Expression And Analysis Of Recombinant Protease Bbepn L-2 From Beauveria Bassiana And Detection Of Expression Profile Of Bbepn Family

Beauveria bassiana,an important entomopathogenic fungus,which has circular conidia and the white colony,can be used as a fungal spore preparation for pest control.The main way of Beauveria bassiana infecting insects is cuticle degrading.The cuticle of insects is composed of protein and chitin,in which protein is the majority and chitin is contained.Protease plays an important role in the process of Beauveria bassiana penetrating insect cuticle.Serine protease,Bacillus subtilis protease(Pr1)and trypsin(Pr2)are known to degrade insect cuticles.When the host is Bombyx mori,the aspartic protease Endothiapepsin-like protein(BbepnL-1)can also degrade the cuticle.In the previous experiment,a strain GXsk1011 with high virulence to Bombyx mori was isolated in our laboratory.When the strain penetrated the cuticle of Bombyx mori,four aspartic protease genes were detected,three of which were up-regulated.After knockout one of the genes(BbepnL-1),the virulence of the strain to Bombyx mori was reduced.The BbepnL-1 expressed by Pichia pastoris expression system can degrade the cuticle of Bombyx mori.In this study,another up-regulated gene,Endothiapepsin-like protein(BbepnL-2),was cloned,bioinformatics analyzed and expressed by the Pichia pastoris expression system.Explore the protease activity of BbepnL-2 in the infection of Bombyx mori and the expression spectrum of the Bbepn genes.The main research methods and results are as follows:1.Cloning and Bioinformatics analysis of the gene BbepnL-2Extract the total RNA of B.bassiana GXsk1011,reverse transcribe the c DNA and clone the BbepnL-2 through the high fidelity PCR.Bioinformatics analysis showed that the total length of the BbepnL-2 was 1197 bp,encoding 398 amino acids.The predicted molecular weight of the protein was 42.9 k Da.There was a signal peptide at the N-terminal and a glycosylation site.BbepnL-2 protein contains the conserved domain of aspergillopepsin-like and pepsin retropepsin superfamily which belongs to the aspartic protease family.There is a highly conserved Asp-Thr-Gly active site at the N-terminal and C-terminal,respectively.There is no mutation in the active site,and it is located in the center of the protein,which catalyzes the hydrolysis of the peptide bond.The homology between BbepnL-2 and Beauveria bassiana A2860 was 97%.2.Exogenous expression of BbepnL-2The recombinant expression plasmid BbepnL-2-p PIC9 K was constructed by one-step cloning method,and then transfered into the competent cell GS115 of Pichia pastoris.Positive transformants were screened by histidine free medium and inoculated in G418 concentration gradient medium to screen high copy strains.Finally,a 2200 bp and a 1700 bp(BbepnL-2 related)band were obtained by PCR with primer5’AOX/3’AOX.After constructing the right strain,expressing it with methanol,and the fermentation supernatant was analyzed by SDS-PAGE.The results showed that there was a target protein band at 45 k Da,and the protein expression was the highest at 96 h.The 45 k Da band was identified as BbepnL-2 by Western blotting.The fermentation supernatant was collected and concentrated by ammonium sulfate.The protein was purified by Ni-NTA resin for subsequent functional verification.3.Activity detection of BbepnL-2The activity of recombinant protease BbepnL-2 was 291.542 under acid condition.SDS-PAGE showed that no small peptide was detected at 15 k Da in the supernatant of interaction between BbepnL-2 and B.mori cuticle.Scanning electron microscope showed that the surface of the B.mori cuticle which treated with recombinant protease BbepnL-2 was smooth,however there was no degradation trace compared with the PBS control group.It was inferred that recombinant protease BbepnL-2 had no degradation effect on the B.mori cuticle.4.Detection of the gene expression profile of the Bbepn familyB.bassiana GXsk1011 was cultured in B.mori cuticle medium.The RNA of B.bassiana was extracted at different time points.The expression of the Bbepn family was detected by q RT-PCR.The expression level of BbepnL-2 was high from 12 h to 96 h,increased from 12 h to 36 h,and reached the highest level at 36 h.The expression level of BbepnL-1 and BBA＿03607 was the highest at 24 h,and then began to decrease.When B.bassiana infected the B.mori for 12 hours,the conidia had germinated and attached to the B.mori cuticle,and the hyphae penetrated into the B.mori blood cavity after 24 hours.During this period,the expression of Bbepn family genes showed an upward trend,indicating that they were activated by the B.mori cuticle and related to the infection of B.bassiana into the Bombyx mori cuticle.In conclusion,the gene BbepnL-2 was successfully cloned in this study,and the structure and function of the protein were preliminarily understood by bioinformatics analysis.Express the BbepnL-2 through the Pichia pastoris and explore the function when the protein acted on the B.mori cuticle.The results showed that the BbepnL-2cannot degrade the cuticle of B.mori.The expression profile of the Bbepn family showed that the three genes showed different expression characteristics with time.