中国各地不同枣树品种上枣疯病植原体的PCR检测及分子变异分析  

Molecular detection and variability of jujube witches'-broom phytoplasmas from different cultivars in various regions of China

作　　者：徐启聪[1] 田国忠[1] 王振亮[2] 孔繁华[3] 李永[1] 王合 

机构地区：[1]中国林业科学研究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 [2]河北林业科学院森林保护研究所,石家庄050061 [3]山东泰安市农业科学研究院,泰安271000 [4]北京市林业保护站,北京100029

出　　处：《微生物学报》2009年第11期1510-1519,共10页Acta Microbiologica Sinica

基　　金：科技部“十一五”林业科技支撑计划项目“商品林重大生物灾害防控技术研究”项目(2006BAD08A113-2);国家质量监督检验检疫总局公益性行业科研专项“重要果树黄化病原鉴定技术标准研制”(200810517-3)~~

摘　　要：【目的】检测不同地区枣树品种上的枣疯植原体侵染及保守基因序列的变异。【方法】利用植原体16S rDNA的通用引物R16mF2/R16mR1、16S-23S间区序列(SR)的通用引物SR1/SR及secY基因引物FD9f/r,通过PCR检测采自国内7个地区14个枣树品种上的32个枣疯病和4个酸枣丛枝病样品。将PCR产物进行直接或克隆测序,结合已报导的测序数据,进行序列同源性和系统进化分析。【结果】所有枣疯病样品中均检测到植原体;皆属于榆树黄化16SrV-B亚组,与我国重阳木丛枝和樱桃致死黄化遗传关系更近,而与国外的其他植原体亲缘关系较远。不同地区和不同抗性品种上的植原体在16S rDNA、SR和secY基因上都存在程度不同的序列差异。直接测序法检出的优势株系分布范围很广。不同株系间的secY基因变异比16S rDNA和SR更明显,某些碱基替换并不影响编码氨基酸种类,但有的株系的碱基缺失使编码框中断。【结论】不同地区不同品种枣树上枣疯植原体间存在着较为丰富的遗传多样性,与PCR产物直接测序法相比,用克隆测序结果能检测到更多的碱基突变,对于鉴别不同植原体株系及研究其系统发育关系更为有用。[ Objective] Jujube witches＇-broom is an important disease in jujube cultivation areas, which causes serious losses in jujube fruit production. To understand the genetic variability and diversity of jujube witches＇-broom phytoplasma population from the different cultivars and various regions of China. [ Method] We collected 32 samples from 14 cuhivars or wild sour jujubes in 7 regions of China and detected them with PCR with the primers R16mF2/R16mR1 for phytoplasma 16S rDNA, SR1/SR for16S- 23SrRNA space region （SR） and FD9f/r for secretion proteins （secY）. The direct sequencing of PCR products and sequencing by cloned PCR products were used for sequence polymorphism and phylogenetic analyses by comparison to the databases of known conserved gene sequences. [ Results] We detected phytoplasmas by PCR amplification of 16SrDNA from all the diseased jujube samples. All the phytoplasma isolates infected various jujube cultivars belonged to subgroup 16SrV-B of elm yellows group and had closer homology with Bischofia polycarpa witches＇-broom and cherry lethal yellows phytoplasmas occurred in China than other 16SrV phytoplasmas in other countries. The sequence polymorphism at different extent in 16SrDNA, SR and secY gene and genetic diversity were revealed in phytoplasma strain population related to different habitats, among which the dominant strains were always detected by the direct sequencing of PCR products in all the diseased areas of China. The degree of variability on secY gene of collected phytoplasma strains was greater than that of 16SrDNA and SR sequences, and some base substitutions could not alter encoded amino acid, however certain single base deletions detected in a Shandong and a Beijing strains may have impact on the gene structure or function. [ Conclusion] Phytoplasma strains from different cultivars and regions show dramatic genetic diversity. Compared with direct sequencing of PCR products, the sequencing by cloning PCR products was more useful for the displaying of variants and phylog

关 键 词：枣疯病 植原体 16S rDNA 16S-23S间区 secY基因 分子变异 

分 类 号：S436.65[农业科学—农业昆虫与害虫防治]