金莲花绿变植原体的分子鉴定  

Molecular identification of Trollius chinensis virescence phytoplasma

作　　者：王蓉[1] 陈炳蔚 李勇[1] 刘琨 王天佑 丁万隆[1] WANG Rong;CHEN Bingwei;LI Yong;LIU Kun;WANG Tianyou;DING Wanlong(Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100193,China;College of Traditional Chinese Medicine,Jilin Agricultural University,Changchun 130118,China)

机构地区：[1]中国医学科学院北京协和医学院药用植物研究所,北京100193 [2]吉林农业大学中药材学院,长春130118

出　　处：《植物保护》2020年第3期152-156,共5页Plant Protection

基　　金：中国医学科学院医学与健康科技创新工程(2016-I2M-3-017);中央本级重大增减支项目(2060302)。

摘　　要：本研究对河北省大面积发生的金莲花绿变病的病原进行检测和鉴定。以金莲花叶片的总DNA为模板,使用植原体16S rDNA和核糖体蛋白(ribosomal protein)基因rp的特异性引物进行PCR扩增,在感病金莲花样品中扩增到植原体的16S rDNA(1 432 bp)片段和rp基因(1 240 bp)片段。序列分析发现,获得的16S rDNA和rp基因片段与洋葱黄化植原体Onion yellows phytoplasma(GenBank登录号:AP006628)的相似度最高,分别为99.9%和99.3%,确定金莲花绿变病的病原为植原体,暂命名为金莲花绿变植原体Trollius chinensis virescence phytoplasma。对金莲花绿变植原体的16S rDNA进行虚拟RFLP分析,发现其酶切图谱与16SrⅠ-B亚组的洋葱黄化植原体的参照图谱完全一致,相似系数1.00。16S rDNA和rp基因的系统发育进化树显示,金莲花绿变植原体与16SrⅠ-B亚组的植原体聚为一支,属于植原体16S rⅠ-B亚组。Trollius chinensis virescence disease widely occurred in Hebei province. In this study, the pathogen of this disease was detected and identified. To determine the pathogen of T.chinensis virescence disease, PCR was performed using total DNA extracted from symptomatic and symptomless leaves of T.chinensis, with primers specifically amplifying 16 S rDNA and ribosomal protein gene(rp) of phytoplasma. A 16 S rDNA fragment of 1 432 bp and a rp fragment of 1 240 bp were amplified from symptomatic samples, while no expected PCR product was obtained from symptomless samples. Sequence analysis showed that 16 S rDNA fragment and rp fragment of T.chinensis virescence shared the highest nucleotide identity(99.9% and 99.3%, respectively) with those of Onion yellows phytoplasma(GenBank accession No.AP006628). Therefore, we confirmed that the pathogen of T.chinensis virescence disease was phytoplasma, tentatively named T.chinensis virescence phytoplasma. The virtual restriction fragment length polymorphism(RFLP) pattern derived from the 16 S rDNA fragment of T.chinensis virescence phytoplasma is identical(similarity coefficient 1.00) to the reference pattern of Onion yellow phytoplasma, a representative member of 16 Sr group Ⅰ, subgroup B. Phylogenetic analysis showed that 16 S rDNA and rp gene of T.chinensis virescence phytoplasma clustered in 16 Sr group Ⅰ, subgroup B.

关 键 词：金莲花 绿变病 植原体 16S RDNA 核糖体蛋白 系统发育进化树 

分 类 号：S435.672[农业科学—农业昆虫与害虫防治]