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为了探索研究柑橘木虱Diaphorina citri与柑橘黄龙病(Huanglongbing,HLB)病原菌的相互作用蛋白,本研究运用RNA转录中5’末端转换机制(Switching Mechanism at 5’End of the RNA Transcript,SMART)技术构建了柑橘木虱的酵母双杂交cDNA文库。以实验室饲养的柑橘木虱为材料,提取总RNA,经反转录后合成ds cDNA,两端添加同源重组序列,并用层析柱纯化;ds cDNA与文库质粒pGADT7-Rec在酵母Y187感受态细胞内发生同源重组,柑橘木虱cDNA重组到文库质粒上,完成酵母双杂交cDNA文库的构建。结果表明,文库容量达到106,扩增文库滴度为2.23×107cfu/mL,cDNA插入片段的平均长度大于750bp,达到试剂盒建库要求。另外,我们利用构建的酵母双杂交文库,以HLB病原菌Candidatus Liberibacter asiaticus(CLas)的两个膜蛋白ORF420和ORF3420作为诱饵进行筛选试验,但是并没有得到阳性克隆。柑橘木虱酵母cDNA文库的构建为开展柑橘木虱与柑橘黄龙病病原菌互作机制的研究奠定了基础。
In order to explore the interaction between Diaphorina citri and Huanglongbing (HLB) pathogenic bacteria, this study constructed the RNA interference system using the 5 ’terminal of the RNA transcription (SMART) Yeast Two-Hybrid cDNA Library of Citrus psyllid. The total RNA was extracted from laboratory-grown citrus psyllids. The ds cDNA was synthesized by reverse transcription and the homologous recombination sequences were added to both ends of the cDNA library. The ds cDNA and library plasmid pGADT7-Rec were expressed in yeast Y187 Homologous recombination occurred in the intracellular state of the cell and the citrus psyllid cDNA was recombined into the library plasmid to construct the yeast two-hybrid cDNA library. The results showed that the library capacity reached 106, the titer of amplified library was 2.23 × 107cfu / mL, and the average length of cDNA insert was more than 750bp. In addition, we used the constructed yeast two-hybrid library to screen the two membrane proteins ORF420 and ORF3420 of HLB pathogen Candidatus Liberibacter asiaticus (CLas) as bait, but no positive clone was obtained. The construction of citrus lice yeast cDNA library laid the foundation for the research on the interaction mechanism between citrus psylla and citrus Huanglongdao pathogen.