| Beauveria bassianais animportant entomopathogenic fungus. In recent years, it is newly classified to the genus Cordyceps, Cordycipitaceae, Hypocreales, Hypocreomycetidae, Sordariomycetes, Pezizomycotina, Ascomycota, Dikarya. B. bassiana not only often causes infection andepidemics of wild insects, but some strains also show pathogenicity to the silkworm, Bombyx mori L. Insect body wall as the first natural barrier play animportant role in defensing against fungal pathogens. In the process of B. bassiana adhering to the body wall and penetrating into it, the silkworm exhibits a series of physiological, pathological and immune response, which are associated with infection-related genes involved the specific response in B. bassiana. Therefore, screening and identifying the related genes in this process and studying the function would be significant for further revealing the pathogenic mechanism of B. bassiana.Based on the previous study of our laboratory about the comparative transcriptomicanalysis of two B. bassianas trains with different pathogenicity, a candiadate gene of infection-related BbppepA00319 gene was selected as target gene from the differential expressed genes. Through extraction of genomic DNA from B. bassiana strain GXsk1011, cloning of gene BbpepA00319 and construction of BbpepA00319 gene knockout mutants using the method of homologous recombination; as well as phenotypic analysis of mutant strain such as colony characteristics, spore production, chemical sensitivity and pathogenicity text were performed. The preliminary biological functions of BbpepA00319 gene in B. bassiana growth and pathogenesis were characterized based on the results of the present study. The main study results are listed as follows: 1. Bioinformational analysis of BbpepA00319 gene in B. bassianaBased on previous results of transcriptomeanalysis, the candidate gene BbpepA00319 for infection related gene was selected as the target gene for subsequent validation analysis. The bioinformatic analysis of the gene sequences showed the gene has a length of 1259 bp open reading frame, which codes 395 amino acids. The molecular weight of encoding protein was 41565.0 u and the isoelectric point was 5.77. Prediction by online softwares showed that the protein had a signal peptide sequence of 18 amino acids in N-terminal, while transmembranedomain was not found. Secondary structure prediction indicated that the structure of the protein is roughly similar with serine protease. By further analyzing the domain of peptidase_S8 encoding by the gene, it is part of the serine peptidase structure domain. The result of homology analysis displayedthe protein encoded by this gene was similar to alkaline protease 1 in B. bassiana strain D1-5(99% identity). Thus it is speculated that the protein encoded by BbpepA00319 gene belongs to serine protease. 2. Construction of knockout vector for BbpepA00319 gene in B. bassiana and screening of mutant strainsThe genomic DNA of B. bassiana strain GXsk1011 was extracted and PCR amplification of 5’ and 3’ flanking sequences(the homologous exchange arms pepA00319 L and pepA00319R) using the specific primers was done, which will be used for knock out of target gene. Then the fragments were recovered and linked with the clone vector pMD19-T respectively, thus obtained two recombinant cloning vector containing the flanking fragments. The skeleton vector pK2-bar plasmid was used for knock-out of target gene, corresponding fragment from the recombinant cloning vector(containing the left and right homologous fragments) were enzymatic cut and linked with pK2-bar vector respectively, then the knock-out vector pepA00319L-pK2-pepA00319 R was constructed successfully. Finally, using B. bassiana GXsk1011 as transformation receptor, the knockout transformants were obtained through the method of AGL-1 agrobacterium-mediated genetic transformation. And the knockout mutant strain ΔBbpepA00319 was screened by PCR amplification. 3. Phenotypic analysis of gene knockout mutant strain ΔBbpepA00319In order to analyzed the phenotypic of the correct knockout mutant strains. First of all, with the wild-type as control group, we comparethe colony characteristics, growth rate and spore production on the normal SDAY medium. The results of phenotypic analysis indicated the two strains have no significant difference in colony character; and for the colony average growth rates of two strains, the colony of mutant reached 3.96 mm/d while wild-type have the rate of 3.79 mm/d, which showed no significant difference between both strains; comparingthe conidial yield of two strains, ΔBbpepA00319 mutantstrain was always higher than the wild-type strain from 8 d to 16 d. At 16 th day, the conidia yield of ΔBbpepA00319 mutant strain reached 3.32×107conidia/cm2, which was significantly higher than that of the wild-type strain. The results suggested BbpepA00319 gene may involved in the conidiation of B. bassiana.In order to detect the sensitivity of the knockout mutant strain to chemicals, six kinds of metal ions, two high osmotic salts and three kinds of cell wall inhibitors were selected. The results indicated the promoting effectof six species metal ion on wild-type strains for 51.3%~94%, on ΔBbpepA00319 mutation strains only for 6.5%~20%, and the Cu2+(3 mM) exhibitedinhibitory effect on ΔBbpepA00319 mutant strain, comparing with the control group, the colony growth rate reduced 8.5%. Based on the results above, ΔBbpepA00319 mutant strain was more sensitive to six kinds of metal ions compared to the wild-type strain. And no apparent effects was detected on the wild- type strain when grown on 1/4SDAY media with two osmotic salt. While the inhibitory effect on the ΔBbpepA00319 mutant strain was examined, the growth rate decreased 33.5% and 48.5% in the media with 1M NaCl and 1M KCl, respectively. The ΔBbpepA00319 mutant strain was more sensitive to two antioxidants than the wild-type strain; and the ΔBbpepA00319 mutant strain and the wild strain have no significant difference in sensitivity to two insecticides and three kinds of cell wall inhibitors.The silkworm at 5th age larva was used as test insect. Through the method of epidermic inoculation, the virulence of mutant strain was measured. The result of pathogenicity assay showed the median lethal time(LT50) of the ΔBbpepA00319 mutant strain at the concentration of 106 conidia/m L was significantly longer than that of the wild-type(p<0.05); and LT50 of ΔBbpepA00319 mutant and the wild-type strain at the concentration of 107 conidia/m L and 108 conidia/m L have no significant difference(p>0.05). The results showed that the peptide gene Bbpep A00319 may participate in early stage of B. bassiana infection, In the low concentration, the pathogenicity of ΔBbpepA00319 mutant strain on silkworm significantly reduced, but no significant difference in pathogenicity was showed between the two strains at higher concentrations. |
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