Analysis And Gene Mining Of The Synthesis Pathway Of Flavonoids From Sanghuangporus Baumii

Flavonoids are widely distributed secondary metabolites in plants.They provide pigments for plant and participate in plant development and plant-environment interaction.Flavonoids are widely used in food,pharmaceutical and nutrition industries because of their important biological activities such as anti-aging,anti-oxidation and anti-inflammation.Sanghuangporus baumii is a medicinal mushroom and has been used as medicine for more than 2,000 years.Modern research has shown that the flavonoids,triterpenoids,polysaccharides and other active components in S.baumii share prominent anti-tumor,anti-oxidation and anti-inflammation effects.However,most of the genes related to the flavonoid synthesis pathway are absence in the genomes of S.baumii.Therefore,it is an open question whether mulberry yellow can synthesize flavonoids.In this study,targeted metabolome technology and transcriptome technology were used to demonstrate the synthesis of flavonoids in S.baumii.The main results are as follows:(1)The culture characteristics of the mycelia of S.baumii were studied and a chemical defined media was designed.Eighty-one flavonoids were detected from the mycelia cultured in chemical defined media,including 25 flavones,23 flavonols,10 flavanols,8 isoflavones,5flavanones,4 flavanonols,3 proanthocyanidins,2 chalcones and 1 isoflavanone.The multiple classes of flavonoids are consistent with the presence of biosynthetic routes that are commonly found in plants.Because the chemical defined media did not contain flavonoids,the detected flavonoids were synthesized by S.baumii.(2)Nine culture methods were used to induce the synthesis of flavonoids in S.baumii.The results showed that aeration could significantly increase the content of flavonoids in mycelia.Targeted metabolome and transcriptome techniques were used to analyze the mycelia of S.baumii after aeration.The targeted metabolome data showed that the contents of 9flavonoids were significantly up-regulated after aeration.The relative contents of 3-O-acetyl flavanone,kaemphenol-3-O-(6"-O-acetyl)glucoside and genistein 7-O-galactoside increased by 48333.33,14233.33 and 12488.89 times,respectively.Transcriptome data showed that aeration induced the biosynthesis of secondary metabolites of S.baumii.However,only 4genes related to flavonoid biosynthesis pathway were found in the transcriptome,of which 3were differentially expressed genes,and most genes in the pathway were still missing.(3)Three differentially expressed genes in flavonoid biosynthesis pathway,phenylalanine Ammonia lyase gene(PAL),4-Coumaroyl Co A ligase gene(4CL),isoflavone reductase gene(IFR),were cloned and analyzed.The results showed that the proteins encoded by the three genes had conserved sites of their respective enzyme families,but the protein sequences differ greatly from that of enzymes in plants.The prokaryotic expression and protein purification of the three genes were carried out using Escherichia coli as host,and experiments were designed to prove that the protein expressed by PAL has the catalytic function of phenylalanine ammonia-lyase.(4)A PEG-mediated genetic transformation system of S.baumii was established,and the transgenic strain overexpressing PAL was successfully obtained through transformation experiments.The PAL transcription level in the transgenic strain was upregulated,along with the increase of PAL enzyme activity in mycelia,and the flavonoid content was increased by48.85%(14.12 mg/g).In conclusion,S.baumii can synthesize 81 flavonoids.The synthesis pathway of flavonoids is different from that of plants,and the missing genes in the pathway may be replaced by distant superfamily genes.PAL plays an important role in the regulation of flavonoid synthesis pathway.Overexpression of PAL in S.baumii can significantly increase the transcription level of PAL gene and PAL enzyme activity,and result in the accumulation of flavonoid.