Aldo-keto Reductase Bbakr1 Was Involved In Hyperosmotic And Heavy Metal Chromium Stress Response Of The Insect Fungal Pathogen, Beauveria Bassiana

Beauveria bassiana is one of entomogenic fungi, which extensively used in biological control of agriculture, forestry and hygiene pests, it is also one model strain for researching filamentous fungi-host and-environment interaction. Aldo-keto reductase is one of NADP- dependent oxidoreductases, which is widely existed in the prokaryotic and eukaryotic organisms, it is involved in carbohydrate glucose metabolism and many kinds of stress responses, however, the research on function of aldo-keto reductase in fungus are rarely reported. Previous studies showed that mitogen-activated protein kinase Bbhog1 in B. bassiana, which is homologous to the yeast Hog1, is an important regulatory pathway of adverse environment stresses and pathogenicity. To reveal potential molecular machanism of Bbhog1 regulation of environmental stress responses, we constructed suppression subtractive hybridization(SSH) library between wide type and ΔBbhog1 under hyperosmotic stress, previously. Fifteen downstream target genes of the Hog1 MAP-kinase from subtraction library were isolated, which were significantly down-regulated in ΔBbhog1. One clone H231 displayed high homology to members of the aldo-keto reductase superfamily in other fungi and named it Bbakr1. In the paper, the strategies of gene knock-out, reverse complement and over expression were used to investigate the relationship between Bbakr1 and development, stress and virulence of B. bassiana. The main results obtained are listed as following: 1. Sequence analysis and expression pattern of Bbakr1Sequence analysis revealed that Bbakr1 contained 930 bp open reading frame, encoding 310 amino acids with a predicted molecular mass of approximately 34.1 KDa. Bbakr1 is highly homologous to AKR proteins in many fungi. The phylogenetic tree analysis of different AKR protein sequences showed that Bbakr1 clustered in Cordyceps militaris, which speculated that Bbakr1 might have closer evolutionary relationships with AKR of Cordyceps militaris.qRT-PCR analysis indicated that the expression of Bbakr1 are induced by hyperosmotic and heavy metal chromium, and the expression level did change with time. It could be inferred that Bbakr1 might be involved in high osmotic stress response and detoxification of heavy metal chromium. 2. Bbakr1 was related to spore production and development of Beauveria bassianaTo clarify the relationship between Bbakr1 and development, stress response and virulence of B. bassiana, in the paper, this study constructed gene knock-out mutant(ΔBbakr1), reverse complement strain(Comp) and over expression transformant(OE-Bbakr1), and a comparative study of them and wide type(WT) had been done, finally found that Bbakr1 mediated spore production and development of B. bassiana.The conidia yield on basic medium of ΔBbakr1 and OE-Bbakr1 had fallen 41%(P < 0.01) and 10%(P < 0.05) from WT, respectively; the conidia yield on eutrophication medium of ΔBbakr1 had decreased by 15% than WT(P < 0.01), while there were no significant difference between OE-Bbakr1, Comp and WT. The results indicated that Bbakr1 mediated conidia yield in B. bassiana.Disruption of Bbakr1 decreased conidia germination rate, median germination time of ΔBbakr1 lowered 0.72 h than WT(P < 0.05), while there were no significant difference between OE-Bbakr1, Comp and WT. The results indicated that Bbakr1 mediated conidia germination in B. bassiana.Bbakr1 related to blastospores yield and hyphae biomass accumulation of B. bassiana. Disruption of Bbakr1 led to a reduction in blastospores yield and hyphae biomass accumulation, while no significant difference was detected in blastospores yield and hyphae biomass accumulation in OE-Bbakr1, Comp and WT strains. 3. Bbakr1 was associated with high osmotic stress response in Beauveria bassianaDisruption of Bbakr1 resulted in an increase in sensitivity to hyperosmotic stress, while no significant difference was detected between OE-Bbakr1, Comp and WT. Fungal cells often accumulate high levels of sugar alcohol to adapt hyperosmotic stess. The intracellular accumulation of sugar alcohol results showed that disruption of Bbakr1 led to a reduction in accumulation of erythritol, while overexpression of Bbakr1 led to an increase in accumulation of erythritol. In addition, disruption or overexpression of Bbakr1 interferred glycerol, arabitol and mannitol accumulation in the fungal cells. It is speculated that Bbakr1 might mediate sensitivity to osmotic stress via regulation of the intracellular accumulation sugar alcohol(especially erythritol). 4. Bbakr1 was involved in the detoxification of heavy metal chromiumDisruption of Bbakr1 caused an increase in sensitivity to heavy metal chromium, whereas no significant difference in the sensitivity was observed in OE-Bbakr1, Comp and WT. The growth rate of ΔBbakr1 was 22% approximately lower than WT(P < 0.01). The toxicity of heavy metal to cells usually causes by the intracellular lipid peroxidation and the accumulation of active aldehyde. The intracellular level of lipid peroxidation and malonaldehyde(MDA) accumulation showed that disruption of Bbakr1 led to a reduction in lipid peroxide(LPO) and an increase in MDA. The LPO of ΔBbakr1 were 43.21% and 12.74% lower than WT, respectively(P < 0.01), and the accumulation of MDA were 6.0 times and 3.4 times as WT, respectively(P < 0.01). It is speculated that Bbakr1 might play an important role of detoxification by reducing active aldehydes which induced by heavy metal chromium. 5. Disruption and overexpression of Bbakr1 did not influence the virulence of Beauveria bassianaThe bioassay results showed that disruption and overexpression of Bbakr1 had no effect on the virulence to Galleria mellonella larvae.