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

• RESEARCH PAPERS • Previous Articles     Next Articles

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

Abstract:
 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