用原位 PCR 法检测致死性黄化病椰子胚中的植原体 DNA。

In situ PCR detection of phytoplasma DNA in embryos from coconut palms with lethal yellowing disease.

机构信息

Plant Pathogen Interactions Division, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK.

出版信息

Mol Plant Pathol. 2003 Mar 1;4(2):99-108. doi: 10.1046/j.1364-3703.2003.00152.x.

DOI:10.1046/j.1364-3703.2003.00152.x

PMID:20569368

Abstract

SUMMARY DNA of the lethal yellowing (LY) phytoplasma was detected in 13 of 72 embryos from fruits of four diseased Atlantic tall coconut palms by polymerase chain reaction (PCR) assays employing phytoplasma universal rRNA primer pair P1/P7, nested LY group-specific rRNA primer pair 503f/LY16Sr or LY phytoplasma-specific nonribosomal primer pair LYF1/R1. Phytoplasma distribution in sectioned tissues from six PCR positive embryos was determined by in situ PCR and digoxigenin-11-deoxy-UTP (Dig) labelling of amplification products. Dig-labeled DNA products detected by colourimetric assay were clearly evident on sections from the same three embryos investigated in detail by in situ PCRs employing primer pairs P1/P7 or LYF1/R1. Deposition of blue-green stain on sections as a result of each assay was restricted to areas of the embryos corresponding to the plumule and cells ensheathing it. By comparison, similarly treated embryo sections derived from fruits of a symptomless Atlantic tall coconut palm were consistently devoid of any stain. Presence of phytoplasma DNA in embryo tissues suggests the possible potential for seed transmission which remains to be demonstrated.

摘要

通过聚合酶链反应（PCR）分析，利用植原体通用 rRNA 引物对 P1/P7、嵌套的致死黄化（LY）组特异性 rRNA 引物对 503f/LY16Sr 或 LY 植原体特异性非核糖体引物对 LYF1/R1，从四个患病大西洋高椰棕果实的 72 个胚胎中的 13 个胚胎中检测到致死黄化植原体 DNA。通过原位 PCR 和 Dig 标记扩增产物，在六个 PCR 阳性胚胎的切片组织中确定了植原体的分布。通过比色法检测到的 Dig 标记 DNA 产物在通过 P1/P7 或 LYF1/R1 引物对进行详细原位 PCR 分析的同一三个胚胎的切片上清晰可见。每个检测产生的蓝绿色染色在与芽和包裹它的细胞相对应的胚胎区域沉积。相比之下，来自无症状大西洋高椰棕果实的胚胎切片经过同样处理后，始终没有任何染色。胚组织中植原体 DNA 的存在表明可能存在种子传播的可能性，这有待进一步证实。