蚕豆萎蔫病毒2号板蓝根分离株全基因组序列测定与分析
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摘要
板蓝根(Isatidis Radix)病毒病害的发生已对其产量和品质造成了严重影响。因此,建立一套灵敏、快速、有效的板蓝根病毒病害检测手段十分重要。本研究利用双链RNA(double-stranded RNA,dsRNA)和非序列依赖PCR扩增(sequence-independent amplification,SIA)等技术对感病板蓝根进行鉴定,确定其被蚕豆萎蔫病毒2号(Broad bean wilt virus 2, BBWV2)所侵染。为明确BBWV2板蓝根分离物(BBWV2-IR)的进化关系,对其全基因组进行序列扩增与分析,获得其RNA1序列全长为5 955 bp,RNA2序列全长为3 602 bp,分别编码由1 870和1 064个氨基酸组成的多聚蛋白质。序列比对发现,BBWV2-IR RNA1与BBWV2-Am分离物的同源性最高,RNA2与BBWV2-SN分离物的同源性最高。全基因组变异情况分析表明,BBWV2-IR RNA1和RNA2分别与其同源性最高的株系存在多个氨基酸变异位点。系统进化分析表明,BBWV2-IR RNA1与BBWV2-Am RNA1聚为一簇,RNA2与BBWV2-SN RNA2聚为一簇,亲缘性最近。本研究获得了BBWV2板蓝根分离株的全基因组序列,并明确其在进化过程中的地位和区域变化情况,为进一步研究BBWV2-IR致病性变异提供一定的理论基础。
The occurrence of the Isatidis Radix virus disease has seriously influences on its yield and quality. Therefore, it is very important to establish a sensitive, rapid and effective detection method for the Isatidis Radix virus disease. In this study, double-stranded RNA(dsRNA) and sequence-independent amplification(SIA) methods were used to identify the pathogens of Isatidis Radix. The results showed that the Isatidis Radix was infected by Broad bean wilt virus 2(BBWV2). In order to clarify the evolutionary relationship of Isatidis Radix isolates of BBWV2, the whole genome sequence was amplified and analyzed. The full length of RNA1 and RNA2 sequences were 5 955 and 3 602 bps, encoding polypeptides of 1 870 and 1 064 amino acids, respectively. The sequence alignment showed that BBWV2-IR RNA1 had the highest homology with BBWV2-Am and RNA2 has the highest homology with BBWV2-SN. Analysis of genome variation showed that BBWV2-IR RNA1 and RNA2 has multiple amino acid variation sites with the most homologous strain, respectively. Phylogenetic analysis showed that BBWV2-IR RNA1 and BBWV2-Am RNA1 clustered together, and RNA2 and BBWV2-SN RNA2 clustered together, which was closely related. In this study, the whole genome sequence of BBWV2-IR was obtained for the first time, and its status and regional changes in the evolutionary process were clarified, which will provided a theoretical basis for further study of pathogenic variation of BBWV2-IR.
The occurrence of the Isatidis Radix virus disease has seriously influences on its yield and quality. Therefore, it is very important to establish a sensitive, rapid and effective detection method for the Isatidis Radix virus disease. In this study, double-stranded RNA(dsRNA) and sequence-independent amplification(SIA) methods were used to identify the pathogens of Isatidis Radix. The results showed that the Isatidis Radix was infected by Broad bean wilt virus 2(BBWV2). In order to clarify the evolutionary relationship of Isatidis Radix isolates of BBWV2, the whole genome sequence was amplified and analyzed. The full length of RNA1 and RNA2 sequences were 5 955 and 3 602 bps, encoding polypeptides of 1 870 and 1 064 amino acids, respectively. The sequence alignment showed that BBWV2-IR RNA1 had the highest homology with BBWV2-Am and RNA2 has the highest homology with BBWV2-SN. Analysis of genome variation showed that BBWV2-IR RNA1 and RNA2 has multiple amino acid variation sites with the most homologous strain, respectively. Phylogenetic analysis showed that BBWV2-IR RNA1 and BBWV2-Am RNA1 clustered together, and RNA2 and BBWV2-SN RNA2 clustered together, which was closely related. In this study, the whole genome sequence of BBWV2-IR was obtained for the first time, and its status and regional changes in the evolutionary process were clarified, which will provided a theoretical basis for further study of pathogenic variation of BBWV2-IR.
引文
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[3] 年士恒, 刘敏. 板蓝根清热解毒作用机理研究进展[J]. 淮海医药(Zhang SH, Liu M. Research progress on mechanism of heat clearing and detoxicating of Radix Isatidis[J]. J Huaihai Med), 2005, 23(5): 428-429
[4] 马毅敏, 李娜, 刘承伟, 等. 板蓝根不同提取部位抗炎镇痛活性比较研究[J]. 中草药(Ma YM, Li N, Liu CW, et al. Comparative studies on anti-inflammatory and analgesic activities of different fractions from Isatidis Radix[J]. Chin Tradit Herb Drugs), 2014, 45(17): 2517-2521
[5] 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
[6] 寇丽莎, 赵慧琪, 王德富, 等. 药用植物柴胡病毒病病原物的分子鉴定[J]. 病毒学报(Kou LS, Zhao HQ, Wang DF, et al. Molecular identification and of pathogens after Bupleurum Chinense infection[J]. Chin J Virol),2017, 33(4): 610-616
[7] 张璇, 赵慧琪, 王德富, 等. 黄瓜花叶病毒(CMV)2个山西分离CP 序列测定及亚组分类分析[J]. 植物病理学报(Zhang X, Zhao HQ, Wang DF, et al. Sequence and subgroup analysis of two new isolates of Cucumber mosaic virus (CMV) in Shanxi[J]. Acta Phytopathol Sin), 2018, 48(1): 35-45
[8] Krajacic M, Ivancic-Jelecki J, Forcic D, et al. Purification of plant viral and satellite double-stranded RNAs on DEAE monoliths[J]. J Chromatogr A, 2007, 1144(1): 111-119
[9] Tzanetakis IE, Martin RR. A new method for extraction of double-stranded RNA from plants [J]. J Virol Methods, 2008, 149(1): 167-170
[10] 郑光宇, Kositratana W, Gumpf DJ. 应用斑点免疫结合法检测植物病毒[J]. 病毒学报(Zheng GY, Kositratana W, Gumpf DJ. Detection of plant viruses using Dot-Immunobiding assay[J]. Chin J Virol), 1988, 4(2): 59-65
[11] 牛颜冰, 姚敏, 王德富, 等. 臭椿病毒病病原鉴定[J]. 植物病理学报(Niu YB, Yao M, Wang DF, et al. Identification of the pathogenic virus on Ailanthus altissima[J]. Acta Phytopathol Sin), 2011, 41(4): 437-440
[12] 童秀英, 杨文权, 寇建村. 药用植物病毒病及其防治[J]. 青海农林科技(Tong XY, Yang WQ, Kou JC. Virus disease of medical plants and its control[J]. Sci Technol Qinghai Agric For), 2005, (2): 33-34
[13] Ferrer RM, Ferriol I, Moreno P, et al. Genetic variation and evolutionary analysis of broad bean wilt virus 2[J]. Arch Virol, 2011, 156(8): 1445-1450
[14] 刘成科, 李燕宏, 王卫兵, 等. 蚕豆萎蔫病毒2号分离物PV131和P158侵染寄主植物病理比较观察[J]. 植物病理学报(Liu CK, Li YH, Wang WB, et al. Comparison of cytopathological changes of plant hosts infected with two Broad bean wilt virus2 isolates PV131 and P158[J]. Acta Phytopathol Sin), 2009, 39(2): 168-173
[15] Lisa V, Boccardo G. Fabaviruses: Broad bean wilt and allied viruses[M]. The Plant Viruses, 1996: 229-250
[16] 刘成科. 蚕豆萎蔫病毒号细胞病理学及VP37蛋白功能的研究[D]. 浙江大学(Liu CK. Study of cytopathology Broad bean wile virus 2 and function of VP37 protein[D]. Zhejiang University), 2008
[17] Stubbs LL. A destructive vascular wilt virus disease of broad bean (Vicia faba L.) in Victoria[J]. J Dept Agric Victoria, 1947, 46: 323
[18] 周雪平, 戚益军, 李凡, 等. 蚕豆萎蔫病毒2中国分离物全基因组结构及可能的基因表达方式[J]. 生物化学与生物物理学报(Zhou XP, Qi YJ, Li F, et al. Complete nucleotide sequence and possible genomic expression strategy of a Chinese isolate of Broad Bean Wilt Virus 2[J]. Chin J Biochem Biophys), 2001, 33(1): 44-52
[19] 牛颜冰, 赵欣, 赵慧琪, 等. 蚕豆萎蔫病毒病2号山西分离物株的全基因组序列测序与分析[J]. 中国生物化学与分子生物学报(Niu YB, Zhao X, Zhao HQ, et al. Identification and analysis of complete genomic sequence of Broad bean wilt virus 2 Shanxi Isolate[J]. Chin J Biochem Mol Biol), 2014, 30(9): 927-932
[20] 匡云波, 叶炜, 李金辉, 等. 太子参蚕豆萎蔫病毒2号CP基因的遗传多样性及分子进化分析[J]. 植物病理学报(Kuang YB, Ye W, Li JH, et al. Genetic polymorphism and phylogenetic analysis of Broad bean wilt virus 2 CP genes isolated from Pseudostellaria heterophylla[J]. Acta Phytopathol Sin), 2017, 47(4): 470-478
[21] 严丹侃, 郑红英, 张海珊, 等. 蚕豆萎蔫病毒2号安徽分离物全基因组序列测定与分析[J]. 植物保护(Yan DK, Zheng HY, Zhang HS, et al. Identification and analysis of complete genomic sequence of Broad bean wilt virus 2 Anhui isolate[J]. Plant Prot), 2018, 44(1): 67-73
[2] 林丽君, 聂黎行, 戴忠, 等. 板蓝根的研究概况[J]. 中国药业(Lin LJ, Nie LX, Dai Z, et al. Research review on Radix Isatidis[J]. China Pharm), 2015, 24(21): 1-4
[3] 年士恒, 刘敏. 板蓝根清热解毒作用机理研究进展[J]. 淮海医药(Zhang SH, Liu M. Research progress on mechanism of heat clearing and detoxicating of Radix Isatidis[J]. J Huaihai Med), 2005, 23(5): 428-429
[4] 马毅敏, 李娜, 刘承伟, 等. 板蓝根不同提取部位抗炎镇痛活性比较研究[J]. 中草药(Ma YM, Li N, Liu CW, et al. Comparative studies on anti-inflammatory and analgesic activities of different fractions from Isatidis Radix[J]. Chin Tradit Herb Drugs), 2014, 45(17): 2517-2521
[5] 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
[6] 寇丽莎, 赵慧琪, 王德富, 等. 药用植物柴胡病毒病病原物的分子鉴定[J]. 病毒学报(Kou LS, Zhao HQ, Wang DF, et al. Molecular identification and of pathogens after Bupleurum Chinense infection[J]. Chin J Virol),2017, 33(4): 610-616
[7] 张璇, 赵慧琪, 王德富, 等. 黄瓜花叶病毒(CMV)2个山西分离CP 序列测定及亚组分类分析[J]. 植物病理学报(Zhang X, Zhao HQ, Wang DF, et al. Sequence and subgroup analysis of two new isolates of Cucumber mosaic virus (CMV) in Shanxi[J]. Acta Phytopathol Sin), 2018, 48(1): 35-45
[8] Krajacic M, Ivancic-Jelecki J, Forcic D, et al. Purification of plant viral and satellite double-stranded RNAs on DEAE monoliths[J]. J Chromatogr A, 2007, 1144(1): 111-119
[9] Tzanetakis IE, Martin RR. A new method for extraction of double-stranded RNA from plants [J]. J Virol Methods, 2008, 149(1): 167-170
[10] 郑光宇, Kositratana W, Gumpf DJ. 应用斑点免疫结合法检测植物病毒[J]. 病毒学报(Zheng GY, Kositratana W, Gumpf DJ. Detection of plant viruses using Dot-Immunobiding assay[J]. Chin J Virol), 1988, 4(2): 59-65
[11] 牛颜冰, 姚敏, 王德富, 等. 臭椿病毒病病原鉴定[J]. 植物病理学报(Niu YB, Yao M, Wang DF, et al. Identification of the pathogenic virus on Ailanthus altissima[J]. Acta Phytopathol Sin), 2011, 41(4): 437-440
[12] 童秀英, 杨文权, 寇建村. 药用植物病毒病及其防治[J]. 青海农林科技(Tong XY, Yang WQ, Kou JC. Virus disease of medical plants and its control[J]. Sci Technol Qinghai Agric For), 2005, (2): 33-34
[13] Ferrer RM, Ferriol I, Moreno P, et al. Genetic variation and evolutionary analysis of broad bean wilt virus 2[J]. Arch Virol, 2011, 156(8): 1445-1450
[14] 刘成科, 李燕宏, 王卫兵, 等. 蚕豆萎蔫病毒2号分离物PV131和P158侵染寄主植物病理比较观察[J]. 植物病理学报(Liu CK, Li YH, Wang WB, et al. Comparison of cytopathological changes of plant hosts infected with two Broad bean wilt virus2 isolates PV131 and P158[J]. Acta Phytopathol Sin), 2009, 39(2): 168-173
[15] Lisa V, Boccardo G. Fabaviruses: Broad bean wilt and allied viruses[M]. The Plant Viruses, 1996: 229-250
[16] 刘成科. 蚕豆萎蔫病毒号细胞病理学及VP37蛋白功能的研究[D]. 浙江大学(Liu CK. Study of cytopathology Broad bean wile virus 2 and function of VP37 protein[D]. Zhejiang University), 2008
[17] Stubbs LL. A destructive vascular wilt virus disease of broad bean (Vicia faba L.) in Victoria[J]. J Dept Agric Victoria, 1947, 46: 323
[18] 周雪平, 戚益军, 李凡, 等. 蚕豆萎蔫病毒2中国分离物全基因组结构及可能的基因表达方式[J]. 生物化学与生物物理学报(Zhou XP, Qi YJ, Li F, et al. Complete nucleotide sequence and possible genomic expression strategy of a Chinese isolate of Broad Bean Wilt Virus 2[J]. Chin J Biochem Biophys), 2001, 33(1): 44-52
[19] 牛颜冰, 赵欣, 赵慧琪, 等. 蚕豆萎蔫病毒病2号山西分离物株的全基因组序列测序与分析[J]. 中国生物化学与分子生物学报(Niu YB, Zhao X, Zhao HQ, et al. Identification and analysis of complete genomic sequence of Broad bean wilt virus 2 Shanxi Isolate[J]. Chin J Biochem Mol Biol), 2014, 30(9): 927-932
[20] 匡云波, 叶炜, 李金辉, 等. 太子参蚕豆萎蔫病毒2号CP基因的遗传多样性及分子进化分析[J]. 植物病理学报(Kuang YB, Ye W, Li JH, et al. Genetic polymorphism and phylogenetic analysis of Broad bean wilt virus 2 CP genes isolated from Pseudostellaria heterophylla[J]. Acta Phytopathol Sin), 2017, 47(4): 470-478
[21] 严丹侃, 郑红英, 张海珊, 等. 蚕豆萎蔫病毒2号安徽分离物全基因组序列测定与分析[J]. 植物保护(Yan DK, Zheng HY, Zhang HS, et al. Identification and analysis of complete genomic sequence of Broad bean wilt virus 2 Anhui isolate[J]. Plant Prot), 2018, 44(1): 67-73
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