| Ganoderma lucidum is a famous Chinese medicinal fungus with a very long history of application.Modern research has found that Ganoderma lucidum has high medicinal value and contains a variety of active substances with pharmacological effects,among which triterpenoids are one of the most important active components of Ganoderma lucidum.In this paper,the effect of exogenous addition of acetic acid as an inducer on the biosynthesis of Ganoderma lucidum triterpenes was investigated using wild species of Ganoderma lucidum G20 as the experimental strain,under the premise that the microorganism can use acetic acid as an available carbon source.Under the condition of fixed treatment duration,the addition of acetic acid could inhibit the growth of Ganoderma lucidum more significantly.Compared with WT,under the condition that the final concentration of exogenous added acetic acid was 8 m M,the triterpene of Ganoderma lucidum significantly increased by 51.3%,the polysaccharide content of Ganoderma lucidum significantly increased by 57.2%,and the biomass decreased by 25.49% compared with WT.Under the premise of significant increase in Ganoderma triterpene content,the expression of key enzyme genes for Ganoderma triterpene synthesis was further determined,and the expression of both 3-hydroxy-3-methylglutaryl-Co A synthase and3-hydroxy-3-methylglutaryl-coenzyme A reductase showed significant increase,while the expression of squalene synthase and oxidosqualene cyclase showed only a small increase.Under the fixed treatment concentration,the triterpene content of mycelium and the treatment duration were positively correlated,and the triterpene content reached 22.58 mg/g at the treatment duration of 72 h,which increased by 54.9% compared with the control group.Under the same conditions,the different duration of acetic acid treatment also affected the content of extracellular polysaccharides of Ganoderma lucidum,and the content of extracellular polysaccharides increased by 40.2% relative to WT under the condition of 72 h treatment,and the degree of increase was positively correlated with the treatment duration.The biomass of mycelium was inversely proportional to the duration of acetic acid treatment,and the biomass of Ganoderma lucidum mycelium decreased in a stepwise manner when the treatment duration exceeded 48 h.Taken together,the above results indicated that the addition of acetic acid could enhance the synthesis of Ganoderma triterpenes,and had obvious concentration and time effects.Based on the screening of acetic acid additions most suitable for the growth of Ganoderma lucidum in liquid fermentation experiments,the induction of exogenous additives was applied to the cultivation of Ganoderma lucidum substrates.Under the conditions of determining the acetic acid used for exogenous additives,the starting time,treatment interval and treatment dose of exogenous added acetic acid treatment were regulated in conjunction with the growth cycle of the substrates,and after rough screening,the treatment periods were the protoplast stage,the opening stage and one week before the disappearance of the yellow edge;the treatment intervals were once a day,twice a week,once a week and once every two weeks;the treatment doses were 2 m M,4 m M,8 m M(20m L of reagent per single package).All acetic acid treatments affected pileus diameter,pileus thickness and stipe length.At the treatment concentrations of 2 m M,4 m M and 8m M,the diameter of the pileus was reduced by 10.78%,10.02% and 11.79%,respectively,in the basal stage,while there was no significant difference between the open stage and the yellow edge disappearance stage.The thickness of the pileus only showed a significant decrease when treated with 8 m M acetic acid in the basal stage,which was 23.18%compared with the control group;the length of the stipe increased by 7.2%,8.42% and 18.7%compared with the control group in the basal stage,and increased by 1.26%,2.23% and6.44% in the opening stage,respectively.Under acetic acid induction,the triterpene content and agronomic traits of Ganoderma lucidum were changed,and the higher the induction treatment started,the higher the increase of triterpene content,and the triterpene content of Ganoderma lucidum and mycorrhizal length were highly positively correlated and negatively correlated with the dry weight of Ganoderma lucidum.This indicates that acetic acid induction during the mushroom emergence period can effectively influence the agronomic traits of Ganoderma lucidum,and the degree of influence is directly related to the concentration of acetic acid treatment,the higher the concentration of acetic acid,the more obvious the influence on the agronomic traits of the fruiting entities.The dry weight of Ganoderma lucidum seeds treated at the beginning of the primary stage decreased the most significantly,compared with the control group,the dry weight of seeds under 8 m M treatment decreased by 48.6%,while the degree of decrease was reduced in the group treated at the beginning of the opening stage,while the seeds treated at the beginning of the week before the disappearance of the yellow edge showed no significant decrease in dry weight.The triterpene content of Ganoderma lucidum seed entities tended to increase and then decrease with the time interval of exogenous acetic acid treatment,and the highest values of triterpene content were concentrated between twice weekly treatment and once weekly treatment,with an increase of 34.02-46.18%.Compared to the original base stage,the increase in triterpene content of Ganoderma lucidum in the open slicing stage decreased to23.56-34.18%.In contrast,among the substrates treated one week before harvesting,only the acetic acid concentration of 8 m M and twice weekly treatment conditions showed a significant increase in the triterpene content of Ganoderma lucidum,which was 21.68%higher than the control.It can be concluded that acetic acid induced increase in triterpene content of Ganoderma seed entities needs to be carried out in the middle of the growth of the seed entities or even earlier,and the induction treatment with exogenous inducer when Ganoderma is almost harvested does not have a significant effect on the triterpene content of the seed entities.In addition to the study of the induction results of acetic acid addition at different time points,it was also found that there were differences in the response of triterpene content to acetic acid in different parts of the ascospores: the increase of triterpene content in the ganoderma of Ganoderma lucidum under acetic acid induction treatment was significantly higher than the increase of triterpene content in the pileus,with the former up to 51.79%and the latter up to 24.51% only.The analysis of the results showed that the effect of acetic acid on the mycorrhiza was more obvious and intuitive when treated as an exogenous inducer.This indicates that the sensitivity of different parts of Ganoderma lucidum substrates to exogenous inducers is different and varies greatly.In addition to the study of the results of acetic acid induction by the addition of acetic acid at different time points,differences were found in the degree of response of triterpene content to acetic acid in different parts of the cotyledons.Under acetic acid induction treatment,the increase of triterpene content in the stipe of Ganoderma lucidum was significantly higher than that in the pileus,and the highest increase of triterpene content in the stipe was 51.79% compared with the control group,while the increase of triterpene content in the pileus was only 24.51%.The analysis of the results showed that the effect of acetic acid on the mycorrhiza was more obvious and intuitive when treated as an exogenous inducer.This indicates that the sensitivity of different parts of Ganoderma testa to exogenous inducers is different and varies greatly.Acetyl coenzyme A is the starting point of the Ganoderma triterpene synthesis pathway,and a series of reactions such as the mevalonate(MVP)pathway generate ganoderic acid analogs.On the premise that acetic acid induction can cause significant expression of key enzyme genes at the front end of the synthesis pathway,silent transformants of the ACS gene were constructed.After acetic acid induction treatment of the silent transformants,no significant changes were found in the content of Ganoderma triterpenes.It showed that the ACS gene was involved in the regulatory process of acetic acid-induced triterpene biosynthesis in Ganoderma lucidum,which provides a basis for further studies.This experiment is the first time to practically apply the exogenous addition of inducer to the cultivation of fungal substrates,proving that the exogenous inducer acetic acid can produce a certain induction effect on substrates,and optimizing the regulation process of exogenous addition of acetic acid in the cultivation of substrates,which can effectively enhance the cultivation effect of substrates and improve the application value of substrates. |
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