea rosea virus 1. In order to further clarify the evolutionary relationship of Althaea rosea isolate, the whole genome of WMV-Tg was amplified and its full-length sequence was 10 046 nucleotides. The reported nucleotide sequence homology of the WMV isolate was 83.3% to 90.2%. The phylogenetic relationship showed that WMV-Tg and WMV-Pg clustered into a cluster, and they are closely related. The analysis of size distribution, 5′-terminal nucleotide preference, strand polarity and hotspot distribution of WMV-Tg-derived small interfering RNAs from Althaea rosea will help us to have a comprehensive understanding of WMV-vsiRNAs and to further study the function of virus-derived small interfering RNAs in antiviral defense. It also lays a theoretical foundation for the prevention and treatment of virus diseases in Althaea rosea.
The occurrence of Althaea rosea(Linn.) virus disease has serious influence on its growth. It is of great significance to clarify the species and mutation evolution of Althaea rosea virus to prevent and control the disease.The small RNA deep sequencing technique was used to identify the leaves of althaea with obvious symptoms of vein clearing and mosaic. It was found that Althaea rosea leaves with typical mosaic symptoms were infected by Watermelon mosaic virus, Mala vein cleaning virus and a new RNA virus, tentatively named Althaea rosea virus 1. In order to further clarify the evolutionary relationship of Althaea rosea isolate, the whole genome of WMV-Tg was amplified and its full-length sequence was 10 046 nucleotides. The reported nucleotide sequence homology of the WMV isolate was 83.3% to 90.2%. The phylogenetic relationship showed that WMV-Tg and WMV-Pg clustered into a cluster, and they are closely related. The analysis of size distribution, 5′-terminal nucleotide preference, strand polarity and hotspot distribution of WMV-Tg-derived small interfering RNAs from Althaea rosea will help us to have a comprehensive understanding of WMV-vsiRNAs and to further study the function of virus-derived small interfering RNAs in antiviral defense. It also lays a theoretical foundation for the prevention and treatment of virus diseases in Althaea rosea.
引文
[1] 宋利娜, 弓传伟, 孙丽萍, 等. 北京地区锦葵科草本观赏植物引种栽培试验[J]. 北京农学院学报 (Song LN, Gong CW, Song LP, et al. Study on introduction and cultivation of Malvaceae herbaceous ornamental plants in Beijing [J]. J Beijing Agric Univ), 2017, 32(3): 89-93
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[3] Marzieh Ahmadi, Abolfazl Khajavi Rad, Ziba Rajaei, et al. Alcea rosea root extract as a preventive and curative agent in ethylene glycol-induced urolithiasis in rats [J]. Indian J Pharmacol, 2012, 44(3): 304-307
[4] 王少平, 林紫玉, 刘改芬. 不同浓度硼酸对蜀葵种子发芽的影响[J]. 北方园艺(Wang SP, Ling ZY, Liu GF. Effect of boric acid with different concentration on seed germination in hollyhock [J]. North Horticult), 2007,2017 (10): 128-130
[5] 吴桐, 李翠兰, 邵泽强, 等. 几种花卉植物对土壤中铅富集特征的研究[J]. 吉林农业大学学报(Wu T, Li CL, Shao ZQ, et al. Enrichment characteristics of several ornamental plants to lead in soil [J]. J Jilin Agric Univ), 2012, 34(3): 305-310
[6] 杨锋, 牛二波, 王德富, 等. 锦葵脉明病毒中国蜀葵分离物cp基因序列分析[J]. 植物病理学报(Yang F, Niu EB, Wang DF, et al. Sequence analysis of cp gene of Malva vein clearing virus Althaea rosea isolates in China [J]. Acta Phytopathol Sin), 2017, 47(4): 458-462
[7] 苏秀, 徐毅, 陈莎, 等. 利用小RNA深度测序和组装技术鉴定紫藤花叶病病原[J]. 植物病理学报(Su X, Xu Y, Chen S, et al. Detection of viruses infecting Wisteria sinensis by deep sequencing and assembly of small RNA[J]. Acta Phytopathol Sin), 2015, 45(1): 88-92
[8] Choi SK, Yoon JY, Ryu KH, et al. First report of Zucchini yellow mosaic virus on Hollyhock (Althaea rosea)[J]. Plant Pathol J, 2002, 18(3): 121-125
[9] Menzel W, Winter S, Richert-poggeler KR. First report of Malva vein clearing virus naturally occurring in hollyhock in Germany[J]. Plant Dis, 2010, 94(2): 276
[10] Abdel-Salam AM, El-Shazly MA, Thouvenel JC. Biological, biochemical and serological studies on hollyhock leaf crumple virus (HLCrV): a newly discovered whitefly transmitted Geminivirus[J]. Arab J Biotech, 1998, 1(1) 41-58
[11] Srivastava A, Kumar S, Raj SK, et al. Association of a distinct strain of hollyhock yellow vein mosaic virus and Ludwigia leaf distortion betasatellite with yellow vein mosaic disease of hollyhock (Alcea rosea) in India[J]. Arch Virol, 2014, 159(10): 2711-2715
[12] Venkataravanappa V, Lakshminarayana Reddy CN, Devaraju A, et al. Association of a recombinant Cotton leaf curl Bangalore virus with yellow vein and leaf curl disease of okra in India[J]. Indian J Virol, 2013, 24(2): 188-198
[13] Bigarré L, Chazly M, Salah M, et al. Characterization of a new begomovirus from Egypt infecting Hollyhock (Althea Rosea)[J]. Eur J Plant Pathol, 2001, 107(7): 701-711
[14] Mi S, Cai T, Hu Y, et al. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′ terminal nucleotide[J]. Cell, 2008, 133(1): 116-127
[15] 谢雪花. 小RNA深度测序鉴定草莓病毒的研究及草莓快繁体系的优化[D]. 湖南农业大学(Xie Xue-Hua.Identification of Strawberry virus by RNA sequencing and Optimization of Strawberry Rapid Propagation System[D]. Hunan Agricultural University),2016
[16] Zhang Y, Singh K, Kaur R, et al. Association of a novel DNA virus with the grapevine vein-clearing and vine decline syndrome[J]. Phytopathology, 2011, 101(9): 1081-1090
[17] Wang Y, Cheng X, Wu X, et al. Characterization of complete genome and small RNA profile of pagoda yellow mosaic associated virus, a novel badnavirus in China[J]. Virus Res, 2014, 188: 103-108
[18] 陈莎. 深度测序鉴定玉米病毒及感病玉米组织中小RNA分析[D]. 浙江大学(Chen Sha. Identification of plant viruses from maize by deep sequencing and analysis of small RNA in virus infected maize[D]. Zhejiang University), 2015
[19] He Y, Cai L, Zhou L, et al. Deep sequencing reveals the first fabavirus infecting peach[J]. Sci Rep, 2017, 7(1): 11329
[20] 牛颜冰, 时晓丽, 赵慧琪, 等. 白术矮化病毒病病原的分子鉴定和部分序列分析[J]. 植物病理学报(Niu YB, Shi XL, Zhao HQ, et al. Molecular identification and partial sequence analysis of pathogen of Atractylodes macrocephala virus[J]. Acta Phytopathol Sin), 2014, 44(4):357-362
[21] Tzanetakis IE, Martin RR. A new method for extraction of double-stranded RNA from plants [J]. J Virol Methods, 2008, 149(1): 167-170
[22] Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs[J]. Genome Res, 2008, 18(5): 821-829
[23] 周健, 顾兴芳, 张圣平, 等. 黄瓜对西瓜花叶病毒病抗性的研究进展[J]. 中国蔬菜(Zhou J, Gu XF, Zhang SP, et al. Research progress in cucumber resistance to watermelon mosaic virus[J]. China Veg), 2012, (5x): 7-13
[24] 付晶晶, 杨彩霞, 韩彤, 等. 西瓜花叶病毒辽宁臭椿分离物的鉴定与全序列分析[J]. 江西农业大学学报(Fu JJ, Yang CX, Han T, et al. Detection and complete viral sequence analysis of watermelon mosaic virus on Ailanthus altissima in Liaoning[J]. Acta Agriculturae Universitatis Jiangxiensis), 2018, (1): 96- 102
[25] Anderson CW. Two Watermelon mosaic virus strains from central Florida[J]. Phytopathology,1954, 44(4): 198-202
[26] Webb RE, Scott HA. Isolation and identification of watermelon mosaic viruses 1 and 2[J]. Phytopathology, 1965, 55(8): 895-900
[27] Park D, Kim H, Hahn Y. Genome sequence of a distinct watermelon mosaic virus identified from ginseng (Panax ginseng) transcriptome[J]. Acta Virol, 2017, 61(4): 479-482
[28] 任春梅, 程兆榜, 缪倩, 等. 江苏省葫芦科作物西瓜花叶病毒的分子鉴定和序列分析[J]. 南方农业学报(Ren CM, Cheng ZB, Miu Q, et al. Molecular identification and sequence analysis for Watermelon mosaic virus in Jiangsu[J]. J Southern Agriculture), 2013, 44(9): 1464-1470
[29] 李大伟, 牛胜鸟, 韩成贵, 等. 西瓜花叶病毒2号山西分离物外壳蛋白基因核苷酸序列分析[J]. 河南农业大学学报(Li DW, Niu SN, Han CG, et al. Nucleotide sequence analysis of coat protein gene of Watermelon mosaic virus-2 Shanxi isolate[J]. J Henan Agric Univ), 2000, 34(4): 337-340
[30] 秦碧霞, 蔡健和, 刘志明, 等. 广西西瓜花叶病病原病毒鉴定[J]. 广西农业科学(Qin BX, Cai JH, Liu ZM, et al. Identification of watermelon mosaic disease in Guangxi[J]. Guangxi Agric Sci), 2001, 2001(6): 296- 297
[31] 王惠哲, 李淑菊, 霍振荣, 等. 利用RT-PCR检测黄瓜上的西瓜花叶病毒[J]. 天津农学院学报(Wang HZ, Li SJ, Huo ZR, et al. Detection of watermelon mosaic viruses on cucumber by RT-PCR[J]. J Tianjin Agric Univ), 2004, 11(4): 20-22
[32] 陈红运, 赵文军, 程毅, 等. 辽中地区西瓜花叶病病原的分子鉴定[J]. 植物病理学报(Chen HY, Zhao WJ, Cheng Y, et al. Molecular identification of the virus causing watermelon mosaic disease in Mid- Liaoning [J]. Acta Phytopathol Sin), 2006, 36(4): 306-309
[33] 张建新, 吴云锋, 王睿, 等. 西瓜花叶病毒中国分离株全基因组核苷酸序列测定[J]. 病毒学报(Zhang JX, Wu YF, Wang R, et al. Complete nucleotide sequence of watermelon mosaic virus China isolate[J]. Chin J Virol), 2007, 23(2): 153-156
[34] 刘金亮, 王凤婷, 魏毅, 等. 侵染南瓜的西瓜花叶病毒和黄瓜花叶病毒CP基因的克隆和序列分析[J]. 中国农学通报(Liu JL, Wang FT, Wei Y, et al. Cloning and sequence analyses of the coat protein genes of watermelon mosaic virus and cucumber mosaic virus from one mix-infected squash plant[J]. Chin Agric Sci Bull), 2010, 26(16): 262-266
[35] 刘卫荣, 向本春. 西瓜花叶病毒2号新疆昌吉分离物外壳蛋白基因核苷酸序列分析[J]. 植物病理学报(Liu WR, Xiang BC. Nucleotide sequence analysis of coat protein gene of Water melon mosaic virus 2 Xinjiang Changji isolates[J]. Acta Phytopathol Sin), 2008, 38(6): 576-581
[2] 姚家喜, 钱彪, 王勤章, 等. 蜀葵根预防和治疗大鼠肾草酸钙结石的效果评价及机制初探[J]. 天津医药(Yao JX, Qian B, Wang QZ, et al. Effects and mechanism of Alcea Rosea roots in ethylene glycol induced nephrolithiasis in rats [J]. Tianjin Med J), 2014, (4): 329-332
[3] Marzieh Ahmadi, Abolfazl Khajavi Rad, Ziba Rajaei, et al. Alcea rosea root extract as a preventive and curative agent in ethylene glycol-induced urolithiasis in rats [J]. Indian J Pharmacol, 2012, 44(3): 304-307
[4] 王少平, 林紫玉, 刘改芬. 不同浓度硼酸对蜀葵种子发芽的影响[J]. 北方园艺(Wang SP, Ling ZY, Liu GF. Effect of boric acid with different concentration on seed germination in hollyhock [J]. North Horticult), 2007,2017 (10): 128-130
[5] 吴桐, 李翠兰, 邵泽强, 等. 几种花卉植物对土壤中铅富集特征的研究[J]. 吉林农业大学学报(Wu T, Li CL, Shao ZQ, et al. Enrichment characteristics of several ornamental plants to lead in soil [J]. J Jilin Agric Univ), 2012, 34(3): 305-310
[6] 杨锋, 牛二波, 王德富, 等. 锦葵脉明病毒中国蜀葵分离物cp基因序列分析[J]. 植物病理学报(Yang F, Niu EB, Wang DF, et al. Sequence analysis of cp gene of Malva vein clearing virus Althaea rosea isolates in China [J]. Acta Phytopathol Sin), 2017, 47(4): 458-462
[7] 苏秀, 徐毅, 陈莎, 等. 利用小RNA深度测序和组装技术鉴定紫藤花叶病病原[J]. 植物病理学报(Su X, Xu Y, Chen S, et al. Detection of viruses infecting Wisteria sinensis by deep sequencing and assembly of small RNA[J]. Acta Phytopathol Sin), 2015, 45(1): 88-92
[8] Choi SK, Yoon JY, Ryu KH, et al. First report of Zucchini yellow mosaic virus on Hollyhock (Althaea rosea)[J]. Plant Pathol J, 2002, 18(3): 121-125
[9] Menzel W, Winter S, Richert-poggeler KR. First report of Malva vein clearing virus naturally occurring in hollyhock in Germany[J]. Plant Dis, 2010, 94(2): 276
[10] Abdel-Salam AM, El-Shazly MA, Thouvenel JC. Biological, biochemical and serological studies on hollyhock leaf crumple virus (HLCrV): a newly discovered whitefly transmitted Geminivirus[J]. Arab J Biotech, 1998, 1(1) 41-58
[11] Srivastava A, Kumar S, Raj SK, et al. Association of a distinct strain of hollyhock yellow vein mosaic virus and Ludwigia leaf distortion betasatellite with yellow vein mosaic disease of hollyhock (Alcea rosea) in India[J]. Arch Virol, 2014, 159(10): 2711-2715
[12] Venkataravanappa V, Lakshminarayana Reddy CN, Devaraju A, et al. Association of a recombinant Cotton leaf curl Bangalore virus with yellow vein and leaf curl disease of okra in India[J]. Indian J Virol, 2013, 24(2): 188-198
[13] Bigarré L, Chazly M, Salah M, et al. Characterization of a new begomovirus from Egypt infecting Hollyhock (Althea Rosea)[J]. Eur J Plant Pathol, 2001, 107(7): 701-711
[14] Mi S, Cai T, Hu Y, et al. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′ terminal nucleotide[J]. Cell, 2008, 133(1): 116-127
[15] 谢雪花. 小RNA深度测序鉴定草莓病毒的研究及草莓快繁体系的优化[D]. 湖南农业大学(Xie Xue-Hua.Identification of Strawberry virus by RNA sequencing and Optimization of Strawberry Rapid Propagation System[D]. Hunan Agricultural University),2016
[16] Zhang Y, Singh K, Kaur R, et al. Association of a novel DNA virus with the grapevine vein-clearing and vine decline syndrome[J]. Phytopathology, 2011, 101(9): 1081-1090
[17] Wang Y, Cheng X, Wu X, et al. Characterization of complete genome and small RNA profile of pagoda yellow mosaic associated virus, a novel badnavirus in China[J]. Virus Res, 2014, 188: 103-108
[18] 陈莎. 深度测序鉴定玉米病毒及感病玉米组织中小RNA分析[D]. 浙江大学(Chen Sha. Identification of plant viruses from maize by deep sequencing and analysis of small RNA in virus infected maize[D]. Zhejiang University), 2015
[19] He Y, Cai L, Zhou L, et al. Deep sequencing reveals the first fabavirus infecting peach[J]. Sci Rep, 2017, 7(1): 11329
[20] 牛颜冰, 时晓丽, 赵慧琪, 等. 白术矮化病毒病病原的分子鉴定和部分序列分析[J]. 植物病理学报(Niu YB, Shi XL, Zhao HQ, et al. Molecular identification and partial sequence analysis of pathogen of Atractylodes macrocephala virus[J]. Acta Phytopathol Sin), 2014, 44(4):357-362
[21] Tzanetakis IE, Martin RR. A new method for extraction of double-stranded RNA from plants [J]. J Virol Methods, 2008, 149(1): 167-170
[22] Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs[J]. Genome Res, 2008, 18(5): 821-829
[23] 周健, 顾兴芳, 张圣平, 等. 黄瓜对西瓜花叶病毒病抗性的研究进展[J]. 中国蔬菜(Zhou J, Gu XF, Zhang SP, et al. Research progress in cucumber resistance to watermelon mosaic virus[J]. China Veg), 2012, (5x): 7-13
[24] 付晶晶, 杨彩霞, 韩彤, 等. 西瓜花叶病毒辽宁臭椿分离物的鉴定与全序列分析[J]. 江西农业大学学报(Fu JJ, Yang CX, Han T, et al. Detection and complete viral sequence analysis of watermelon mosaic virus on Ailanthus altissima in Liaoning[J]. Acta Agriculturae Universitatis Jiangxiensis), 2018, (1): 96- 102
[25] Anderson CW. Two Watermelon mosaic virus strains from central Florida[J]. Phytopathology,1954, 44(4): 198-202
[26] Webb RE, Scott HA. Isolation and identification of watermelon mosaic viruses 1 and 2[J]. Phytopathology, 1965, 55(8): 895-900
[27] Park D, Kim H, Hahn Y. Genome sequence of a distinct watermelon mosaic virus identified from ginseng (Panax ginseng) transcriptome[J]. Acta Virol, 2017, 61(4): 479-482
[28] 任春梅, 程兆榜, 缪倩, 等. 江苏省葫芦科作物西瓜花叶病毒的分子鉴定和序列分析[J]. 南方农业学报(Ren CM, Cheng ZB, Miu Q, et al. Molecular identification and sequence analysis for Watermelon mosaic virus in Jiangsu[J]. J Southern Agriculture), 2013, 44(9): 1464-1470
[29] 李大伟, 牛胜鸟, 韩成贵, 等. 西瓜花叶病毒2号山西分离物外壳蛋白基因核苷酸序列分析[J]. 河南农业大学学报(Li DW, Niu SN, Han CG, et al. Nucleotide sequence analysis of coat protein gene of Watermelon mosaic virus-2 Shanxi isolate[J]. J Henan Agric Univ), 2000, 34(4): 337-340
[30] 秦碧霞, 蔡健和, 刘志明, 等. 广西西瓜花叶病病原病毒鉴定[J]. 广西农业科学(Qin BX, Cai JH, Liu ZM, et al. Identification of watermelon mosaic disease in Guangxi[J]. Guangxi Agric Sci), 2001, 2001(6): 296- 297
[31] 王惠哲, 李淑菊, 霍振荣, 等. 利用RT-PCR检测黄瓜上的西瓜花叶病毒[J]. 天津农学院学报(Wang HZ, Li SJ, Huo ZR, et al. Detection of watermelon mosaic viruses on cucumber by RT-PCR[J]. J Tianjin Agric Univ), 2004, 11(4): 20-22
[32] 陈红运, 赵文军, 程毅, 等. 辽中地区西瓜花叶病病原的分子鉴定[J]. 植物病理学报(Chen HY, Zhao WJ, Cheng Y, et al. Molecular identification of the virus causing watermelon mosaic disease in Mid- Liaoning [J]. Acta Phytopathol Sin), 2006, 36(4): 306-309
[33] 张建新, 吴云锋, 王睿, 等. 西瓜花叶病毒中国分离株全基因组核苷酸序列测定[J]. 病毒学报(Zhang JX, Wu YF, Wang R, et al. Complete nucleotide sequence of watermelon mosaic virus China isolate[J]. Chin J Virol), 2007, 23(2): 153-156
[34] 刘金亮, 王凤婷, 魏毅, 等. 侵染南瓜的西瓜花叶病毒和黄瓜花叶病毒CP基因的克隆和序列分析[J]. 中国农学通报(Liu JL, Wang FT, Wei Y, et al. Cloning and sequence analyses of the coat protein genes of watermelon mosaic virus and cucumber mosaic virus from one mix-infected squash plant[J]. Chin Agric Sci Bull), 2010, 26(16): 262-266
[35] 刘卫荣, 向本春. 西瓜花叶病毒2号新疆昌吉分离物外壳蛋白基因核苷酸序列分析[J]. 植物病理学报(Liu WR, Xiang BC. Nucleotide sequence analysis of coat protein gene of Water melon mosaic virus 2 Xinjiang Changji isolates[J]. Acta Phytopathol Sin), 2008, 38(6): 576-581