[1]胡敏,汪文华,何恩铭,等.快速简单组织定位甘蔗RSD致病菌Lxx的原位PCR方法[J].华侨大学学报(自然科学版),2018,39(2):221-226.[doi:10.11830/ISSN.1000-5013.201709055]
　HU Min,WANG Wenhua,HE Enming,et al.Fast and Simple In Situ PCR Method for Localizing RSD Pathogen Lxx in Sugarcane Tissue[J].Journal of Huaqiao University(Natural Science),2018,39(2):221-226.[doi:10.11830/ISSN.1000-5013.201709055]
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快速简单组织定位甘蔗RSD致病菌Lxx的原位PCR方法()
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《华侨大学学报（自然科学版）》[ISSN:1000-5013/CN:35-1079/N]

卷:
第39卷
期数:
2018年第2期
页码:
221-226
栏目:
出版日期:
2018-03-20

文章信息/Info

Title:
Fast and Simple In Situ PCR Method for Localizing RSD Pathogen Lxx in Sugarcane Tissue
文章编号:
1000-5013(2018)02-0221-06
作者:
胡敏1;  汪文华2;  何恩铭2;  孟红岩2;  郭莺1; 2
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
Author(s):
HU Min1;  WANG Wenhua2;  HE Enming2;  MENG Hongyan2;  GUO Ying1; 2
1. College of Chemical Engineering, Huaqiao University, Xiamen 361021, China; 2. Fujian Key Laboratory of Subtropical Plant Physiology and Biochemistry, Fujian Institute of Subtropical Botany, Xiamen 361006, China
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
Keywords:
ratoon stunting disease;  Leisonia xyli subsp. xyli;  frozen sections;  in situ polymerase chain reaction
分类号:
Q945.8
DOI:
10.11830/ISSN.1000-5013.201709055
文献标志码:
A
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.
Abstract:
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 excellent in specificity.

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备注/Memo

备注/Memo:
收稿日期: 2017-09-20
通信作者: 郭莺(1981-),女,副研究员,博士,主要从事植物病害检测和抗病分子育种的研究.E-mail:47402221@qq.com.
基金项目: 国家自然科学基金青年科学基金资助项目(31301381)http://www.hdxb.hqu.edu.cn
更新日期/Last Update: 2018-03-20