Expression And Bioactivity Detection Of Two Biocontrol Proteins Harpin_Xooc And AiiA

Pesticides, with a history of over 100 years, played an important role in plantprotection, greatly improved the agricultural development, and brought large economicprofits to human creatures. However, the impropriate application of pesticides also hassome negative effects, such as, the drug resistance of plant disease and insects and weeds,direct poison of non-target creatures and the environmental pollution, which now becomethe most urgent problems to resolve. Fortunately, the appearance of gene engineeringtechniques in 1970s provides new solutions and methods to solve these problems.Harpins, a group of non-specific protein elicitors secreted by plant pathogenic bacteriathrough typeⅢsecretion system, can induce plant hypersensitive reaction (HR) responseand systematic acquired resistance (SAR) in non-host plants. The hrf2 gene was amplifiedfrom genome of Xanthomonas oryzae pv. oryzicola RS105 by PCR and inserted into thedownstream of the a-factor signal sequence of the P. pastoris expression vector pPICZαA.The linearized recombinant plasmid was transformed into P. pastoris strain X-33 byelectroporation. The recombinant strains were chosen by YPDS plates containing Zeocinand identified by PCR. SDS-PAGE analysis proved a special band about 18 kD andWestern blot demonstrated good antigenicity of the expressed protein. The results ofbioactivity detection showed that the recombinant harpinxooc had higher activity to inducehypersensitive response and promote tobacco growth than harpin_Xooc expressed inEscherichia coli.AiiA protein can degrade the AHL signal molecules, which regulate the quorumsensing process in bacteria, by hydrolyzing the lactone bonds, thus attenuate plant diseasesymptoms caused by bacterial pathogens. A 753 bp gene sequence was amplified fromBacillus subtilis by PCR using the specific primers designed based on the reportedαiiAgene. The blast result of this fragment demonstrates its similarity over 85% with thereportedαiiA genes from Bacillus genus. The PCR fragment was inserted into expressionvector pET30 (a) to construct the recombinant vector pET30-αiiA. Then vector pET30-αiiA was transformed into E. coli BL21 (DE3) to express the recombinant protein under theIPTG inducing condition. SDS-PAGE results showed that the expression product existed asinclusive bodies inside bacterial cells and had a molecular weight of about 28 kD.