利用Gateway技术筛选Candidatus Liberibacter asiaticus致病相关基因研究
Screening of pathogenicity-related genes from Candidatus Liberibacter asiaticus using Gateway technology
查看参考文献21篇
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文摘
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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,CaLas)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Pal_(Ca Las)、Mot_(Ca Las)和Hly_(Ca 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,Pal_(Ca Las) ,、Mot_(Ca Las)和Hly_(Ca 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OD_(600)值介于0.6 ~ 0.8,测试本生烟苗龄6周左右,Pal_(Ca 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Mot_(Ca Las)和Hly_(Ca Las)均不能引发类似反应。由此证实,Gateway技术可成功用于筛选CaLas致病相关基因,Pal_(Ca Las)基因可能在CaLas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CaLas致病相关基因功能研究,为后续开展CaLas病原寄主互作分子机制研究奠定了基础。 |
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其他语种文摘
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Citrus Huanglongbing is one of the most devastating diseases threatening the healthy development of global citrus industry. The causal agent is a phloem-limited bacterium. Candidatus Liberibacter asiaticus ( CaLas) is the only pathogen associated with Citrus Huanglongbing in China. Due to the difficulty of in vitro culture,very little is known about the molecular mechanism of pathogenicity of CaLas. In the present study, three putative pathogenicity-related genes of Pal_(Ca Las) ,Mot_(Ca Las) and Hly_(Ca Las) were selected from the whole genome sequence of CaLas ( Psy62: NC_012985.1) according to the characteristics of pathogenicity-related genes available to other similar plant pathogenic bacteria reported. The above 3 genes were constructed to the plant expression vector psk103 via BP and LR reaction respectively based on the homologous recombination of Gateway technology. Agrobacterium-mediated transient expression method was used to analyze the phenotype induced by 3 genes in Nicotiana benthamiana. Our result showed that Pal_(Ca Las) ,Mot_(Ca Las) and Hly_(Ca Las) were correctly constructed to psk103 vector as confirmed by PCR products cloning and sequencing. Transient expression in N. benthamiana indicated: PalCaLas construct successfully induced a strong hypersensitive response at 4dpi when inoculated with Agrobacterium OD_(600) in the range of 0.3-0.8 to 6-week old N. benthamiana. None of the other two constructs of Mot_(Ca Las) or Hly_(Ca Las) gave similar reaction under the same circumstance. With time progressed,stronger HR response was observed. And the whole leaf became necrosis at 12 dpi after infiltration of PalCaLas . In conclusion, Gateway is a powerful technique for the successful screening of pathogenicity-related genes from CaLas. And this is the first report nation-wide for quick screening of pathogenicity-related genes from CaLas which laid a basic foundation for future research on the molecular mechanism of host-pathogen interactions. |
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来源
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植物病理学报
,2017,47(6):816-823 【核心库】
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DOI
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10.13926/j.cnki.apps.000112
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关键词
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黄龙病菌
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Candidatus Liberibacter asiaticus
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致病相关基因
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Gateway技术
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地址
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华中农业大学植物科学技术学院, 湖北省作物病虫害检测与安全控制重点实验室, 武汉, 430070
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语种
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中文 |
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文献类型
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研究性论文 |
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ISSN
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0412-0914 |
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学科
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植物保护 |
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基金
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中央高校基本科研业务费专项资金资助项目
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文献收藏号
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CSCD:6190626
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