侵染滇黄精的菜豆普通花叶病毒的检测与鉴定

doi:10.19586/j.2095-2341.2018.0140

生物技术进展 ›› 2019, Vol. 9 ›› Issue (2): 122-128.DOI: 10.19586/j.2095-2341.2018.0140

• 植物-微生物互作专栏 • 上一篇下一篇

侵染滇黄精的菜豆普通花叶病毒的检测与鉴定

陈泽历1,杨林毅2,陈潞2,孙雁2,魏朝霞2,李永忠3,赵明富2*,文国松1*

1.云南农业大学农学与生物技术学院, 昆明 650201；

2.云南农业大学, 农业生物多样性与病虫害控制教育部重点实验室, 昆明 650201;

3.云南农业大学烟草学院, 昆明 650201

收稿日期:2018-12-29 出版日期:2019-03-25 发布日期:2019-01-24
通讯作者: 赵明富，副教授，博士，主要从事植物病理学研究。E-mail：zhaomingfu@163.com；文国松，研究员，博士，主要从事中药学研究。E-mail：wengs@163.com
作者简介:陈泽历，硕士研究生，研究方向为药用植物资源评价与种质创新。E-mail：18387112694@163.com。
基金资助:
云南省农业综合开发省级科技推广示范项目资助。

Detection and Identification of BCMV Infecting Polygonatum kingianum

CHEN Zeli, YANG Linyi, CHEN Lu, SUN Yan, WEI Zhaoxia, LI Yongzhong, ZHAO Mingfu, WEN Guosong

1.College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, China;

2.Key Laboratory of Agrobiodiversity and Pest Management of the Ministry of Education, Yunnan Agricultural University, Kunming 650201, China;

3.College of Tobacco Science, Yunnan Agricultural University, Kunming 650201, China

Received:2018-12-29 Online:2019-03-25 Published:2019-01-24

摘要/Abstract

摘要： 滇黄精作为一种重要的中药材，应用领域广泛。随着其市场需求量的增加，野生滇黄精已较为稀少，因而人工种植产业得到发展，但大面积的单一种植导致滇黄精病害发生较为严重。目前对于滇黄精真菌病（如叶斑病、炭疽病、黑斑病等）的研究较多，但尚未有其病毒病的报道。为了明确侵染滇黄精并引起其花叶、叶片皱缩症状的病毒病原，对采集于云南文山的滇黄精病样采用透射电子显微镜观察以及双抗体夹心酶联免疫吸附法（double antibody sandwich enzyme-linked immunosorbent assay，DAS-ELISA）、反转录PCR（reverse transcription PCR，RT-PCR）技术检测。对侵染症状明显的滇黄精病叶进行病毒粒子提纯，在电子显微镜下可观察到线状病毒粒子，大小为(670~760) nm×(9~12) nm。通过DAS-ELISA检测出3份滇黄精病样（DHJ1、DHJ2、DHJ3）汁液与菜豆普通花叶病毒（Bean common mosaic virus，BCMV）抗体呈强阳性反应。随后提取DAS-ELISA检测呈阳性的DHJ1病样的总RNA，利用马铃薯Y病毒科特异性简并引物（Sprimer/M4T）进行RT-PCR扩增，并对扩增产物进行测序分析。结果表明，所得序列长为1 609 bp，其中包含588 bp的部分核内含体蛋白b基因（NIb）、864 bp的完整外壳蛋白（coat protein，CP）基因和157 bp的3′端非翻译区（3′-untranslated region,3′-UTR）。而同源性分析显示该序列与马铃薯Y病毒科(Potyviridae)马铃薯Y病毒属（Potyvirus）的BCMV大豆分离物（KJ807813）的核苷酸序列和CP氨基酸序列的同源性分别高达99%和100%，与BCMV花生分离物（HM776124）的同源性分别为97%和99%。基于CP氨基酸序列的系统发育分析表明，该病毒与已报道的BCMV中国大豆分离物（KJ807813）、花生分离物（HM776124）亲缘关系最近，聚类为一簇。这是首次报道病毒侵染黄精属植物，将其命名为菜豆普通花叶病毒滇黄精分离物（BCMV-DHJ1），表明菜豆普通花叶病毒能自然侵染滇黄精。

关键词: 滇黄精, 菜豆普通花叶病毒, 检测与鉴定

Abstract: As an important Chinese medicinal material, Polygonatum kingianum is widely used in many fields. With the increase of its market demand, wild resources of Polygonatum kingianum are already scarce, as a result, the artificial planting industry has been developed. However, the large-area monoculture leads to the serious disease of Polygonatum kingianum. At present, there are many studies on fungal diseases of Polygonatum kingianum, such as leaf spot, anthracnose and black spot, but no report of its viral diseases. In order to ascertain the viral pathogen that infects Polygonatum kingianum and causes the symptoms of mosaic leaf and leaf shrinkage, the diseased samples of Polygonatum kingianum collected from Wenshan in Yunnan province were observed by transmission electron microscope (TEM) and detected with DAS-ELISA and RT-PCR. The leaves of Polygonatum kingianum with obvious infection symptoms were purified to obtain their virus particles, and the shapes of virus were filamentous as observed by a TEM, with the size of (670~760) nm × (9~12) nm. The juice of Polygonatum kingianum samples (DHJ1，DHJ2，DHJ3) with infection symptoms were strongly positive against Bean common mosaic virus (BCMV) antibody detected by DAS-ELISA. Whereafter, total RNA of DHJ-1 which was a positive sample detected by DAS-ELISA was extracted, and amplified by RT-PCR with genera-specific degenerate primers of Potyviridae (Sprimer/M4T), and then the amplified products were sequenced and analyzed. The results showed that the length of obtained sequence was 1 609 bp, including partial nucleosome inclusion body protein b gene (NIb) (588 bp), full-length coat protein (CP) gene (864 bp) and 3′-UTR (157 bp). BCMV belongs to the genus Potyvirus in the family Potyviridae, and the results of homology analysis indicated that the sequence of DHJ1 was 99% and 100% homologous with the nucleotide sequence and CP amino acid sequence of the BCMV Glycine max isolate (KJ807813), respectively, and 97% (at nt level) and 99% (at aa level) homologous with BCMV Arachis hypogaea isolate (HM776124). Phylogenetic analysis based on CP amino acid sequences revealed that the virus DHJ-1 was closely related to the Chinese Glycine max isolate (KJ807813) and Arachis hypogaea isolate (HM776124), and they clustered in one group. It was the first report of virus infecting the genus Polygonatum, which was named as BCMV-DHJ1, indicating that BCMV could naturally infect Polygonatum kingianum.

Key words: Polygonatum kingianum, Bean common mosaic virus, detection and identification

陈泽历,杨林毅,陈潞,孙雁,魏朝霞,李永忠,赵明富,文国松. 侵染滇黄精的菜豆普通花叶病毒的检测与鉴定[J]. 生物技术进展, 2019, 9(2): 122-128.

CHEN Zeli1, YANG Linyi2, CHEN Lu2, SUN Yan2, WEI Zhaoxia2, LI Yongzhong3, ZHAO Mingfu2*, WEN Guosong1*. Detection and Identification of BCMV Infecting Polygonatum kingianum[J]. Curr. Biotech., 2019, 9(2): 122-128.

侵染滇黄精的菜豆普通花叶病毒的检测与鉴定

Detection and Identification of BCMV Infecting Polygonatum kingianum

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