The Role Of Crz1 Transcription Factor Of Cryptococcus Humicola In Abiotic Stress And Its Interaction With Calcineurin Catalytic Subunit CNA

Calcium ion is an important intracellular signal,which plays a vital role in some cellular processes and its homeostasis regulation.When the intracellular calcium ion increases,it will activate downstream signals pathway-related proteins including calcium/calcineurin.This signaling pathway is widely found in fungi,mammals,and parasitic protozoa.Calcium(Ca²⁺)-calcineurin cascade signals have been extensively studied in eukaryotic systems.Studies have shown that calcium/calcineurin signaling pathways are involved in abiotic stress including oxidative stress,osmotic stress,p H stress,cold and thermal stimuli(temperature stress).C₂H₂ type transcription factor Crz1 is a downstream substrate of the calcium/calcineurin signalling pathway.Calcineurin dephosphorylates Crz1 to make it possible for Crz1 nuclear translocation and target gene transcription.Crz1 is involved in regulating cell wall integrity,membrane transport,redox processes,carbohydrate metabolism,protein kinases,encoding vesicle-mediated transport,and other metabolic and biological processes.C₂H₂ Zinc finger transcription factor Crz1 is also present in Cryptococcus humicola.In order to further verify the role and function of calcineurin and the transcription factor Crz1 in the resistance of yeast to abiotic stress,in this study C.humicolawas used as an experimental material and the following results were obtained:C.humicola was used to perform plate experiments under stress conditions of different calcium and cadmium concentrations,and the results showed that the growth of C.humicola was significantly inhibited.In order to further verify the role of calcineurin and its downstream substrate Crz1 in the growth and resistance to abiotic stresses of C.humicola,the strain was grown in a time gradient containing 50 mM calcium ions and 300?M cadmium ion concentration.Quantitative PCR was performed on the regulatory subunits B of calcineurin(CNB)and Crz1,and the expression of genes under calcium and cadmium stress was analyzed at the molecular level.The results showed that calcium and cadmiumstress improved the expression of Crz1 and CNB.The yeast two-hybrid technique was used to verify the interaction between the calcineurin catalytic subunit A(CNA)and Crz1 of the calcineurin pathway.The constructed bait expression vector and prey expression vector were respectively transformed into Y2HGold and Y187 yeast competent cells,and tested for two-hybrid verification.The results showed that there was an interaction between CNA and Crz1.Transgenic Saccharomyces cerevisiae INVSc1-pYES3-Crz1 was used to detect the role of Crz1 gene in stress tolerance including hydrogen peroxide,calcium,cadmium and aluminum.The results of the plate test showed that with the increase of stress concentration,the growth of the transgenic yeast pYES3/CT-Crz1 was significantly higher than the control yeast containing the empty vector pYES3/CT.Hydrogen peroxide can promote yeast growth within a certain concentration range,and the concentrationexceeds 600?M,growth is inhibited.Plate stress experiments with calcium,cadmium and aluminum showed that Crz1 can promote the growth of strains under stress.The growth curves of several transgenic strains under different concentrations of hydrogen peroxide,calcium,cadmium,and aluminum were measured.The growth curve of the strains with hydrogen peroxide and aluminum stress showed an extended lag period;however,the lag period of the transgenic strains with calcium stress and cadmium stress was not significantly different from that without stress.The rest of the results are consistent with the plate experiment,under the same growth conditions,the transgenic yeast grows better than the control yeast.Under the same growth conditions,the transgenic yeast grows better than the empty yeast.