The Molecular Biological Function Of Tudor Protein BbTdp1 In Of Beauveria Bassiana

As an entomopathogenic fungus,Beauveria bassiana has important pest control functions and has been widely used in agricultural production all over the world.At the same time,B.bassiana is also an important model fungus,which is of great significance in studying the growth of fungi and the mechanism of interaction between B.bassiana and its host.Through literature review,our research group found that the Tudor protein gene BbTdp1 in the genome of B.bassiana has a high homology with the human cancer protein SND1,which has been proved to be mainly related to the growth and development of cells.This study takes gene BbTdp1 as the research object,by means of homologous recombination and ectopic integration built knockout and covering strains,and the function analysis,to explore the influence of gene for growth,development and the virulence of B.bassiana,which will provide the theory basis for pathogenic mechanism of B.bassiana and help improve the pathogenic regulation network of B.bassiana.The main contents and results of this study are summarized as follows:1.Biological analysis of BbTdp1 gene of B.bassiana and construction of knock-out and complementary strainsBioinformatics analysis showed that gene BbTdp1 is a conserved transcription factor,which is composed of four homologous staphylococcal endonucase domains（SNc）and Tudor in tandem.The full length ORF of BbTdp1 gene is 2652 bp,encoding 883 amino acids.Phylogenetic analysis showed that BbTdp1 protein had high homology in a variety of entomopathogenic fungi,animal pathogenic fungi,plant pathogenic fungi and yeast fungi,but no homology gene was found in Saccharomyces cerevisiae.The genome of wild type（WT）of B.bassiana was used as a template,and the knockout strain（ΔBbTdp1）and complement strain（ΔBbTdp1/BbTdp1）of B.bassiana were obtained by homologous recombination,heterotopic insertion,and Agrobacterium-mediated fungal transformation.2.BbTdp1 gene of B.bassiana mediates colony growth and hyphal extensionIn order to investigate the influence of different nutrition on the colony growth of WT,ΔBbTdp1 andΔBbTdp1/BbTdp1,the strains were inoculated on the SDAY and CZA culture plates.After 10 days of culture,the colony area ofΔBbTdp1 was～24.7%larger than that of WT,but the mycelial biomass was～37.7%less than that of WT in the SDAY medium.In CZA medium,the colony area ofΔBbTdp1 was～71.8%smaller than that of WT,and the biomass was also～50.0%less.The number of mycelium septa of WT strain was significantly more than that ofΔBbTdp1 strain.3.BbTdp1 gene of B.bassiana regulates conidial growth,development and stress resistanceLaser confocal microscopy and flow cytometry showed smaller conidia size of the knockout strain.The sporulation ofΔBbTdp1 strain was 64.1%～87.7%lower than that of WT,and the half germination time(GT₅₀)was more prolonged～39.3%.The hydrophobicity ofΔBbTdp1 strain was～36.4%lower than that of WT;Stress sensitivity tests showed thatΔBbTdp1 strain was～45.6%,～54.3%,～33.2%and～4.4%more sensitive to osmotic stress,oxidative stress,cell wall destructing agent and chemical pesticides than WT,respectively.4.BbTdp1 gene of B.bassiana regulates cell growth,size and cycle of blastosporesIn order to investigate the growth,development and cell cycle of blastospores,blastospores were cultured in NLB medium.The results showed that the spore production ofΔBbTdp1 strain was 71.4%～96.3%lower than that of WT.The blastospore size ofΔBbTdp1 strain was～33.8%smaller than that of WT;The cell cycle of blastospores was detected by flow cytometry.Compared with WT,the S phase ofΔBbTdp1 strain was prolonged by～65.6%,the G0/G1 phase and the G2/M phase were shortened by～12.6%and～54.4%,respectively.ELISA assay showed that the P-CDK1 protein concentration ofΔBbTdp1 strain was～31.5%lower than that of WT,but there was no significant difference in the protein concentration of NP-CDK1 among the three strains.5.The BbTdp1 gene of B.bassiana is an essential element for fungal virulenceTo investigate the regulation of BbTdp1 on virulence of Galleria mellonella,two methods of in vitro immersion and in vivo injection were used.The results showed that the half lethal time(LT₅₀)of the infected insects soaked byΔBbTdp1 was～24.7%slower than that infected by WT,while when injected with infection,it was～40.0%slower.Confocal laser microscopy was used to observe the body fluids of infected insects.It was found that the blastospores of infected insects infected withΔBbTdp1 strain were significantly less and shorter.After 5 days of continuous culture,the mycelia on the body surface of the insects infected by the control strain were denser than those infected byΔBbTdp1 strain.All above proved that the BbTdp1 gene of B.bassiana is an essential element for fungal virulence.6.Transcriptional profile changes mediated by BbTdp1 gene of B.bassianaThe mycelia grown on SDAY medium for 6 days were frozen and ground to extract total RNA,which was used for transcriptome sequencing.Sequencing results showed that9305 genes were shared betweenΔBbTdp1 strain and WT strain,when threshold was set to|log2ratio|>2和q-value<0.05,4094 differentially expressed genes（DEGs）were screened out,including 3499 down-regulated genes and 595 up-regulated genes,which caused huge fluctuations in the global transcriptional map.The differentially expressed genes were analyzed by KEGG,GO and Fun Cat,indicating that the gene BbTdp1 is involved in carbohydrate,amino acid and lipid metabolism,protein synthesis and transport,and signal transduction in B.bassiana.