Exploring The Developmental Mechanism Of The Medicinal Fungus Polyporus Umbellatus Sclerotia Based On Transcriptomic

Polyporus umbellatus(Per.s)Fr.,a traditional Chinese precious medicine as long been used for eliminating dampness,which medicinal part is the sclerotium located in the underground part.Currently,the research on Polyporus umbellatus is mainly focused on the sclerotial cultivation technique,the culture and optimization of hyphae,the study on the content difference and pharmacological activity of active ingredients,the physiological conditions and molecular mechanisms of artificially growing hyphae to form sclerotia and so on.However,due to the slow development of the sclerotia and the relatively harsh environmental conditions,the development of the Polyporus sclerotia has been stuck in the study of structure and development history,the cloning and expression analysis of functional genes.But the developmental process and the molecular mechanism of metabolic processes are relatively lagging behind.In this study,for determining the color change of the epidermis in the development of sclerotia whether is the deposition of melanin,we did the extraction,purification,explored the physicochemical properties to the black sclerotium epidermis.The reason for the deposition of melanin was analyzed by the transcriptome of the Polyporus sclerotia at different stages.Screened differentially expressed genes,combined with the difference in the content of active ingredients and metabolic pathways,and preliminarily elucidated the developmental process of the sclerotia and the molecular mechanism of metabolic processes.The main conclusions are as follows: 1.The black substance was extracted and purified from the epidermis of the black Polyporus sclerotia,and did the analysis to trial the solubility,chemical characterization,the spectral properties,and analysis of physicochemical properties such as stability and oxidation resistance.The results indicated that the substance doesn't dissolve in water and common organic solvent.It precipitated in the acidic solution and dissolved in the alkaline solution.It doesn't have the properties of tannins and flavonoids.In the range of 200-800 nm,the absorbance decreases exponentially with increasing wavelength.The wavelength is plotted against the absorbance,and the slope is negative.After the sample is placed for one month,the slope becomes smaller.All of the above are typical properties of melanin.Infrared spectroscopy identified the presence of functional groups,such as-OH,aliphatic-CH,C=C and C=N skeletons of the aromatic ring,C=O and COO-functional groups of carboxylic acid and etc.All the peaks position and waveform is similar to the infrared spectrum of DHN melanin.The increase in temperature and the increase in pH have a color enhancement effect,and the color reduction effect is caused by freezing and illumination.The substance has significant scavenging activity on hydroxyl radicals and superoxide anion radicals,and has certain reducing ability.The polyphenol content of 1g of this substance is equivalent to 224.48±11.46 mg gallic acid.Based on the above results,it was determined that the color changed of the epidermis during the development of the Polyporus sclerotium was caused by the deposition of melanin.2.To explore the differences in different stages of the Polyporus sclerotia,we utilized de novo transcriptome assembly analysis technology.Finally,we obtained 587 381 230 cleanreads,including 88.12 Gb sequence and containing 85 235 unigenes(?200?bp)assembled.We annotated the 85 235 unigene successfully from the seven database.The transcriptome results showed that the aerobic respiration of the white Polyporus sclerotia is weaker than that of the ash and black Polyporus sclerotia,and the activity of the flavin dehydrogenase associated with electron transfer was decreased.It is speculated that the white Polyporus sclerotia lead to the formation of oxidative stress may because of the exposed to environmental irritations,such as temperature differences in the natural environment.Based on the differentially expressed genes,we hypothesized that the white Polyporus sclerotia ensure the active material exchange,information transmission,and energy production and output through high expression of membrane transporters and ergosterol.To initiate its own defense mechanism by regulating the high expression of WD40 protein.In GL and HL,the polar growth and morphological development of filamentous hyphae were achieved by up-regulating the expression of small G protein RhoA,CYP450 and phenylpropanoid.Besides,accompany with the synthesis of melanin,polysaccharides and chitin,the epidermal of the Polyporus sclerotia becomes blacken and the cell wall becomes thicken.3.To determine the content of Polyporus umbellatus polysaccharide and ergosterol in BL,GL and HL.The results showed that,with the development matures,the polysaccharide content and ergosterol content gradually increased,but the synthesis rate reduced.Screened out for the key enzyme genes involved in polysaccharide synthesis,including HK,TNV,GPI,pgm,MPI,galU,UDGH,UXS1 and GSase48,etc.Most of the enzymes are distributed in the upstream pathway of polysaccharide synthesis,forming key intermediates.It is indicated that the Polyporus polysaccharides rapidly synthesized in the early stage of sclerotia development,because the rate of synthesis decreased as it matures.Similarly,both ergosterol synthase ERG4 and ERG5 are down-regulated,it is suggested that the early development is in response to external stimuli and accelerated the synthesis of ergosterol to ensure the normal function of transmembrane transport of cell membranes.