摘要
从四川攀枝花芒果园中表现为丛枝、小叶和黄花等症状的坡柳植株发病样品中,利用植原体16S rDNA基因的通用引物R16m F2/R16m R1和R16F2n/R16R2,对发病植株总DNA进行巢式PCR检测,同时设计植原体抗原膜蛋白基因（AntMP）的保守引物AntMP-F/AntMP-R进行PCR验证。结果显示,坡柳样品巢式PCR的第一轮、第二轮DNA条带大小分别为1 400 bp和1 200 bp左右,经NCBI序列相似性比较均为植原体16S rDNA序列,Gen Bank登录号为KT957205和KT957206;PCR结果显示抗原膜蛋白基因大小约600 bp,与目标基因大小一致。将测得的16S rDNA基因序列与Gen Bank数据库中登录的16SrⅠ～ⅩⅤ组植原体16S rDNA序列进行同源性比对,构建系统进化树,结果显示四川坡柳丛枝植原体（DOVI-SC）属于16SrⅠ组（即翠菊黄花组）,与已报道的5个16SrⅠ组（AY101386,AY566302,AY389822,L33760和KP662119）同属一个组。利用植原体亚组分类鉴定软件iPhy Classfier对获得的2条植原体16S rDNA序列进行虚拟RFLP分析,结果显示KT957205,KT957206与16SrⅠ-B亚组洋葱黄化植原体（NC-005303）相似度分为1.0、0.97,归属于16SrⅠ-B亚组。本研究对引起四川坡柳丛枝的植原体病原进行检测,为坡柳植原体病害的早期诊断、快速检测以及预防措施制定提供科学线索。
By using the common primers R16mF2/R16mR1 and R16F2n/R16R2, the nested PCR was applied to detect the pathogen DNA of infected plants of Dodonaea viscosa L. with symptons as witches＇ broom, small leaves and yellow flowers from mango growing area in Panzhihua, Sichuan province. The conserved primer pairs,AntMP-F/AntMP-R, of antigen membrane protein gene（AntMP） were also designed for PCR validation. The results showed that the DNA band sizes about 1 400 bp and 1 200 bp were a mplified from the first round and the second round of nested PCR, respectively. Both sequences were phytoplasma 16 S rDNA sequences by NCBI blast search. The sequences were deposited in Gen Bank（Accession number KT957205 and KT957206, respectively）. PCR results showed that the size of AntMP was about 600 bp, which was the same as the target gene size. The fragment of antigen membrane protein gene also showed that Dodonaea viscosa L. was infected by the phytoplasma. The16 S rDNA gene sequence was compared with 16 S rDNA gene sequences of 16 Sr Ⅰ ～ ⅩⅤ phytoplasma groups recorded in Gen Bank database and was used for phylogenetic tree construction. The results showed that this phytoplasma belonged to 16 SrⅠgroup（aster huanghua group）, and together with other five phytoplasmas（AY101-386, AY566302, AY389822, L33760 and KP662119） belonged to the same group. The virtual RFLP analysis of16 S rDNA sequences of two phytoplasmas was carried out by using phytoplasma subgroup classification and identification software iPhy Classfier. The results showed that the similarity between KT957205, KT957206 and16 Sr Ⅰ-B subgroup of onion yellows phytoplasma（NC-005303） was 1.00 and 0.97, respectively, belonging to16 SrⅠ-B subgroup. The pathogen of phytoplasma causing witches＇ broom on Dodonaea viscosa L. was detected in this study, which might provide the scientific clues for early diagnosis, rapid detection and establishment of prevention methods against phytoplasmadisease of Dodonaea viscosa L.
作者
笈小龙
吴育鹏
谢慧敏
余乃通
王健华
张秀春
刘志昕
Ji Xiaolong;Wu Yupeng;Xie Huimin;Yu Naitong;Wang Jianhua;Zhang Xiuchun;Liu Zhixin(Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, 570228;Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101;Quality and Safety Inspection Station of Agricultural Products, Wenchang, 571300)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2018年第6期2423-2429,共7页
Genomics and Applied Biology
基金
海南省重大科技计划项目(ZDKJ2017003)
海南省热带果树生物学重点实验室开放课题(KFZX2017002)
中国热带作物学会青年人才托举工程项目(CSTC-QN2017004)共同资助
关键词
坡柳
植原体
分子鉴定
系统进化树
Dodonaea viscosa L.
Phytoplasma
Molecular identification
Phylogenetic tree
