The Mechanism Of Calcineurin And C₂H₂ Transcription Factor Asr1 In Cryptococcus Humicola To Resist Aluminum

Aluminum toxicity is a major limiting factor in acid soil crop yields.The study of aluminum resistance mechanism is the prerequisite and basis for solving the problem of aluminum poisoning in acidic soil.Calcium ion is a ubiquitous signal in the cell.Increased concentrations of calcium in the cytoplasm can activate many proteins,including Calmodulin and calcium/Calmodulin-dependent protein Calcineurin.Calcineurin is conserved from yeast to humans(except plants).Many Calcineurin target proteins have been identified.C2H2 transcription factor exists in many fungi,amoeba and lower eukaryotes.Studies have shown the functions of calcineurin pathway,including the ion balance in cell,the form of fiber in cell wall,are conserved in various creatures.Crzl is a zinc finger transcription factor that response to regulation of Calcineurin.Activation of Calcineurin can upregulate multiple gene expression,however,expression of these genes in ?crzl will be reduced.This is a full proof that the main function of Crzl is to regulate the expression of genes via Calcineurin.The C2H2 transcription factor gene invovled in aluminum stress response(Asrl)in the Cryptococcus humicola.To research the function of Calcineurin and Asrl in yeast under aluminum resistance,the following results were obtained using Cryptococcus humicola as experimental material:Real-time PCR was performed to test transcriptional level of genes from Calcineurin pathway,including:Calmodulin(cam)?Calcineurin catalytic subunit A(cna);C2H2 transcription factor Asrl gene and Cell wall composition synthetase-related gene:endo-1,3(4)-beta-glucanase(1-G5)?chitin synthase(2-E5)?chitin deacetylase-like mannoprotein(7-C12)?capsular associated protein(7-C10).The results showed that Calcineurin pathway related genes cam?cna;asrl gene and cell wall component synthetase-related genes are affected by aluminum ions.The Asrl prokaryotic expression vector was constructed by using the prokaryotic expression system of Escherichia coli.The recombinant protein was induced by low temperature and purified.We have constructed Asrl Saccharomyces cerevisiae expression vector.Studies have shown that transgenic yeast was still well grown on plates containing 5 mM aluminum ion,but the growth of control yeast was significantly inhibited.By measuring the remaining active aluminum in the medium,it was found that transgenic yeast had stronger adsorption of aluminum than control yeast.The asrl gene was knocked out by homologous recombination.The growth of the mutant,wild type and complementary strain was characterized on plates containing aluminum.The results showed that wild type and complementary strain lived much better on 200 mM aluminum plates,while,the growth of ?asrl was inhibited.By measuring the amount of residual active aluminum in the medium,the capacity of ?asrl strain to absorb aluminum has significantly reduced.