›› 2013, Vol. 56 ›› Issue (3): 228-233.doi:

• 研究论文 • 上一篇    下一篇


马晓芳, 陈国庆, 张学潮, 徐海君   

  1. (1. 浙江大学昆虫科学研究所, 杭州 310058; 2. 浙江省柑桔研究所, 浙江台州 318020)
  • 出版日期:2013-03-20 发布日期:2013-03-20

Construction and evaluation of yeast two-hybrid cDNA library of Diaphorina citri (Hemiptera: Psyllidae)

(1. Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China; 2. Zhejiang Orange Research Institute, Taizhou, Zhejiang 318020, China)   

  • Online:2013-03-20 Published:2013-03-20

摘要: 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2.23×107 cfu/mL, cDNA插入片段的平均长度大于750 bp, 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。

关键词: 柑橘木虱, 柑橘, 黄龙病, 酵母双杂交cDNA文库, 膜蛋白, 蛋白质互作

 In order to study the interacting proteins between the Asian citrus psyllid (Diaphorina citri) and Candidatus Liberibacter asiaticus (CLas) which is the pathogenic bacterium causing Huanglongbing, yeast two-hybrid cDNA library of D. citri was constructed using the Switching Mechanism at 5′ End of the RNA Transcript (SMART) technique. The total RNA was isolated from the citrus psyllid adults bred in the laboratory and subjected to reverse transcription, and the double-strand cDNAs (ds cDNAs) were synthesized. The ds cDNAs were ligated with homologous adapter and purified by the chromatography column. By using homologous recombination reaction, the ds cDNAs were transformed into the Y187 competent cell with the library plasmid pGADT7-Rec to construct yeast two-hybrid cDNA library. Detection of the library indicated that it contained more than 106 independent clones, the titer of the amplified library was 2.23×107 cfu/mL, and the average size of inserts was above 750 bp in the cDNA library. These results demonstrate that the library meets the requirements of the standard cDNA library. Moreover, two membrane proteins, ORF420 and ORF3420, from (CLas) were used as bait proteins to screen the interacting proteins in the library, but no positive clone was screened in the tests. The yeast two-hybrid cDNA library of D. citri will be useful for the research on the interaction between insect vectors and C. Liberibacter asiaticus in the future.

Key words: Diaphorina citri, citrus, Huanglongbing (HLB), yeast two-hybrid cDNA library, membrane protein, protein interaction