Bacillus Licheniformis W10 Antibacterial Protein And Its Synergistic Disease Resistance With Harpin Protein

Bacillus licheniformis W10 is a biocontrol bacterium screened from plant rhizosphere.It can inhibit various plant pathogenic fungi well,and has significant control effect on these diseases in the field.The biocontrol mechanism of W10 is mainly to produce an antifungal protein.In order to further understand the biocontrol mechanism and apply W10,the antifungal protein of W10 was purified and identified,and the coding gene was cloned and recombinant protein expression was performed.At the same time,in order to improve the biocontrol effect,this paper also preliminarily studied the synergistic disease resistance of the antifungal protein with the harpin protein Hpalxoo from Xanthomonas oryzae pv.oryzae.Thereby,we can understand the interaction mechanism of these two types of proteins,with two different signaling pathways,and lay the foundation for the application of the two proteins.The crude antifungal protein was obtained from the culture supernatant filtrate of Bacillus licheniformis W10 by using ammonium sulfate precipitation and dialysis,and then was purified through a low pressure liquid chromatography system(AKTA purifier)with a Sephacryl S-100 HiPrep 16/60 High Resolution column.After that,the W10 antifungal protein was identified as a serine protease by 5800 matrix-assisted laser desorption ionization-time of flight MS-MS(MALDI-TOF/MS-MS)and Mascot Ions Search analysis.The full-length gene sequence was obtained from W10 genome by primers designed and PCR.After sequencing and analysis,the sequence contains one ORF encoding a gene of 1347 bp.By bioinformatics analysis,the antifungal protein gene encodes 448 amino acid residues with a molecular weight of 48794.16 Da and isoelectric point of 6.04.It is a kind of hydrophilic protein.In the secondary and tertiary structure,the proportion of a-helix and ?-sheet is similar,and it contains the conserved domain of Peptidase_S8.In this paper,the encoding gene of W10 antifungal protein was cloned and expressed in Escherichia coli and Bacillus subtilis.Among them,the recombinant protein failed to express in the E.coli system,but it expressed in B.subtilis system successfully.However,the molecular weight of the expressed protein is slightly different from the above,and it doses not have the antifungal activity.It is presumed that the amino acid structure may be changed during expressing,which affects the molecular weight and the antifungal activity.At the same time,the serine protease inhibitor PMSF had a certain inhibition effect on the extracted antifungal protein,which indicated that W10 antifungal protein is a serine protease.In addition,the synergistic disease resistance of W10 antifungal protein and Hpa1xoo protein was also studied.The combination of the two proteins could significantly increase the control effect on tomato grey mould compared with their respective treatments.The defense response-related genes analysis revealed that W10 antifungal protein induced the expression of hsr203J,COI1,Ntexp2 and Ntexp6 genes in different degrees in Nicotiana tabacum L."Xanthi",which involved HR,jasmonic acid signaling pathway and plant growth regulation.The expression of hsr203J,aoxl,Ntexp2 and Ntexp6 genes in different degrees were induced by Hpalxoo protein in Nicotiana tabacum L."Xanthi",and HR,active oxygen metabolism and plant growth regulatory genes were involved.The combination of W10 antifungal protein and Hpa1xoo protein induced the expression of all the above mentioned genes in Nicotiana tabacum L."Xanthi",and the expression intensity was increased compared with the treatment alone.In addition,it was found that W10 antifungal protein induced the expression of hsr203J,Ntexp2,Ntexp6,COI1,and Chia5 genes in different degrees in transgenic hpalxoo gene tobacco.Therefore,these two types of proteins have a synergistic disease resistance and increase the disease control effect by increasing the relevant signaling pathways and related gene expression.This will improve the biocontrol effect by the integration of biocontrol resources and provide a theoretical basis for the new application of Bacillus licheniformis W10.