Study On The Biological Function Of A Novel Zn（Ⅱ）₂Cys₆ Regulator Bb Tpc1 In Beauveria Bassiana

Beauveria bassiana is a typical representative of entomopathgenic fungi which is widely used in biological control of pest.However,in the process of infecting,the effect of biocontrol always affected by environmental factors,such as temperature,humidity,chemicals.These results shows that the biocontrol of fungus insecticidal was slow and unstable.Therefore,exploring the physiological mechanism of stress resistance of biocontrol fungi is of great significance to the genetic improvement of strains aiming at improving the potential of biocontrol and the rational application of fungus preparation.In this study,ΔBbTpc1 andΔBbTpc1/BbTpc1 strain were constructed via the Agrobacterium-mediated transformation procedure and compared with the wild-type strains（WT）to exploring the function of BbTpc1 in B.bassiana.Revealed that BbTpc1plays an important role in fungal cell growth and development and stress resistance,especially in the chitin synthesis.The main research and results of this study are as follows:（1）Biological function of BbTpc1 in B.bassiana1)The deletion of BbTpc1 had an effect on vegetative growth.On nutrient-rich SDAY,1/4SDAY and nutrient-deficient CZA medium,the colony area of gene knockout strains was significantly smaller than that of control strains（wild type and complementary type）.Decreased by 40.1%,52.5%and 66.8%,respectively.Compared with the control strains,the cell ofΔBbTpc1 were shorter and compact.After culturing in nitrogen-limited liquid medium,abundant autophagic bodies were detected in all the strains,whereas the larger lysosomes were observed in cells derived from theΔBbTpc1.These results suggested that the differentiation of hyphal cells became abnormal in the deletion mutant.2)The capacity of conidium production of BbTpc1 knockout strain showed serious defects.From the 5th to 7th day on SDAY medium,the conidia yield decreased by 68.5%,50.5%and 48.8%,respectively.At the same time,the blastospoer yield of BbTpc1knockout strain also decreased significantly,which decreased by 34.2%and 75%at 72 h and 96 h,respectively.Finally,the results of Flow cytometry showed that the size of conidia and blastospore ofΔBbTpc1 decreased by 12.3%and 39.6%,respectively.However,there was no difference in germination rate and speed between BbTpc1knockout strains and control strains.These results suggest that BbTpc1 plays an important role in the production of conidia and blastospore3)A sensitive concentration of chemicals was used to assess the effects of multiple chemical stresses on the growth of fungal colonies on CZA medium at 25°C.Based on the relative survival rate,the deletion mutant showed a notable elevation in cell sensitivity to high osmolarity（Na Cl）,fungicides（CAR）,cell wall perturbation（SDS and CR）and oxidation（H₂O₂）.The sensitivity ofΔBbTpc1 to the above chemical stresses was enhanced by 45.6%,32.8%,39.8%,52.6%and 18.2%,respectively.Conidial thermotolerance was assayed by exposing conidia to wet-heat stress at 45°C.The median lethal time(LT₅₀,min)for estimating the conidial tolerance to high temperature was distinctly reduced by 11.8%inΔBbTpc1 compared with the estimates in WT.These results indicated that BbTpc1 plays a crucial role in sustaining the tolerance of B.bassiana to multiple chemical stresses and high temperatures.4)The third instar larvae of Galleria mellonella were infected with the conidia of WT andΔBbTpc1 strains by topical application and intrahemocoel,the LT₅₀ of the knockout strain was delayed by 36.3%and 66.7%,respectively.There was almost no mycelium in the blood of the infected larvae,and the fungus growth was sparse on the body surface of the cadavers.These results indicated that BbTpc1 is a necessary factor for the virulence of B.bassiana.（2）Deletion of BbTpc1 significantly affected the global transcriptomeThe deletion of BbTpc1 resulted in significant changes in the expression level of1553 genes,including 1166 down-regulated genes and 387 up-regulated genes.KEGG enrichment analysis showed that 1553 different genes were mainly enriched in fatty acid degradation,amino sugar,nucleotide sugar metabolism and MAPK signaling pathway-yeast.GO analysis of 1553 differential genes showed that BbTpc1 knockout mainly affected membrane-related genes,and BbTpc1 regulated fungal asexual reproduction through different genetic pathways.The knockout of BbTpc1 significantly affected the transcriptional map of B.bassiana.（3）Effect of BbTpc1 on cell wall integrity of gungiThe thickness of the conidia cell wall of the knockout strain was observed by the Transmission Electron Microscope（TEM）.The conidia cell wall of the knockout strain was less than that of the control strain.The cell wall composition of the knockout strain was measured by acid hydrolysis.The content of chitin in the cell wall of the knockout strain decreased,but there was no significant difference in the content of glucan.The transcriptional level of chitin synthase family genes was quantified by q RT-PCR.The results showed that the expression of seven chitin synthase genes was downregulated in the knockout strains.These results show that BbTpc1 can regulate the expression of chitin synthase,thus affecting the content of chitin in the cell wall,resulting in the cell wall of the knockout strain being thinner than that of the control strain.Finally,through yeast single hybrid technology,it is proved that BbTpc1 can not directly regulate chitin synthase gene.