Cloning And Functional Study Of BbchiA2 Gene From Beauveria Bassiana

As an entomogenous fungus,Beauveria bassiana is capable of infecting more than 700species of insects in 521 genera,149 families,in 15 orders.Because of its high pathogenicity,high adaptability and wide host range,Beauveria bassiana has gradually become a major role in biological control in recent years,replacing chemical pesticides.However,due to its wide host range,economic animals such as silkworm are also affected by B.bassiana,resulting in irreparable economic losses.Therefore,it is particularly important to study the pathogenic mechanism of B.bassiana and the different physiological activities it produces when dealing with different hosts.In our laboratory,a strain GXsk1011 with high virulence was isolated and identified in the silkworm population,and a gene Bb Chi A2（BBA＿05353）with low expression in the silkworm epidermis and high expression in the cotton bollworm epidermis was obtained through transcriptomic analysis of its response to different insect epidermis.In this study,the gene had been cloned and bioinformatics analyzed.And the knockout,complement and overexpression mutants of this gene were constructed by using related genetic engineering methods.In order to investigate the infection characteristics of chitinase Bb Chi A2 of B.bassiana by comparing the differences of each mutants strain in dealing with two representative hosts,bombyx mori and cotton bollworm.At the same time,the chitinase gene was exogenically expressed by pichia pastoris expression system to further explore the function of the gene expression product and the difference in its degradation of insect epidermis from the molecular perspective.1.Cloning and biological analysis of target geneMain research results:na was extracted and the target gene BbchiA2 was cloned by high-fidelity PCR system.Bioinformatics analysis of BbchiA2 gene by related software and database showed that BbchiA2 gene belonged to the glycoside hydrolase 18 family（GH18）gene,which had 99.01%homology with Beauveria bassiana A2860 strain in NCBI database and did not contain introns.The nucleotide length of the gene was1008bp.Encoded metabolites with a size of 35.47 KDa;The metabolite contained a n-terminal signal peptide at amino acid position 1-18,which was preliminarily identified as secretory protein.And an N-glycosylation site at the amino acid position 151.2.Construction of mutant strainsThe knockout,complement and overexpression vectors of the target gene BbchiA2were respectively constructed and the transformation of the wild-type Beauveria bassiana GXs K1011 was realized by agrobacterium transformation method,and the positive transformants of the corresponding mutant were obtained:ΔBbchiA2（knockout strain）,Com BbchiA2（replacement strain）and OEBbchiA2（overexpression strain）.3.Comparison of biochemical properties of mutant strainsThe colony morphology,growth rate,sporulation rate,spore germination rate,stress resistance and virulence of B.bassiana mutant and Wild-type（WT）strain against silkworm and cotton bollworm were investigated.The results were as follows:1)Compared with the wild strain,the colonies of the knockout strain were characterized by short mycelium and spreading outwards;The colony morphology of the replacement strain was similar to that of the knockout strain,but a small number of long mycelia grew close to that of the wild strain.The overexpression strain showed completely different phenotypes from the knockout strain and the replacement strain,and the aerated mycelia were significantly longer than those of the knockout strain and the wild strain.2)In terms of sporulation,the sporulation of knockout plant was significantly higher than that of replacement plant,wild plant and overexpressed plant,among which the overexpressed plant showed a significant decline in sporulation;3)From the point of view of spore germination rate（activity）,it was found that the germination ability of overexpressed strain was significantly higher than that of wild strain,knockout strain and complementary strain.There was no significant difference among wild strains,knockout strains and replacement strains.4)From the effect of stress factors on mutant strains,the growth of overexpressed strain was significantly inhibited by the action of the cell wall inhibitor congored,but the knockout strain and the replacement strain grew better than the wild strain under the action of various stress factors.5)In terms of the body surface infection results of the fifth generation bombyx mori and cotton bollworm,both the knockout strain and the replacement strain showed lower virulence than the wild strain,and the over-expressed strain showed the weakest virulence against the silkworm.Even at the conidia concentration of 1×10⁸ conidia/m L,the mortality rate was only about 35%,but still did not reach 50%.However,the overexpressed strain showed higher virulence than the knockout strain and the complementary strain.The LT₅₀ was 180h when the conidia concentration was 1×10⁷ conidia/m L,and the LT₅₀ was 144 h when the conidia concentration was 1×10⁸ conidia/m L.In addition,there was a great difference between infecting silkworm by injection and body wall infecting.In general,the virulence of the overexpressed strain was significantly higher than that of other strains infected by injection,and the virulence of the wild strain was higher than that of the knockout strain and the replacement strain.The LT₅₀ of overexpressed strain was 80 h at 1×10⁶ conidia/m L,60h at 1×10⁷ conidia/m L,and 40 h at 1×10⁸ conidia/m L.From the virulence experiment,the interaction between different mutant strains and different insect body walls was indeed different,which directly affected the virulence of the strain to different insects.4.Exogenous expression of target genesIn order to further explore the effect of target gene BbchiA2 on insect body wall,the eukaryotic expression vector PPIC9K-Chi A2 was constructed by one-step cloning method,and successfully transferred into the receptor state of Pichia pichia by electric transformation method to obtain a highly expressed strain.At the same time,the fermentation conditions of the expressed strain were explored and the optimal fermentation time was 5 days and the optimal concentration of imidazole eluent was 50 m M.Finally,the target gene BbchiA2 protein product was obtained by fermentation and purification and concentration.5.Activity determination of recombinant BbchiA2 proteinThe concentration of the exogenously expressed protein product was 3.5 mg/m L,and the highest enzyme activity was 45.16 U/g at 40℃.In addition,BbchiA2 protein was degraded in the epidermal degradation experiments of silkworm and cotton bollworm.Finally,in the 200μL system with 0.01 g epidermal as substrate,the amount of reducing sugar produced by the epidermal degradation of silkworm was 0.823μg after 1 hour reaction at40℃.However,the amount of reducing sugar produced by the epidermal degradation of cotton bollworm was 1.554μg,so the degradation effect of BbchiA2 protein on cotton bollworm was significantly higher than that on silkworm.In conclusion,in this study,the knockout strains,complement strains and overexpression strains of chitinase BbchiA2 gene of B.bassiana were constructed,and the biological phenotypes of mutant strains and wild strains were investigated respectively.The virulence of the knockout strain to bombyx mori and bollworm was lower than that of the wild strain.The virulence of the overexpressed strain to the epidermal infection of Bombyx mori decreased significantly,but increased to the cotton bollworm.Heterokaryotic expression of this protein on the epidermis,the degradation of cotton bollworm epidermis was significantly higher than silkworm epidermis.The results showed that the differential expression regulation of the chitinase family gene BbchiA2 may be an important molecular mechanism for B.bassiana to adapt to different insect epidermal infection.