Transcriptome Analysis Of Aluminum Stress Response Genes In Cryptococcus Humicola And Preliminary Study On Aluminum Resistance Mechanism Of Cell Wall

Aluminum is the most abundant metal element in the earth’s crust.Normally,it exists in the soil as a non-toxic binding state,but when the pH<5,the binding state of aluminium ions will change into a toxic ionic state.In recent years,environmental pollution has been severer and severer,and the rainfall of acid rain has increased year by year,as a result,the acidification of soil has become increasingly serious.The free aluminum ion becomes the main way of the presence of aluminum,which seriously endangers the growth of crops,reduces the yield of crops,and degrades forests.Therefore,it is imperative to solve the problem of aluminum poisoning.Ca²⁺is an important second messenger in cells,which participates in many physiological processes.Calmodulin（CAM）is its downstream target protein,which is activated after binding to Ca²⁺and then binding to itselves downstream target protein.Calcineurin（CN）,which makes its downstream transcription factors dephosphorylate and then enter to the nucleus,will regulate the expression of a series of genes.Cryptococcus humicola is a new type of high aluminium-tolerant Cryptococcus yeast which can tolerate 200 mM aluminium concentration and it belongs to our laboratory.To explore the specific mechanism of aluminum tolerance,we analyzed the transcriptome of C.humicola under aluminum stress after Aluminum stress,FK506（Calcineurin inhibitor）treatment,aluminum stress and FK506 co-treating.The results obtained are as follows:Through transcriptome sequencing,we obtained the specific functional classification,protein functional classification,metabolic pathways involved and differential expression of genes under the three treatments.Through GO analysis,we obtained the number of genes expressed in cell components,molecular functions,and biological processes.The number of genes involved in biological processes was the highest,followed by cell components and molecular functions.Through KOG analysis,we obtained gene expression in various biological processes,among which 873 genes were successfully annotated by Energy Production and Conversion,and 832 genes were successfully annotated by RNA processing and modification.Through KEGG analysis,we obtained the pathway of genes participation,and KEGG annotated 3751 genes.Under the condition of aluminum stress,4760 significant differentially expressed genes were obtained,of which 3066 were up-regulated and 1694 were down-regulated.Compared with the control,there were 5,612significant differentially expressed genes after FK506 treatment,of which 2086 were up-regulated and 3526 were down-regulated.Under the stress of Al and FK506 treatment,4157 significant differentially expressed genes were obtained,of which 1445 were up-regulated and 2712 were down-regulated.The expression of CN and its downstream transcription factors was up-regulated under Al stress,suggesting that genes in CN pathway play an important role in cell resistance under Al stress.Under the treatment of FK506,the CN pathway was blocked,the expression of CAM and CN-related genes was down-regulated,and the C₂H₂ transcription factor gene was also down-regulated.This indicates that the C₂H₂ transcription factor is regulated by the CN signaling pathway;when Al+FK506 is used to treat the C.humicola,the MAPK pathway gene expression is up-regulated,indicating that the CN pathway is blocked under aluminum stress,and the MAPK pathway plays a role in aluminum resistance.C.humicola was treated with 0 mM,20 mM,50 mM,100 mM Al ions and treated with50 mM Al ions for 0 h,2 h,4 h,8 h,12 h and 24 h before cell wall extraction.The phenol-sulfuric acid method was used to determine the content of the cell wall’s dextran and mannan.It was found that the content of cell wall mannan and dextran increased first and then decreased;Aluminum content in cell wall also increased first and then decreased,and the change trend of both was the same.After treated with tunicamycin,it was found that C.humicola’s OD₆₀₀00 decreased when the content of tunicamycin increased.This indicates that these two polysaccharide play a certain role in the aluminum tolerance process of C.humicola.Infrared spectrum data showed that the main components of cell wall had obvious absorption peaks under Al stress,which indicated that cell wall did play a role in the process of Al tolerance of C.humicola.