Preliminary Study On Salt Tolerance Mechanism Of The Halophilic Fungus Aspergillus Montevidensis ZYD4

Halophilic microorganisms have adaptively evolved the special genetic mechanism,physiological structure and metabolic types under high-salt conditions,and these halophiles have special salt-tolerant genes which can produce special metabolites.However,the salt-tolerant mechanism of halophiles is still mainly focused on halophilic bacteria and halophilic archaea,while halophilic fungi are rarely studied.In this study,Aspergillus montevidensis ZYD4,a halophilic fungus isolated from the sediment of Hua-ma salt lake in northern Shaanxi,was used to preliminarily explore the fungal salt-tolerant mechanism.The main research results are as follows:1.Through the preliminary investigation of the biological characteristics of halophilic fungus ZYD4,it was found that the strain ZYD4 grew in media with the optimum NaCl concentration of 1.5 mol/L.When sucrose was used as carbon source and peptone as nitrogen source,strain ZYD4 showed the best salt tolerance and the best carbon to nitrogen ratio was 10:1.Under the influence of different carbon and nitrogen sources,with the increase of salt concentration,the whole colony of halophilic fungus ZYD4 gradually changed from yellow to livid and black.At the same time,the halophilic fungus ZYD4 mainly produces the cleistothecium structure for sexual reproduction in the absence of salt,while in the high salt environment,it mainly produces conidiophore structure for asexual reproduction.The results showed that strain ZYD4 alleviated salt stress by pigmentation and changing the reproduction mode.2.A total of 19,729 Unigenes were obtained by transcriptome analysis of the halophilic fungus ZYD4.Notes were compared in 5 databases: 18,486(NR: 93.7%),14,882(KEGG: 75.43%),15,008(SwissProt: 76.07%),14,991(UniProt: 75.94%),and 12,880(KOG: 65.28%).The functional annotations in the KOG database are grouped into 25 KOG groups.A total of 230 Unigenes were significantly expressed(including 125 up-regulation and 105 down-regulation).The DEGs were significantly enriched 25 pathways in KEGG,including transporters,DNA repair and recombination proteins,energy,and alanine,aspartate,and glutamate metabolism.Most significant DEGs are related to the morphological,physiological,and genetic responses of the halophilic fungus ZYD4 at high salinity.According to the functions of these differentially expressed genes(P<0.0001),they can be roughly divided into control of ion transport,amino acid transport and metabolism,soluble sugar accumulation,lipid metabolism,energy metabolism and stress response.The results showed that the halophilic fungus ZYD4 could survive and grow in highly permeable habitats by ion homeostasis strategy,and at the same time by accumulating amino acids such as alanine,ornithine and proline as well as a large number of soluble sugars to resist the environment of high osmotic pressure.3.Through the detection of intracellular trehalose and amino acid of halophilic fungus ZYD4,it was found that the content of trehalose showed an obvious tendency of salt regulation,while the content of 17 amino acids in halophilic fungus ZYD4 varied with different salt concentrations.Among them,cystine was only detected at 3 mol/L salt concentration.The contents of lysine,aspartic acid and tyrosine were low and showed no significant change.The change trend of threonine and glycine contents was the same.At 3 mol/L salt concentration,their content increased first and then decreased with time.The contents of histidine,methionine,isoleucine and phenylalanine increased with the fermentation time.The contents of glutamate,serine and proline were positively regulated with salt concentration.However,arginine and valine content showed a reverse regulation trend with salt concentration.The results showed that the trehalose and amino acid synthesized by strain ZYD4 in high-salt environment were related to the reduction of salt stress.The results showed that the halophilic fungus ZYD4 could synthesize trehalose in the high salt environment,regulate the high osmotic environment by the sensitive changes of amino acids,and the specific accumulation of amino acids such as alanine,proline,glutamate and leucine.4.By adding exogenous compounds,it was found that the addition of metabolites such as new hesperidin,diuret,aspartic acid,alanine,proline and ornithine significantly promoted the growth and morphological adaptation of halophilic fungus ZYD4 in the high-salt environment(P<0.05).Tolerance to salt was demonstrated by significant morphological changes.The results showed that halophilic fungus ZYD4 mainly used metabolites such as ornithine,new hesperidin and proline to replace glycerol as the osmotic regulatory solute in high salt environment.In summary,morphological shifts,carbon and nitrogen metabolism for solute accumulation,and ion equilibrium are of great importance for halophilic fungi against high-salinity stresses.