Cloning And Functional Study Of The Aspartic Proteinase-Endothiapepsin Gene BbeapA Of Beauveria Bassiana

Beauveria bassiana is an important insect pathogenic fungus,not only possesses a very important application value in the field of pest control,but also has been used as a model to study the interaction between pathogenic fungi and insect hosts.Therefore,studying the growth pattern of Beauveria bassiana and the interaction with insects has a dual significance of theory and practice.Beauveria bassiana infection is started with body surface invasion,thus host insect epidermis degradation is the key step in this process.Proteins are one of the main components of insect epidermis,therefore the proteases that are secreted by Beauveria bassiana may play an important role in the degradation process.In our previous study,we have obtained the differential expression genes（DEGs）in early infection stage of Beauveria bassiana by comparative transcriptomic analysis between hyper-and hypo-virulent strains.Among these DEGs,an aspartic protease synthesis gene BbeapA has triggered our interest which was analyzed in this study.To explore the potential biological role of BbeapA,we have constructed gene knockout mutants by homologous recombination method,and evaluated both growth and virulence of the mutants.This study provides an important reference for the pathogenicity mechanism of Beauveria bassiana.The main contents of this paper are as follows: 1.Bioinformatics analysis of BbeapA gene of Beauveria bassianaThe genome of Beauveria bassiana GXsk1101 was used as a template to clone the BbeapA gene fragment.Through the analysis of biological information,it is shown that the BbeapA gene ORF is 1131 bp in length that encodes 376 amino acids with a molecular weight of about 39.9kDa and a theoretical isoelectric point of 5.00.BbeapA protein is stable and the peptide chain is rich in hydrophilic group that may be a soluble protein.BbeapA protein contains 17 amino acid signal peptide sequences in the N-terminal and has no obvious transmembrane region.In the secondary structure,the random curl is the main components.Through the analysis of the domain,it is shown that the protein contains an aspartic acid domain belonging to the aspartic acid family pepsin class with two active sites.Therefore,it is speculated that BbeapA protein is an aspartic acid hydrolase.2.Construction of BbeapA gene knockout and complement strainsThe fragment of BbeapA ORF was coloned from the genome of Beauveria bassiana,and ligating with the vector pk2-bar to construct the knocker expression vector.The gene BbeapA of Beauveria bassiana was replaced by the expression element in the vector via homologous recombination to inactivate the target gene.According to this method,B.bassiana GXsk1101 was used as transformation receptor,that was subjected to AGL-1 agrobacterium-mediated genetic transformation to obtain the knockout transformants.Then the knockout strains were verified by PCR and RT-PCR techniques.For constructing gene complement strain,the upstream and downstream fragment of gene BbeapA from the genome of Beauveria bassiana GXsk1101 were cloned,and ligated to vector pk2-sur to construct a complement expression vector.Then the knockout strain was used as transformation receptor to obtain BbeapA gene complement strain by using the method mentioned above.3.Study on the Function of gene BbeapABbeapA gene was involved in the regulation of the growth of Beauveria bassiana.The knockout mutant and wild type strains were inoculated in the SDAY medium and CZA medium respectively,and the results showed that the growth rate of the knockout mutant was lower than that of the wild type strain.The morphological difference of fungal colony was also observed.In the SDAY medium,the colony of wild type strain appeared regular ring-shape with a uplifted spherical center,while knockout mutant formed white snowflakes shape with a sunken center.These results indicated that BbeapA gene may be involved in the regulation of growth and development of Beauveria bassiana.BbeapA gene affected sporulation of Beauveria bassiana.The knockout mutant and wild type strains were cultured in the SDAY medium.The results showed that the conidia production of the mutant was higher than that of the wild type strain.The maximum difference was observed after 8 days,and the sporulation of the knockout strain was 3.1×105 conidia/mm²,that was 5.2 times higher than that of the wild strain which was 0.5×105 conidia/mm².BbeapA gene affected carbon and nitrogen sources metabolism of Beauveria bassiana.By comparing the growth of knockout mutant and wild type strains on SDAY medium,CZA medium and modified CZA medium which contained 10 different carbon and nitrogen sources,we found that while olive oil was used as the sole carbon source,the growth rate of knockout mutant was higher than that of the wild type strain.In addition,the similar phenomenone was observed while gelatin and proline were used as the sole nitrogen source.BbeapA gene affected the growth of Beauveria bassiana under different stress conditions.Both BbeapA gene knockout mutant and wild type strains were cultured in the 1/4 SDAY medium which respectively included six kinds of metal ions which were Zn²⁺,Mg²⁺,Mn²⁺,Cu²⁺,Fe³⁺,Ca²⁺ as stress factors.The results showed that Ca²⁺ and Mg²⁺ stress reduced the growth rate of knockout mutant.Under high permeability conditions（1M NaCl）,the growth rate of the knockout mutant was lower than that of wild type strain.Under the condition of H₂O₂ stress,the growth rate of knockout mutant was higher than that of wild strain.Under the conditions of cell wall inhibitor Congo red（CR）,fluorescent whitening agent（CFW）and SDS stress,the growth rate of knockout strain decreased 20%,8.3% and 7.7% respectively.These results suggested that Beauveria bassiana BbeapA gene might be involved in stress response against several environmental factors.BbeapA gene affected virulence of Beauveria bassiana.To evaluate whether BbeapA gene affected virulence of Beauveria bassiana,1×108 conidia/mL of conidia suspension was inoculated on silkworm cuticule.The results showed that the virulence of the gene knockout mutant was significantly lower than the wild type strain,that the median lethal time（LT50）was extended for 1.39 d（p<0.05）.Both gene knockout mutant and wild type strains reduced the feeding activity of silkworms quickly after infection.However,the mycelial growth of knockout and wild strains was different on the surface of the dead silkworms.By continuous culturing dead silkworm for 2 d,it could be easily observed that the aerial mycelia of the knockout mutant were much higher than that of the wild type strain.