Mechanism Of TORC1 Signaling Pathway Mediated By Free Amino Acids From Dried Lutjanus Sanguineus On Regulation Of Growth And T-2 Toxin Synthesis In Fusarium

Dried Lutjanus sanguineus is a dry aquatic product with high protein and low water content that easily contaminated by environmental Fusarium sp.and produces mycotoxin during processing and storage,which poses a serious threat to food safety.During long-term storage,protein from dried fish can be decomposed into free amino acids(FAA)and used as nutrients for fungi to grow and reproduce.Target of rapamycin C1(TORC1)signaling pathway is an important pathway for fungi to response the concentration of amino acid in extracellular environment and regulate cell growth and metabolism.Key upstream response gene(Gtr1,Gtr2)and downstream regulation gene(Sch9,Tap42)play important roles in absorption and utilization of external nutrients by Fusarium sp..Therefore,this study was carried to investigate the mechanism of FAA from dried fish on regulating growth and metabolism of Fusarium,and further analyze the mechanism of TORC1 signaling pathway mediated by specific amino acid on regulation of growth and T-2 toxin synthesis in Fusarium.The main research results are as follows:1.Changes of free amino acids related factors and microorganism of dried L. sanguineus during processing and storageDuring dried fish processing,high salinity addition reduced the surface moisture,turned free water into bound water,decreased the biogenic amines and TVB-N production.Lower salinity treatment caused increase of total number of bacteria,biological amines(cadaverine,putrescine and histamine)and TVB-N content.During storage,decreased in moisture content,increased in ash content,lipid oxidation,increased in total FAA and decreased in L-Histidine(L-His)and L-Alanine(L-Ala)were detected.Microbial succession showed the common marine bacteria were dominant before storage,bacteria with strong stress resistance such as Halomonas sp.,Bacillus sp.,Bacillus sp.and Alkalibacillus sp.were dominant after storage.Penicillium sp.became dominant fungi after storage.2.Isolation and T-2 toxin production capacity identification of Fusarium from dried fishAfter isolation,25 fungi strains with different phenotypes belonged to twelve species were obtained from dried fish.By molecular identification,Fusarium sp.,Aspergillus sp.and Penicillium sp.,were highly contaminated in dried fish.Seven Fusarium species such as F.oxysporum,F.incarnatum,F.avenaceum,F.nelsonii,F.verticillioides,and F.equiseti were isolated from dried fish.After twice T-2production analysis,Fusarium oxysporum Fo17(GDMCC 60824)showed the higher T-2 production capacity.It can produce small conidia and grow well in PDA medium(28~oC,p H>7).3.Utilization effect of F.oxysporum on free amino acids from dried fish by subtraction methodAfter analysis,higher contents of L-His,L-Proline(L-Pro)and L-Ala in FAA were extracted from dried fish.During addition of FAA as the only nitrogen source,F.oxysporum growth was significantly inhibited with smaller colony,less aerial hyphae and red pigment production.High dose of FAA increased sporulation and T-2 toxin production.By detecting the FAA content in GYM medium before and after culture,the utilization rate of high levels of L-His was 94.1%,L-Pro and L-Ala were 46.6%and 26.7%,respectively.However,the utilization rate of L-Threonine(L-Thr),L-Arginine(L-Arg),L-Methionine(L-Met),L-Phenylalanine(L-Phe),L-Isoleucine(L-Ile),L-Leucine(L-Leu)and L-Lysine(L-Lys)was 100%.Utilization rate of L-Glutamic acid(L-Glu),L-Valine(L-Val)were 99.6%and 98.9%,respectively.Utilization rate of L-Serine(L-Ser)and L-Tyrosine(L-Tyr)were 82.2%and 84.7%,respectively.4.Knockout and complement of Gtr1,Gtr2,Sch9 and Tap42 gene in TORC1pathway of F.oxysporumGene knockout strains?FoGtr1,?FoGtr2,?FoSch9,and?FoTap42 were obtained by the method of double-crossover homologous recombination.Through cell transformation,plasmid DNA extraction,protoplasmic preparation,gene knockout vector construction,transformant screening and PCR verification,related gene complement strains were obtained.The gene complement strains were obtained by vector construction,Agrobacterium tumefacien transformation,A.tumefacien and fungi co-culture,transformed screening and PCR verification.By cultured analysis,?FoGtr1,?FoSch9,and?FoTap42 grew normally on PDA medium,while?FoGtr2 grew slowly with less aerated mycelia.Rapamycin significantly inhibited the growth of the wild-type and gene knockout strains,but less inhibition to?FoSch9.Curly mycelium and long spore morphology were observed in?FoGtr1 and?FoSch9,while straight mycelium in?FoTap42.Except?FoGtr2,they all produce spores.5.Regulation of amino acids on growth and metabolism of F.oxysporum TORC1gene knockout and complement strainsStudy showed that neutral/alkaline amino acids[glycine(Gly),L-His,L-Pro and L-Thr]activated the growth of F.oxysporum.Acidic amino acids[L-Aspartic acid(L-Asp),L-Glu]and sulfur-containing amino acids[L-Cysteine(L-Cys)and L-Met]inhibited the growth of F.oxysporum.Amino acids addition inhibited?FoGtr2growth and made?FoSch9 filaments defective.The growth inhibition of F.oxysporum mainly performed as hyphae apex growth inhibition,branching and spore hypha production.Compared with wild-type,sporulation capacity of?FoGtr2,?FoSch9 and?FoTap42 was significantly reduced.L-Arg could promote sporulation of F.oxysporum,but L-Asp,L-Cys and L-Glu inhibited sporulation.L-Pro and L-Cys activated T-2 toxin production,while L-Asp and L-Glu inhibited T-2 synthesis.6.Detection proteins interacting with F.oxysporum TORC1-Tap42 by co-immunoprecipitation and mass spectrometryCo-immunoprecipitation and mass spectrometry was used to detect the interacting protein with F.oxysporum TORC1-Tap42 under the effects of L-Thr,L-His and L-Asp.High doses of L-Thr and L-His significantly elevated T-2 production of wild-type,?FoTap42 and?FoTap42-C,but with lower Tri5 expression.Lower dose of L-Asp activated T-2 production,while higher L-Asp addition inhibited T-2 synthesis and Tri5 expression.During p ET28a(+)-TAP42 expression vector construction,prokaryotic protein expression and purification,combined mass spectrometry analysis,results showed the protein interacting with Tap42 were:DNA topoisomerase 2,Glyceraldehyde-3-phosphate dehydrogenase 2,heat shock 70 k Da protein,elongation factor EF-1 alpha,actin,plasma membrane ATPase,enolase,ATP synthase subunits beta,enolase,elongation factor 2,etc.,the associated control metabolic pathway mainly belonged to the metabolism,genetic information processing,celluar processes and environmental information processing.This study indicates that amino acids had significant regulatory effects on the TORC1 signaling pathway mediated on growth,sporulation capacity and toxin synthesis of F.oxysporum.These results provide an important basis for further exploring the metabolic pathway of amino acids to the downstream hierarchical functional factors of TORC1 pathway.