林木外生菌根真菌彩色豆马勃巢式PCR检测-应用与环境生物学报.PDFVIP

2012-04-25 DOI: 10.3724/SP.J.1145.2012.00298 / Chin J Appl Environ Biol 2012 18 ( 2 ) : 298~302 应用与环境生物学报 ， 林木外生菌根真菌彩色豆马勃巢式PCR检测* ** 周爱东 吴小芹 叶建仁 （南京林业大学森林资源与环境学院 南京 210037） 摘 要 为探讨林木外生菌根真菌的分子检测方法，利用真菌通用引物ITS1-F/ITS4-B扩增了外生菌根真菌彩色豆马勃 的核糖体DNA 内转录间隔区并进行了序列测定. 通过序列比较，设计了一对彩色豆马勃特异性引物PtF/PtR. 利用该对 特异性引物与ITS1-F/ITS4-B组合进行巢式PCR ，能从供试的彩色豆马勃10个菌株中特异性地扩增出1条347 bp的条带， 而供试的其他6个参比菌株未出现扩增产物. 经分析，该巢式 PCR检测技术的灵敏度可达到 10 fg的DNA ，是常规PCR 检测的 1 000倍. 利用该技术从马尾松苗木菌根中检测到目的外生菌根真菌. 这表明采用本研究设计的特异性引物，利 用巢式PCR技术可以灵敏、准确地从林木外生菌根中检测出彩色豆马勃. 图4 参20 关键词 外生菌根真菌；彩色豆马勃；巢式PCR ；分子检测 CLC Q948.122.3 : Q78 Detection of Forest Ectomycorrhizal Fungi-Pisolithus tinctorius by Nested PCR* ZHOU Aidong, WU Xiaoqin** YE Jianren (College of Forest Resources and Environment, Nanjing Forestry University, Nanjing 210037, China) Abstract For investigating a suitable molecular approach to detect ectomycorrhizal fungi (EMF), the internal transcribed spacer (ITS) region of the EMF Pisolithus tinctorius was amplified with universal primers ITS1-F/ITS4-B and sequenced. Based on the conserved sequence determined by alignment using Clustal W, a pair of specific primers named PtF/PtR were designed by Primer premier 5.0. The specificity of the primer pair was then analyzed in 10 P. tinctorius strains together with 6 reference strains, and a 347 bp product was amplified from all P. tinctorius strains but there was no product from the 6 reference strains. A nested PCR procedure using ITS1-F/ITS4-B as the first-round primers followed by PtF/PtR increased detection sensitivity 1 000-fold to 10 fg DNA template. The target EMF from the mycorrhiza of Pinus massoniana potted seedlings inoculated with P. tinctorius