基于PCR对16S rRNA基因以外的多个基因进行序列分析以区分植原体

PCR-Based Sequence Analysis on Multiple Genes Other than 16S rRNA Gene for Differentiation of Phytoplasmas.

作者信息

Martini Marta, Bottner-Parker Kristi D, Lee Ing-Ming

机构信息

Department of Agricultural, Food, Environmental and Animal Sciences (DI4A), University of Udine, Udine, Italy.

Molecular Plant Pathology Laboratory, USDA, ARS, Beltsville, MD, USA.

出版信息

Methods Mol Biol. 2019;1875:97-115. doi: 10.1007/978-1-4939-8837-2_8.

DOI:10.1007/978-1-4939-8837-2_8

PMID:30361998

Abstract

Differentiation and classification of phytoplasmas have been primarily based on the highly conserved 16S rRNA gene, for which "universal" primers are available. To date, 36 ribosomal (16Sr) groups and more than 150 subgroups have been delineated by RFLP analysis of 16S rRNA gene sequences. However, in recent years, the use of moderately conserved genes as additional genetic markers has enhanced the resolving power in delineating distinct phytoplasma strains among members of some 16Sr subgroups.This chapter describes the methodology of amplification, differentiation, and classification of phytoplasma based on less-conserved non-ribosomal genes, named rp and secY. Actual and virtual RFLP analyses of amplicons obtained by semi-universal or group-specific rp and secY gene-based primers are used for finer differentiation of phytoplasma strains within a given group. The rp and secY gene-based classification not only readily resolves 16Sr subgroups within a given 16Sr group, but also provides finer differentiation of closely related phytoplasma strains within a given 16Sr subgroup.

摘要

植原体的分化和分类主要基于高度保守的16S rRNA基因，针对该基因有“通用”引物可用。迄今为止，通过对16S rRNA基因序列进行限制性片段长度多态性（RFLP）分析，已划分出36个核糖体（16Sr）组和150多个亚组。然而，近年来，使用中度保守基因作为额外的遗传标记提高了在某些16Sr亚组成员中区分不同植原体菌株的分辨能力。本章描述了基于保守性较低的非核糖体基因rp和secY对植原体进行扩增、分化和分类的方法。通过基于半通用或组特异性rp和secY基因的引物获得的扩增子的实际和虚拟RFLP分析用于在给定组内对植原体菌株进行更精细的分化。基于rp和secY基因的分类不仅能轻松分辨给定16Sr组内的16Sr亚组，还能在给定16Sr亚组内对密切相关的植原体菌株进行更精细的分化。