Molecular Mechanism Of Blue Light Regulating Lysine Biosynthesis Mediated By Spermidine In Flammulina Filiformis

Flammulina filiformis is also known as "Intelligence-enhancing mushroom" because it is rich in lysine.In industrialized cultivation,the use of blue light irradiation can regulate the growth and development of F.filiformis fruiting body and induce the biosynthesis of lysine to improve the nutritional quality of F.filiformis.The previous study of the research group found that spermidine was involved in the process of blue light regulating the biosynthesis of lysine in F.filiformis,but the specific regulation mode and mechanism were not clear.On this basis,this study deeply studied the mode of action of blue light and receptor WCC in regulating spermidine and the molecular mechanism of spermidine regulating lysine biosynthesis through its metabolite GABA.The specific research results are as follows:Firstly,for blue light and its photoreceptor FfWCC to positively regulate the expression of spermidine synthase gene FfSpds,yeast onehybrid verification was used,and the results showed that FfWCC cannot directly regulate the expression of FfSpds gene by binding to the FfSpds gene promoter.Then,by constructing FfWcc gene silencing(FfWc1-kd2 and FfWc2-kd2)and overexpressing(FfWc1-oe5 and FfWc2-oe4)transformants of F.filiformis,it was screened that FfWCC can indirectly regulate spermidine biosynthesis through NO signaling molecules.At the same time,the NO synthesis gene FfNr silencing transformants FfNr-kd3 and FfNr-kd4 were constructed.It was detected that spermidine and lysine content in the FfNr-kd transformants decreased significantly.When exogenous NO donor(SNP)was replenished,the content of spermidine and lysine increased significantly,approaching the level of WT.These results indicate that blue light and its photoreceptor FfWCC regulate the level of intracellular NO signaling molecules by acting on the FfNr gene,thereby regulating the metabolism and content of spermidine.The effects of silencing of FfWcc and FfNr on the growth and development of F.filiformis and the lysine content of fruit bodies were further examined.The results showed that silencing of FfWcc gene in PDA medium affected the disorder and sparseness of mycelium,but did not affect the growth rate of mycelium,while silencing of FfNr gene inhibited the growth of mycelium.Fruiting experiments in cultivation medium showed that overexpression of FfWcc could increase the number of primordia,promote the elongation of the stipe of F.filiformis,and increase the content of spermidine and lysine in the fruiting body,while FfWcc silencing was the opposite.FfNr gene silencing reduced the number of primordia,shortened the stipe,and decreased the content of spermidine and lysine in the fruiting body,showing a similar phenotype to FfWcc silencing.The results showed that the blue light receptor WCC and NO signal can positively regulate the biosynthesis of spermidine and lysine in F.filiformis,and also play an important role in the photomorphogenesis and development of F.filiformis fruiting body.Secondly,according to the previous results that spermidine positively regulates the biosynthesis of lysine in F.filiformis,the downstream products of spermidine were further analyzed,and it was found that its metabolite γ-aminobutyric acid(GABA)would affect the biosynthesis of lysine in F.filiformis.Exogenous addition of GABA can significantly induce the biosynthesis of lysine in F.filiformis and increase the lysine content.By constructing GABA transaminase(GABA-T)gene FfGaba T silencing transformants FfGaba T-kd4 and FfGaba T-kd42,it was found that GABA content increased after FfGaba T silencing,and lysine biosynthesis and lysine content were increased.The results indicated that spermidine could positively regulate the biosynthesis of lysine in F.filiformis through its metabolite GABA.The effects of silencing of spermidine synthase gene FfSpds and GABA transaminase gene FfGaba T on the growth and development of F.filiformis and lysine content in fruiting body were further examined.The results showed that on the PDA medium,FfSpds was silenced,spermidine was reduced,and the growth rate of F.filiformis was significantly decreased,while FfGaba T was silenced,GABA was increased,and the mycelial growth rate was increased,indicated that spermidine and its metabolite GABA positively regulated the growth of mycelium.Fruiting experiments were carried out in the cultivation medium.Silencing of the FfSpds gene reduced the number of primordia of F.filiformis,shortened the stipe,and decreased the content of spermidine,GABA and lysine in the fruiting body.However,when FfGaba T was silenced,the content of spermidine,GABA and lysine in the fruiting body increased,the number of primordia increased significantly,and the stipe was elongated.The results showed that spermidine and its metabolite GABA were also positively regulating lysine biosynthesis in the fruiting body,and also played an important role in the photomorphogenesis and development of F.filiformis fruiting body.In summary,the blue light receptor WCC of F.filiformis can positively regulate the level of signal molecule NO by acting on FfNr gene to positively regulate the biosynthesis of spermidine after receiving blue light signal.And spermidine and its metabolite GABA also positively regulate the biosynthesis of lysine in F.filiformis.The results revealed the molecular mechanism of light signal affecting lysine biosynthesis in F.filiformis by regulating spermidine metabolism.It lays a foundation for constructing the regulation network of light on the growth,development and material metabolism of edible fungi,and also provides a basis for the rational use of blue light in production to increase the lysine content of edible fungi.