Identification And Functional Analysis Of A Novel Protein Elicitor PeBL2 From Brevibacillus Laterosporus A60

Brevibacillus laterosporus has various biological activities,such as insecticidal activity,antimicrobial effect,and biodegradation as well as biotransformation.Therefore,it shows great potential value in microbial agent development.In this paper,a protein elicitor PeBL2 from the culture supernatant of B.laterosporus was identified and purified.PeBL2 can induce a hypersensitive response in tobacco.The peBL2 gene was cloned and expressed in Escherichia coli.The results follow as below:A novel secreted protein elicitor(PeBL2)was isolated from the B.laterosporus A60.Through a purification process consisting of ammonium sulfate precipitation,ion-exchange chromatography and high-performance liquid chromatography(HPLC),we isolated and purified a protein elicitor,PeBL2(Protein elicitor from B.laterosporus 2),by the culture filtrate of B.laterosporus strain A60.PeBL2 was identified by electrospray ionization quadrupole time of flight tandem mass spectrometry(ESI–Q-TOF-MS/MS).We obtained the best-matching hypothetical protein from B.laterosporus,with 156 amino acids and theoretical molecular mass of 17.22 kDa.The hypothetical gene was cloned with 471 bp,named as peBL2.The peBL2 sequence was cloned into the pET28a-TEV/LIC vector for expression.We got the recombinant protein PeBL2 which could also induce the HR in tobacco leaves.The tobacco treated by PeBL2 can trigger the defence response of ROS burst.Six-week-old Nicotiana tabacum leaves were treated with 20 ?M of recombinant PeBL2,using the BSA as a control.The disease severity was recorded by measuring lesion diameter of Botrytis cinerea lesion at 3 days post-inoculation.10 random injuries were measured for each treatment.Inhibition showed significant differences between PeBL2 and BSA,indicating PeBL2 have effective in induced disease resistance,with a diameter in PeBL2(14.11mm)as compared to the control(22.5mm).The result indicated that PeBL2 could induce disease resistance against B.cinerea in the tobacco plant.