Study On The Relationship Between Sclerotial Formation And Antioxidant Enzymes Of Pleurotus Tuber-regium Under Abiotic Stress

Sclerotia is the mycelia aggregate produced by fungi under adverse environmental stress.Pleurotus tuber-regium is a kind of edible and medicinal fungus that can produce sclerotia,mostly growing in tropical and subtropical areas,and its sclerotia has high nutritional and medicinal value.Studies have found that abiotic stress can promote the differentiation of sclerotia,such as low temperature,undernutrition,H₂O₂ stimulation,etc.It is worth noting that abiotic stress can also activate the enzymatic response,which relies on antioxidant enzymes to clear ROS in vivo.Therefore,it is reasonable to speculate that antioxidant enzymes are involved in the regulation of the sclerotial formation of P.tuber-regium.At present,research on the sclerotia of P.tuber-regium mostly focus on its nutritional value and physiological active ingredients,while the mechanism of sclerotial formation is paid little attention.Therefore,this paper takes antioxidant enzymes as the breakthrough point to explore the related mechanism of sclerotinia formation under abiotic stress.The main research results are as follows:（1）The relationship between sclerotial formation and antioxidant enzymes activities under five abiotic stress conditions including temperature,acid base,sorbitol,nitrogen restriction and H₂O₂ stimulation was explored.First,we found that 25℃was the key temperature for sclerotial formation.When cultured at 25℃,p H 8,2.5%and7.5%sorbitol,and 3 mmol/L and 4 mmol/L H₂O₂ stimulation could promote the sclerotia differentiation,and the H₂O₂ stimulation had the most obvious promoting effect,and the diameter of sclerotia under 4 mmol/L H₂O₂ was the largest.Under these abiotic stress conditions,the activities of antioxidant enzyme were detected simultaneously.It was found that under the stress conditions that were suitable for sclerotial formation,the overall activities of four antioxidant enzymes were higher.（2）Transcriptomic analysis was performed by comparing the mycelia under the relative low temperature of 25℃for three days and the mycelia without stress.A total of 264 DEGs were detected.The DEGs were mainly involved in carbohydrate metabolism,catalytic activity,oxidoreductase activity and molecular function.Almost all the DEGs in carbohydrate metabolism were glycohydrolases,and all of them were up-regulated.Cytochrome P450 genes accounted for 30%of the DEGs,all of which were down-regulated.It is speculated that in the early stage of 25℃stress,P.tuber-regium activated carbohydrate metabolism（especially glycohydrolase）and proteins related to transmembrane transport,and weakened oxidoreductase activity（especially cytochrome P450 enzyme）.（3）The changes of antioxidant enzymes and gene expression during 24 days of25℃stress were monitored.It was found that SOD mainly active in the first 6 d,and the activity of POD was higher in the first 12 d.As for CAT,it mainly worked in the middle stages（6 d-18 d）.The expression level analysis of five antioxidant oxidase genes showed that POD.401 was active in the later stage of stress,while POD.535 was more highly expressed in the middle stage,which was significantly up-regulated and down-regulated in sclerotia respectively.The expression level of SOD.193 was higher in the early stage of stress,and was significantly down-regulated by 2.8 times in sclerotia.Both CAT.1115 and CAT.330 acted on the middle and late stages of stress,but CAT.1115 lasted longer and its expression level in sclerotia was the highest among these several antioxidant oxidase genes.（4）The effects of overexpression of CAT.1115 on stress tolerance and sclerotial formation of P.tuber-regium was investigated.The catalase CAT.1115 gene was cloned and the expression vector p G-CAT was constructed.After Agrobacterium tumefaciens transformation（ATMT）,three transformants,pg CAT-1,pg CAT-2 and pg CAT-3,were obtained through hygromycin（hyg）resistance screening,PCR verification,fluorescence microscope examination and gene expression level detection.The expression level of CAT.1115 was 1.6 times,2.8 times and 3 times of that in untreated strain,respectively.It was found that the transformants had better tolerance to 25℃relative low temperature and H₂O₂ stimulation.Under the stimulation of 1 mmol/L H₂O₂,the overexpressed strain not only increased the activity of CAT,but also promoted the activity of SOD and POD to jointly resist external stress.In the verification of sclerotial formation,it was found that overexpression of CAT.1115inhibited the sclerotial formation of P.tuber-regium.