Cloning Of Quorum-sensing Related Gene And Applying To Detect In Hami Melon Fruit Blotch

Hami melon bacteria fruit blotch,caused by Acidovorax avenae subsp, citrulli, is a disease whichinfluences the qualities and productions of Hami melon seriously. As so far few reports focused onmolecular genetics of this pathogen. Quorum-sensing system of bacteria is one of the hot topics offoreign and domestic researching recently, and the production of virulence factors in manyphytopathology bacteria is regulated by QS, this paper focuses on the preliminary study of quorumsensing in A. avenae subsp, citrulli.A. tumefaciens NTL4 (pZLR4) was used as AHLs reporter strain, detected the 19 strains ofA.avenae subsp, citrulli,18 strains which were found to produce signal molecules. The luxI and IuxRwhich were two components of QS system, were cloned from genomic library of Acidovorax avenaesubsp, citrulli by heterologous expression in E.coli DH5α. In this experiment, A. tumefaciensNTLA(pZLR4) was used as AHLs reporter strain. The clones p5ACC14 and p5ACC40 from thegenomic library was transformed into P. fluorescens 2P24△I by electric shocks, and it recovered theability of poroducting signals. P5ACC14 was further cloned to get 5.5Kb fragment. It was found thatone ORF was related to A.acidovoraxAACOO-1NCO08752 luxI with homologous coefficients higher to100%. We constructed the aacI prokaryotic gene expression vector, transformed it into E.coli DH5α,and detected the product of signal using reporter plate. According to regions of luxI where divergentfrom other luxI-homolog reported, specific primers IS and IX were designed to identify A. avenae subsp.citrulli. Tested A. avenae subsp, citrulli strains were successfully identified using the primers from arange of closely related bacteria. And it was proved to be highly sensitive with 3×10~3 colony pormingunits.