应用PCR-RFLP技术鉴别松材线虫与拟松材线虫  

Identification of Bursaphelenchus xylophilus and B.mucronatus by PCR-RFLP Technique

作　　者：陈凤毛[1] 叶建仁[1] 汤坚[2] 吴小芹[1] 

机构地区：[1]南京林业大学森林资源与环境学院,江苏南京210037 [2]安徽省林业厅,安徽合肥230031

出　　处：《南京林业大学学报（自然科学版）》2006年第4期5-9,共5页Journal of Nanjing Forestry University：Natural Sciences Edition

基　　金：安徽省林业厅与南京林业大学合作研究项目(2002-2005);江苏省博士后基金计划项目(2005-2007)

摘　　要：应用PCR-RFLP技术分析松材线虫与拟松材线虫之间的差异。实验扩增出松材线虫rDNA-ITS区片段长度约为890 bp,拟松材线虫rDNA-ITS区片段长度约为930 bp。用5种限制性内切酶对两种线虫的ITS区扩增产物进行酶切,结果表明:(1)DraI酶切松材线虫群体均产生两个长度约510 bp和380 bp的片段,而拟松材线虫均不能被DraI酶切;(2)所有的松材线虫群体与拟松材线虫群体的ITS区扩增产物均不能被ApaI酶切;(3)用MspI酶切松材线虫群体,除GZ02不能够被酶切外,其余样本能够产生两条长度分别为530 bp和360 bp的谱带。拟松材线虫群体,都能产生3条一致的亮带,长度分别为340 bp2、90 bp、180 bp;(4)所有松材线虫样本均不能被SalI酶切,而拟松材线虫群体均能够被酶切为两个720 bp和220 bp的片段;(5)XhoI酶切松材线虫ITS区为两条大小分别为520 bp和370 bp的谱带。而拟松材线虫产生两条大小分别为530 bp和400 bp的谱带。因此,内切酶DraI、SalI可以用于检测松材线虫与拟松材线虫。MspI、ApaI、XhoI不宜用于鉴别松材线虫与拟松材线虫。A ploymerse chain reaction-restriction fragment Length polymorphism （PCR- RFLP） analysis was used for discrimination of isolates of Bursaphelenchus xylophilus and B. rnucronatus. The amplication of isolates of B. xylophilus yielded one fragment of approximately 890 bp. But that of B. mucronatus was about 930 bp. Digestion of amplified products of each nematode isolate with five restriction endonucleases revealed the results as follows：（1） Dra I digestion of its products of B. xylophilus populations yielded two fragments, 510 and 380 bp. But Dra Ⅰ couldn＇t digest ITS products of B. mucronatus populations. （2）Sal Ⅰ couldn＇t digest the ITS products of all B. xylophilus populations. But it could digest that of B. mucronatus populations to two fragments,which were 720 and 220 bp; （3）Digesting production of four B. xylophilus populations by MspⅠ yielded two fragments,530 and 360 bp,except GZ02 which couldn＇t be digested. But B. mucronatus populations yielded three fragments,340,290 and 180 bp; （4） All populations of B. xylophilus and B. mucronatus could not be digested by Apa Ⅰ; （5） Digestions of amplified products of B. xylophilus and B. mucronatus with Xho Ⅰ yielded two fragments respectively,520 and 370,530 and 400 bp. The restriction endonucleases, Dra Ⅰ and Sal Ⅰ, could be used for identification of B. xylophilus and B. rnucronatus. The results of digestion of B. xylophilus and B. mucronatus were such sharp difference that it was very easy to identify and it was very convenient to be applied,Msp Ⅰ and Xho Ⅰ were not fit for identification of B. xylophilus and B. mucronatus; Apa Ⅰ could not distinguish and identify B. xylophilus and B. mucronatus.

关 键 词：松材线虫 拟松材线虫 核糖体基因 PCR—RFLP 鉴定 

分 类 号：S763[农业科学—森林保护学] S41[农业科学—林学]