| Shiraia bambusicola is a pathogenic fungus of Brachystachyum densiflorum Keng.Its fruit bodies have been used as a valuable Chinese traditional medicine.Hypocrellins,perylenequinoid pigments in S.bambusicola are of excellent antitumor antibacterial and antiviral.Owe to the low production of hyprocrellins in nature,as usual,asexual mycelial culture was used to produce hypocrellin.At present,the research concerning NO signal in fungi has received a wide attention.NO can participate in the regulation of spore germination,fruit body development and hyphal growth,and is also an important cell signal regulating secondary metabolism in fungi.In this study,the exogenous NO donor was added to promote the biosynthesis of hypocrellinand the regulation mechanism of NO was further analyzed through the transcriptome.Our results provided a foundation for the subsequent large-scale production of hypocrellin and a theoretical reference for regulation of secondary metabolites.The main research is as follows:(1)The optimal induction conditions were optimized on different concentrations of sodium nitroprusside(SNP)and different addition times in culture.It was found that 0.02 mM SNP was added on day 3 of the culture.SNP not only increased 2.78-fold the content of HA in mycelia,but also stimulated the release of HA into the medium with the highest total production of HA(89.66 mg/L),2.27-fold of control.(2)By measuring the physiological and biochemical characteristics after SNP induction,we found that NO had no significant effect on the residual sugar and pH in the medium.Secondly,SNP stimulation did not affect colony morphology,but significantly reduced the number of spores.Under NO treatment,the number of spores was decreased by 55%compared with the control,and was dose-dependent.After 9 h of growth,the spores began to swell and germinate,and the germ tube developed and elongated further.The spores appeared to branch at 24 h,and the hyphae began to entangle after 36 h.Under SNP treatment,the spore germination rate was decreased by 42%,36%,and 45%at 9,12,and 15 h,respectively,indicating that SNP inhibited spore germination.In addition,SNP resulted in the decreased pellet diameter,fluffier pellets,and intracellular pigments are significantly accumulated and released into the medium.(3)According to the RNA-seq sequencing analysis,a total of 24,804 unigene sequences were assembled.By analyzing the differential expression levels of between treatment group and the control group,a total of 571 differentially expressed unigenes(DEGs)were identified,among which 355 unigenes were up-regulated while 216 unigenes were down-regulated.GO classification of DEGs revealed that 'transport(GO:0006810)','membrane(GO:0016020)','integral to membrane(GO:0016021)','intrinsic to membrane(GO:0031224)','membrane part(GO:0044425)' and 'oxidoreductase activity(GO:0016491)' were all affected by NO significantly.(4)We detected reactive oxygen species(ROS)generation in S.bambusicola.Compared to the control,SNP stimulation increased H2O2 and O2-contents in mycelium by 1.71 and 1.59-fold,respectively,on day 7.The activity of NADPH oxidase was continuously increased,and the expression of the enzyme gene was up-regulated by 3.71-fold compared with the control.The enzyme activities of catalase and superoxide dismutase were enhanced,while the expression of the enzyme gene was up-regulated by 1.85 and 1.64-fold.It indicated that SNP stimulated ROS burst,which caused oxidative stress to further promote the accumulation of hypocrellin.(5)Subsequently,we investigated the transcriptional changes of genes involved in transmembrane transport.As a result,30 unigenes encoding major facilitator superfamily(MFS)and 2 unigenes encoding ATP-binding cassette(ABC)transporters were up-regulated after SNP treatment.After the addition of SNP,the proportion of unsaturated/saturated fatty acids in the mycelium was increased.The permeability of mycelial cells was further analyzed by using SYTOX Green,a fluorescent dye stained with high efficiency nucleic acids.It indicated the occurrence of fungal membrane permeation,which lead to the hypocrellin excretion to the extracellular medium.(6)By using transcriptome technology,we screened candidate genes involved in HA biosynthesis in DEGs up-regulated after SNP treatment,including the annotation of polyketide synthase(PKS)gene,50-methyltransferase(o-Methyltransferase,Omef)gene,15 monooxygenase(Mono),3 FAD/FMN-dependent oxidoreductase(FAD),2 Multicopper oxidase(MCO),30 MFS(major facilitator superfamily)transporters and 2 ABC(ATP-binding cassette)a transporter encoding gene.In addition,the NO signal may also regulate upregulation of the cytochrome P450 protein family gene associated with pigment biosynthesis.In this study,we optimized the culture conditions for exogenous SNP to induce the production of hypocrellin by the mycelial culture of the bamboo sclerotium.The NO signal not only promotes the accumulation of HA in the hyphae,but also the transport of HA into the medium,which increases the production of HA by 2.27-fold.Combined with microscopic observation,physiological index determination,q-RT PCR and transcriptome analysis,we investigated the effects of SNP on the growth of mycelium and the regulation of HA biosynthesis.It was found that NO signal can inhibit spore germination and mycelial growth with reduce pellet diameter.Meanwhile,it also affeected the level of redox in the hyphae,increased the expression level of the membrane transport genes,and promoted the expression level of key enzyme genes related to HA biosynthesis,which indicated that NO signal can further regulate the induction of HA accumulation.Our research provides a strategyfor the regulation of synthetic HA production in mycelial cultures.In addition,it provides a theoretical basis and reference for the regulation of NO signal secondary metabolic biosynthesis in fungi. |
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