萝卜花变叶病病原鉴定及赤霉素合成与代谢途径中关键酶基因的表达

Pathogen Identification of Raphanus sativus Phyllody Disease and the Expression Level of Key Enzyme Genes in Gibberellin Synthesis and Metabolism Pathway

【目的】明确云南元谋地区发生的萝卜花变叶病病原以及赤霉素合成途径中关键酶基因表达量的变化。【方法】对自然表现花变叶、丛枝和节间缩短的萝卜感病植株总DNA进行植原体16S rRNA基因扩增、克隆、测序及系统进化树构建,并采用实时荧光定量PCR技术分析赤霉素合成及代谢途径中关键酶基因(GA20ox1、GA3ox1和GA2ox1)表达量的变化。【结果】该植原体株系与16SrⅡ-A亚组相似性最高(0.98),并与16SrⅡ-A亚组中的其他植原体株系明显形成一个进化支。【结论】萝卜花变叶植原体(Raphanus sativus phyllody,RsPh-YNym)为植原体16SrⅡ-A亚组成员,且与候选种Candidatus Phytoplasma aurantifolia相关,明确了该株系的分类地位;感病植株茎中3种关键酶基因GA20ox1、GA3ox1和GA2ox1表达水平发生了变化,表达量均下调,说明植原体的侵染造成了植株赤霉素稳态被破坏,从而导致植株表型改变。

[Purpose]To figure out the causal agent of Raphanus sativus phyllody disease occurred in Yuanmou of Yunnan Province and changes of expression level of key enzymes genes in gibberellin synthesis.[Method]The phytoplasmal 16S rRNA gene sequence was amplified from total DNAs extracted from natural R. sativus plant showing phyllody, witches broom and shortened internodes symptoms. Then the amplicons were cloned, sequenced and phylogentic tree were constructed.Real-time fluorescence qualification RT-PCR was performed to detect changes of the expression level of key enzyme genes(GA20ox1, GA3ox1, GA2ox1) of gibberellin synthesis and metabolism pathway in this experiment.[Result]The phytoplasma strain had the highest similarity of 0.98 with the phytoplasma subgroup 16SrⅡ-A, and formed an evolutionary branch with other phytoplasma stranis which were classified in 16 SrⅡ-A subgroup.[Conclusion]The R. sativus phyllody phytoplasma(RsPh-YNym) strain belongs to phytoplasma 16SrⅡ-A subgroup and is related to Candidatus phytoplasma aurantifolia. Meanwhile, we found that the expression levels of GA20ox1, GA3ox1 and GA2ox1 genes in the susceptible plants were down-regulated compared with the healthy plants and showed that the phytoplasma infection destroyed the homeostasis of gibberellin and changed the phenotypes of Raphanus sativus.