三七病原根结线虫核糖体基因ITS区段的克隆测序及其在检测中的应用

Sequence of the Internal Transcribed Spacer of the Ribosomal Gene of Root Knot Nematode of Panax notoginseng and Its Application in Detecting the Pathogen

根据NCBI基因库中根结线虫属rDNA序列,设计通用引物M18s/M28s,对采自云南文山、砚山、马关、蒙自等地的4个三七病原根结线虫种群(Meloidogyne hapla)rDNA区段进行聚合酶链式反应(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序列分析,结果表明:4个种群rDNA同源性为100%,ITS区序列长度为479 bp,其中ITS1序列长度为213 bp,5.8S序列长度为159 bp,ITS2序列长度为107 bp。根据此序列设计-对特异性寡聚核苷酸引物Mhf/Mhr,应用PCR技术,以北方根结线虫(Meloidogynehapla)、南方根结线虫(Meloidogyne incognita)、花生根结线虫(Meloidogyne arenaria)和爪哇根结线虫(Meloido-gyne javanica)全基因组DNA为对照,对三七病原根结线虫全基因组DNA进行特异性扩增。结果表明,该对引物能从供试的北方根结线虫和三七病原根结线虫种群全基因组DNA中扩增到462 bp长度的分子片段,而南方根结线虫、花生根结线虫和爪哇根结线虫则无扩增产物。该引物可用于三七病原根结线虫的检测。

A pair of universal primer M18s/M28s was designed according to the ribosomal DNA （rDNA） sequence of Meloidogyne spp from NCBI, and the primer was used to amplificate the rDNA of nematode isolated from Panax notoginseng （Wenshan population, Yanshan population, Maguan population and Mengzi population）. The PCR products were cloned into pGEM - T vector. The analysis of the sequences showed that all the 4 populations shared 100% similarity of the sequence, and the internal transcribed spacer （ITS） was 479 bp, containing 213 bp of ITS1, 159 bp of 5. 8S, and 107bp of ITS2. On the basis of DNA sequence information, a pair of oligonucleotide primer Mhf/Mhr specific for the pathogen was designed. Primer Mhf/ Mhr produced an amplification product of 462 bp in length with DNA from M. hapla, Wenshan population, Yanshan population, Maguan population and Mengzi population. However, no amplification product was produced with DNA from other Meloidogyne populations. The pair of primer could be used to detect root knot nematode of P. notoginsengt.