Construction of a reporter plasmid containing bacterial luciferase

• 1、 The Yuzhou District People’s Government of Yulin City, Yulin 537000
• 2、School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004
• 3、College of Life Science and Technology, Guangxi University, Nanning 530004
• 4、State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi, Nanning 530004

Abstract： The luciferase encoded by luxAB gene catalyzes the bioluminescent reaction of bacteria. Type III secretion system (T3SS) is the virulence determinant of many phytopathogenic bacteria. For high-throughput screening (HTS) the small molecular compounds which control T3SS, luxAB gene was cloned into promoterless pLAFR6, acquired the recombinant plasmid pLuxAB. Subsequently, the promoter and signal area sequence of XC0241 gene, a know type III effector (T3SE) of Xanthomonas campestris pv. campestris (Xcc), were cloned into pLuxAB and generated the reporter plasmid pLux0241. The reporter plasmid was transferred to the wild type 8004, mutant strains D3076 and D3077, while pLuxAB was imported into the wild type 8004 as control. After adding the substrate of n-decanal, 8004/pLux0241 produced a high luciferase activity, while in strains D3076/pLux2041, D3076/pLux2041, and the reference strain 8004/pLuxAB, no bioluminescence was detected. Successfully, we constructed a new reporter for high-throughput screening the small molecule compounds specifically controlling T3SS of Xcc.

Keywords： luxAB Xanthomonas campestris pv. campestris T3SS reporter plasmid the high-throughput screening