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柑橘黄龙病是危害全球柑橘产业健康发展的最严重病害之一,病原为韧皮部限制性细菌,我国的柑橘黄龙病由亚洲种(Candidatus Liberibacter asiaticus,Ca Las)引起。由于该病原菌难以获得离体纯培养,从而限制了其致病分子机制的研究。本试验根据已经报道的植物病原细菌致病相关基因特点,从黄龙病菌亚洲种Psy62菌株全基因组序列(Psy62:NC_012985.1)中选取3个假定致病相关基因,分别标记为PalCa Las、MotCa Las和HlyCa Las,利用Gateway技术的同源重组原理,将3个基因通过BP反应和LR反应,分别构建到植物表达载体psk103中,接种本生烟(Nicotiana benthamiana)后,通过瞬时表达筛选可诱导N.benthamiana产生过敏性坏死反应的功能基因。经过PCR克隆和测序验证,PalCa Las,、MotCa Las和HlyCa Las基因均成功构建至植物表达载体上。本生烟瞬时表达试验证实:当含有目的基因的根癌农杆菌菌悬液OD600值介于0.6-0.8,测试本生烟苗龄6周左右,PalCa Las基因在接种4 dpi时可引发明显的HR(hypersensitive reaction)反应,12 dpi时HR反应更为强烈。同等条件下MotCa Las和HlyCa Las均不能引发类似反应。由此证实,Gateway技术可成功用于筛选Ca Las致病相关基因,PalCa Las基因可能在Ca Las致病过程中发挥重要作用,该报道为国内首次采用Gateway技术进行Ca Las致病相关基因功能研究,为后续开展Ca Las病原寄主互作分子机制研究奠定了基础。
Citrus Huanglongbing is one of the most serious diseases endangering the healthy development of the global citrus industry. The pathogen is phloem-restricted bacteria. Citrus Huanglongbing in China is caused by Candidatus Liberibacter asiaticus (Ca Las). As the pathogen is difficult to obtain in vitro pure culture, thus limiting the pathogenic molecular mechanism of the study. In this study, three putative pathogenicity-related genes were selected from Psy62: NC_012985.1, an Asian species of Pseudomonas solanacearum according to the reported pathogenicity-related genes of plant pathogenic bacteria, which were respectively labeled as PalCa Las, MotCa Las And HlyCa Las, using the homologous recombination principle of Gateway technology, the three genes were respectively constructed into the plant expression vector psk103 through BP reaction and LR reaction. After being inoculated with Nicotiana benthamiana, the three genes were inoculated through inducible N Functional genes that cause allergic necrosis. After PCR cloning and sequencing, PalCa Las, MotCa Las and HlyCa Las genes were successfully constructed into plant expression vectors. The instantaneous expression of Nicotiana benthamiana in tobacco showed that when the OD600 value of the Agrobacterium tumefaciens suspension containing the target gene was between 0.6 and 0.8, the test of Nicotiana benthamiana seedling age was about 6 weeks, the PalCa Las gene at 4 dpi showed significant HR (hypersensitive reaction) reaction, HR reaction more strongly at 12 dpi. Under the same conditions, neither MotCa Las nor HlyCa Las triggered a similar reaction. Thus, Gateway technology can be successfully used to screen Ca Las pathogenicity-related genes, PalCa Las gene may play an important role in Ca Las pathogenicity, the report for the first time using Gateway technology for Ca Las disease-related gene function , Which laid the foundation for the follow-up study on the molecular mechanism of Ca Las pathogen host interaction.