我国几种植物植原体的快速分子鉴别与鉴定的研究

Rapid molecular differentiation and identification of different phytopla smas from several plants in China

选用桑萎缩病(M u lberry dw arf,MD)、枣疯病(Ju jube w itches-broom,JW B)、酸枣丛枝病(W ild ju jube w itches-broom,W JW B)、泡桐丛枝病(Pau lownia w itches-broom,PaW B)和苦楝丛枝病(Chinaberry tree w itches-broom,CW B)5种不同植物植原体和来源于3个不同地区PaW B和JW B材料进行16S rDNA和23S rDNA PCR扩增、异源双链迁移率分析(HMA)、PCR产物的RFLP分析和16S rDNA的克隆和测序等比较研究,建立了一种快速确定未知植原体种类和分类地位的分子鉴别与鉴定优化程序;并可对田间采集的各种植物植原体样品进行快速鉴定和鉴别。16S rDNA PCR产物HMA分析结果显示,JW B与CW B、MD和PaW B皆可形成明显的异源杂交双链;而CW B、MD和PaW B植原体之间未能形成异源双链。JW B和PaW B不同地区样品之间、JW B和W JW B之间也未发现异源杂交双链的形成。而23S rDNA PCR产物HMA分析则可以将MD与PaW B区分开。进一步对未知分类地位的CW B序列测定及与其它植原体16S rDNA的RFLP和同源性比较结果显示,CW B与PaW B同源性为99.5%,其中与MD的同源性高达99.7%,因而应将CW B归为翠菊黄花组16S r I-B,16S r I-B(rp-B)。

An efficient procedure for rapid identification and differentiation of phytoplasmas was established by comparative studies of 16S rDNA and 23S rDNA PCR, heteroduplex mobility assay （ HMA）, RFLP and sequence analysis of PCR-amplified products （16S rDNA） to mulberry dwarf （MD）, jujube witches＇-broom （ JWB ）, wild jujube witches＇-broom （ WJWB ）, paulownia witches＇-broom （ PaWB ） and chinaberry tree witches＇-broom （CWB） phytoplasmas as well as different samples of paulownia witches＇-broom and jujube witches＇-broom-phytoplasmas from three different areas in China. It was demonstrated that 16S rDNA PCR-amplified product of JWB-phytoplasma produced distinct heteroduplex mobility bands with CWB, MD and PaWB while CWB, MD and PaWB were not distinguished by HMA. No obvious heteroduplex mobility band was formed between different paulownia witches＇-broom and jujube witches＇-broom-phytoplasma samples as well as between JWB and WJWB-phytoplasmas. However, HMA of 23S rDNA PCR products seemed to distinguish MD with PaWB. The results of sequencing and homologuous comparison with other phytoplasmas further showed that CWB shared 99. 5 % with PaWB, 99. 7% with MD. Thus CWB might be sorted into aster yellows group, 16St I-B, 16St I-B （ rp-B）.