新疆小叶白蜡丛枝病植原体的鉴定及16S rRNA基因序列分析

The identification and sequence analysis of 16S rRNA gene of Fraxinus sogdianaBunge witches’ broom phytoplasma

摘要:

【目的】对新疆小叶白蜡丛枝病植原体进行检测，通过其16S rRNA基因分析确定其分类地位。【方法】利用苯胺蓝和4′,6-二脒基-2-苯基吲哚(DAPI)染色，在荧光显微镜下观察新疆小叶白蜡嫩茎横切片；采用植原体16S rRNA基因的通用引物对P1/P7和R16F2n/R16R2 进行直接和巢式PCR扩增，对得到的16S rRNA基因的序列进行RFLP和构建系统进化树分析。【结果】表现丛枝病症状的新疆小叶白蜡中存在植原体，暂命名为Fraxinus sogdianaBunge witches’ broom phytoplasma (Fraxinus sogdianaBunge WB)；其16S rRNA基因的序列GenBank登录号为KF061042，RFLP图谱与16Sr V-B亚组的枣疯病植原体相同，系统进化地位与枣疯病菌株AB052876相同。【结论】新疆小叶白蜡丛枝病植原体为16Sr V-B亚组成员。

Abstract:

[Objective] To identify and classify the Fraxinus sogdianaBunge witches′ broom phytoplasma. [Methods] Horizontal slices of tender stem from Fraxinus sogdianaBunge were stained with aniline blue and 4,6-diamidino-2-phenylindole (DAPI), and then observed under flurescence microscopy. 16S rRNA gene sequences were amplified using direct-PCR and nested-PCR with universal primers P1/P7 and R16F2n/R16R2 for phytoplasmal 16S rRNA gene sequence. Amplicons were analyzed by restriction fragment length polymorphism (RFLP) and a phylogenetic tree was constructed based on the sequences of 16S rRNA gene. [Results] A phytoplasma was identified from Fraxinus sogdianaBunge which with symptoms of witches’ broom and was tentatively named as Fraxinus sogdianaBunge witches’ broom phytoplasma (Fraxinus sogdianaBunge WB). The 16S rRNA gene sequence was deposited in GenBank with the accession number of KF061042. Its RFLP patterns of 16S rRNA gene were the same as jujube witches′ broom phytoplasma which belong to 16Sr V-B subgroup, and it had the same phylogenetic position with jujube witches’ broom phytoplasma strain AB052876. [Conclusion] Fraxinus sogdianaBunge witches′ broom phytoplasma was identified as the member of 16Sr V-B subgroup.