臭矢菜丛枝病植原体的分子鉴定研究  

Molecular identification of cleome witches′-broom phytoplasma

作　　者：李永[1] 田国忠[1] 徐启聪[1] 朴春根[1] 汪来发[1] 郭民伟[1] 

机构地区：[1]中国林业科学院森林生态环境与保护研究所国家林业局森林保护学重点实验室,北京100091

出　　处：《植物病理学报》2009年第4期377-384,共8页Acta Phytopathologica Sinica

基　　金：国家科技基础条件平台建设项目(2005DKA21207);国家自然科学基金项目(30471393)

摘　　要：本实验采用DAPI荧光显微镜、PCR、克隆和测序等技术,对海南臭矢菜丛枝病样进行了检测和鉴定。以染病臭矢菜总DNA为模板应用3对植原体特异性引物进行PCR扩增,获得PCR产物为16S rDNA(1 430 bp)、16S-23S rDNA(358bp)、rpDNA(1 294 bp)。应用DNA回收试剂盒获得了3个PCR扩增片断的纯化产物,并克隆到DH5α大肠杆菌中测序。应用DNAMAN和MEGA软件对获得的序列与NCBI数据库中植原体序列进行同源性分析和构建系统发育树。结果显示臭矢菜丛枝病植原体与花生丛枝病植原体序列同源性最高,16S rDNA的序列同源性为99.9%,16S-23S rDNA高达100%,rp为99.7%,因而将臭矢菜丛枝病植原体归为花生丛枝组(16SrⅡ),根据16S rDNA的RFLP分析,将其归为16SrⅡ-A亚组。Cleome witches＇-broom phytoplasma （CWB） collected from Hainan Province, was detected and identified using DAPI fluorescence microscopy, PCR, clone and sequencing techniques. Three different type u- niversal primer pairs of phytoplasma were used to amplify 1.43 kb fragment of 16S rDNA, 0.36 kb fragment of 16S-23S rDNA and 1.3 kb fragment of rp DNA. Then PCR-amplified products were cloned and sequenced. The results of sequencing and homologuous comparison with other phytoplasmas showed that cleome witches＇-broom phytoplasma shared 99.9 % similarity with peanut witches＇ -broom phytoplasma in 16S rRNA gene, 100% in 16S-23S rRNA gene and 99.7% in rp gene. Cleome witches ＇-broom phytoplasma might be sorted into peanut witches＇-broom group（ 16SrⅡ ）, 16Sr Ⅱ - A according to the results of RFLP and sequencing of 16S rDNA.

关 键 词：臭矢菜丛枝病 植原体 分子鉴定 序列测定 

分 类 号：S432.4[农业科学—植物病理学]