葡萄金黄化植原体16SrDNA检测与RFLP分析  

Detection and RFLP Analysis of 16S Ribosomal DNA of Phytoplasma Flavescence dorée

作　　者：乾义柯[1,2] 张祥林[1] 马春春[2] 王翀[1] 

机构地区：[1]新疆出入境检验检疫局,乌鲁木齐830063 [2]新疆农业大学农学院,乌鲁木齐830052

出　　处：《新疆农业科学》2010年第5期915-920,共6页Xinjiang Agricultural Sciences

基　　金：国家质检总局质检公益性行业科研专项项目(200810517);国家质检总局科研项目(2008IK243)

摘　　要：【目的】对多种植原体16SrDNA基因序列进行限制性片段长度多态性(RFLP)分析比较,以期区分葡萄金黄化植原体不同亚种,从而为葡萄金黄化植原体的鉴定提供新依据。【方法】用植原体通用引物R16mF2/R16mR1扩增7种不同植原体16SrDNA基因序列,得到约1.5 kb的DNA片段,将此片段克隆到PGM-T载体,并通过酶切鉴定,对重组阳性克隆进行核酸序列测定及同源性分析,利用限制性内切酶XmnⅠ、XspⅠ、TaqⅠ和RsaⅠ对目的片段进行酶切电泳分析。【结果】所扩增2个不同亚种的葡萄金黄化植原体D型和C型序列同源性最高,达99.72%,但利用限制性内切酶可以将上述2个亚种从植原体榆树黄化组中区分。【结论】利用限制性内切酶分析通用引物R16mF2/R16mR1所扩增的基因片段,可以将葡萄金黄化植原体区分。【Objective】The phytoplasma 16SrDNA gene sequences were compared through RFLP analysis,based on the similarity of RFLP patterns,Flavescence dorée would be differentiated from other strains,provide a new thereunder for detecting Flavescence dorée.【Method】A 1.5 kb DNA fragment was amplified by PCR with primer R16mF2/R16mR1 from 16SrDNA of 7 different phytoplasmas.This amplified fragment was ligated into PGM-T vector and the clones were verified by restriction enzyme digestion,sequenced and homology analysis.The amplified fragments were analyzed with XmnⅠ,XspⅠ,TaqⅠ or Rsa Ⅰand separated by electrophoresis.【Result】The result showed that the Flavescence dorée D and Flavescence dorée C strains had the highest similarity（99.72%）,but this tow strains could be differentiated from Elm Yellows group by restriction enzyme digestion.【Conclusion】Phytoplasma Flavescence dorée could be differentiated through RFLP analysis of 16SrDNA amplified with primer R16mF2/R16mR1.

关 键 词：葡萄金黄化植原体 16SrDNA 限制性片段长度多态性 

分 类 号：S463.631[农业科学—植物保护]