Functional Analysis Of GCR2as The Putative Receptor For N-acylhomoserine Lactones In Plant Receptor

N-acyl-homoserine lactones (AHLs) was an important bacterial quorum-sensing signalmolecule which secreted by Gram-negative bacteria. It reported that AHLs could also beperceived by their host plants, and regulated eukaryotic gene expression and cellularresponses. According to previous studies, we predicted that plant cells may useheterotrimeric G protein signaling transduction system to transduct AHLs. The extracellularsignals could be perceived by G protein-coupled receptors, and then it passed signals to theG protein and effectors, thereby regulating downstream pathways.In this paper, we use the transgenic plants of GCR2to study the role of GCR2in plantsresponse to AHL. GCR2homozygous transgenic plants were Screened and identified. Inthe previous study, we found the Arabidopsis mutants gcr2-2show insensitive to3OC6-HSL. Addition of1μmol·L-13OC6-HSL in the medium significantly promoted theroot growth of wild type Arabidopsis thaliana and Arabidopsis mutants GCR2OE/(gcr2-2/GCR2). The root length growth of the Arabidopsis mutants GCR2OE wassignificantly higher than the Arabidopsis mutants gcr2-2/GCR2. The total protein ofdifferent Arabidopsis were extracted. ELISA was used of analysis the binding capacitybetween3OC6-HSL and total protein of different Arabidopsis. Wild type Arabidopsisthaliana and the mutants GCR2OE/(gcr2-2/GCR2) showed more insensitive to3OC6-HSLthan the GCR2-deletionmutants gcr2-2. The evidences suggested that GCR2might play animportant role in Arabidopsis root responsed to3OC6-HSL. And GCR2protein may be thereceptors of3OC6-HSL.The GCR2gene were cloned into expression vector pET28a to recombinate E.coliBL21(pET-28a-GCR2). Recombinant protein was expressed under IPTG inducingcondition. SDS-PAGE and Western Blot analysis showed that recombinant GCR2proteinwas successfully expressed. The result on radioautography analysis showed that3H-3OC6-HSL could specificity bind to GCR2. We used His-tag to obtain the purifiedprotein GCR2, afterwards analysed the receptor dynamics between purified protein GCR2and3OC6-HSL by ELISA and MST.