快速简单组织定位甘蔗RSD致病菌Lxx的原位PCR方法  

Fast and Simple In Situ PCR Method for Localizing RSD Pathogen Lxx in Sugarcane Tissue

作　　者：胡敏 汪文华[2] 何恩铭[2] 孟红岩[2] 郭莺 HU Min;WANG Wenhua;HE Enming;MENG Hongyan;GUO Ying(College of Chemical Engineering,Huaqiao University,Xiamen 361021,China;Fujian Key Laboratory of Subtropical Plant Physiology and Biochemistry, Fujian Institute of Subtropical Botany,Xiamen 361006,China)

机构地区：[1]华侨大学化工学院,福建厦门361021 [2]福建省亚热带植物研究所福建省生理生化重点实验室,福建厦门361006

出　　处：《华侨大学学报（自然科学版）》2018年第2期221-226,共6页Journal of Huaqiao University(Natural Science)

基　　金：国家自然科学基金青年科学基金资助项目(31301381)

摘　　要：提出对甘蔗冰冻切片中甘蔗宿根矮化病(RSD)致病菌Lxx(Leisonia xyli subsp. xyli)进行定位的原位多聚酶链式反应(in situ PCR)方法,为Lxx与甘蔗互作致病机理研究提供有效的实验手段.以染RSD甘蔗品种Badila及其健康株为材料取样制作冰冻切片,切片经溶菌酶消化后,利用病原菌Lxx 16S^23S rDNA内部转录间隔区(ITS)特异性引物进行原位PCR扩增,并对随机参入扩增片段的与地高辛-11-dUTP进行免疫组化,建立甘蔗样品冰冻切片中直接原位PCR方法.结果表明:直接原位PCR对RSD蔗株茎样品切片显色为阳性,阳性信号出现于甘蔗茎部的木质部、韧皮部,而健康蔗株茎样品无信号,方法特异性较好.An in situ polymerase chain reaction(in situ PCR)method was developed for visualizing the localization of the pathogen Lxx(Leisonia xyli subsp.xyli)in sugarcane frozen section with ratoon stunting disease(RSD),providing a tool for the studyon RSD in sugarcane,especiallyon the mechanism of Lxx and sugarcane interaction.Frozen sections were obtained from both RSD infected and uninfectedsugarcane plants(cultivar Badila).After the digestion with lysozyme,in situ PCR was performed directly on the sections by amplifying Lxx 16S^23S rDNA internal transcriptional spacer(ITS)using the specific primers,the resulting products,which contained the random Digoxigenin 11 dUTP in the amplified fragments,were visualized based on immunohistochemistry,enabling a direct in situ PCR detectionfor Lxx on sugarcane frozen sections.The results showed that the positive signalsin the stem of RSD infected samples were detected and localized on sugarcane stem xylem and phloem,while no signals was found in the healthy samples,suggesting this method was

关 键 词：甘蔗宿根矮化病 革兰氏阳性苛养致病菌 冰冻切片 原位多聚酶链式反应 

分 类 号：Q945.8[农业科学—植物病理学]