根肿病菌核糖体基因ITS区段的克隆测序及其在检测中的应用

Cloning and Sequencing the Internal Transcribed Spacer of the Ribosomal Gene of Plasmodiophora brassicae and Applying it in Detecting the Pathogen

应用真菌核糖体基因ITS区段通用引物ITS1和ITS4,对十字花科蔬菜根肿病菌(Plasmodiophorabrassicae)rDNA进行PCR扩增,并将扩增到的目的片段克隆到pGEM-T载体上。对重组克隆进行测序和碱基编码结构特点分析,结果表明:十字花科蔬菜根肿病菌ITS1区长141bp,5 8S区长160bp,ITS2区长187bp,rDNA总长为488bp.根据此序列设计一对根肿病菌特异性寡聚核苷酸引物(前引物:5'AGGTGAACCTGCGGAAGGAT3'和后引物:5'TTCAGCGGGTAATCCTACCT3'),应用聚合酶链式反应(polymerasechainreaction,PCR)技术,对分离自十字花科蔬菜(白菜、青菜、甘蓝、芥蓝、花椰菜等)根肿病菌全基因组DNA进行特异性扩增试验。试验结果表明,该对引物能从十字花科根肿病菌全基因组DNA中扩增到约500bp长度的分子片段,而对照菌株和健康对照则无扩增产物。该实验结果对有效追踪根肿病菌在田间的发生动态监测和预测预报提供了重要的信息和手段。

Polymerase Chain Reaction ( PCR) amplification of the internal transcribed spacer (ITS) of the ribosomal DNA of Plasmodiophora brassicae extracting from cruciferae (Chinese cabbage, cauliflower, broccoli) have done with universal primer ITS1 and ITS4, and the PCR products were cloned into pGEM-T vector. We analysise the sequences and found that the internal transcribed spacer of the ribosomal DNA of Plasmodiophora brassicae were composed of 488?bp, 141?bp are ITS1,160?bp are 5.8?S, 187?bp are ITS2 respectively. On the basis of DNA sequence informations, a pair of oligonucleotide primer set specific for the pathogen was designed. The pair of primer produced an amplification product of approximately 500 bp in length with DNA from Plasmodiophora brassicae extracting from Brassica pekinensis, Brassica chinensis, Brassica oleracea var. capitata., Brassica caulorapa, Brassica alboglabra, Brassica oleracea var. botrytis L. However, no amplification product was produced with DNA from Meloidogyne, Synchtrium endobioticum,Phytophthora parasitica var. and healthy roots. Sequence information and detecting technique were provided for the molecular detection and infecting mechanism of plant diseases caused by Plasmodiophora brassicae according to this research result.