The Effect Of Aspergillus Niger Elicitor On The Growth And Tropane Alkaloid Metabolism Of Atropa Belladonna L.hairy Roots

Atropa belladonna L.is an herbaceous plant of the genus Atropa in the Solanaceae family.The whole plant can be used for medicine.It is the only medicinal plant listed in the Chinese Pharmacopoeia of Tropine alkaloids（TAs）.Hyosyamine and scopolamine are its main medicinal active ingredients,which have clinical anesthetic and analgesic effects,and are used to relieve visceral colic and treat Parkinson’s.The market demand is great.This experiment is based on the established Atropa belladonna L.hairy root system,using Atropa belladonna L.hairy roots as the test material,by adding different concentrations of Aspergillus niger fungal elicitor to the medium to study the effect of Aspergillus niger elicitor on belladonna hairy root Root biomass and the content of hyosyamine and scopolamine,and then analyzed the different concentrations(10 mg·L^-1,50 mg·L^-1,100 mg·L^-1,200 mg·L^-1)of Aspergillus niger elicitor on the physiological indicators,nitrogen metabolism,H₂O₂,and NO content during the growth of Atropa belladonna L.hairy roots,as well as precursors in the TAs anabolic pathway The effects of changes in substance levels and the expression of key enzyme genes are preliminarily exploring the regulatory mechanism of Aspergillus niger elicitor on the growth of belladonna hairy roots and the metabolism of tropane alkaloids.The main findings are as follows:1.The effects of different concentrations of Aspergillus niger elicitor on the growth of Atropa belladonna L.hairy roots and the content of TAs were studied.The results showed that:50 mg·L^-1Aspergillus niger elicitor can maximize the growth of Atropa belladonna L.hairy roots and increase hyoscyamine and scopolamine.The output.2.Analysis of the effects of different concentrations of Aspergillus niger elicitor on the physiological indicators and nitrogen metabolism of Atropa belladonna L.hairy roots found that the addition of Aspergillus niger elicitor can increase the content of total soluble sugar in Atropa belladonna L.hairy roots and reduce catalase（CAT）,Superoxidase（SOD）and peroxidase（POD）activities;compared with the control group,the content of nitrate nitrogen and ammonium nitrogen in the hairy roots of Atropa belladonna L.in the test group increased to a certain extent,soluble protein,The content of free amino acids increased significantly.Nitrate reductase（NR）and glutamine synthetase（GS）activities were significantly increased,and glutamate dehydrogenase（GDH）activity was low throughout the process,and the changes were relatively stable.It shows that the elicitor of Aspergillus niger can alleviate the carbon source shortage brought by hairy root growth and maintain the balance of active oxygen metabolism,promote the absorption and utilization of inorganic nitrogen by Atropa belladonna L.hairy roots,enhance the ability of nitrogen assimilation,and grow hairy roots.Provide more nutrients and provide more substrate synthesis raw materials for secondary metabolic pathways.3.The effects of different concentrations of Aspergillus niger elicitor on the content of signal molecules H₂O₂and NO in Atropa belladonna L.hairy roots were analyzed.The results showed that:H₂O₂and NO content had an accumulation effect with the growth time of Atropa belladonna L.hairy roots;the addition of Aspergillus niger elicitor significantly increased the H₂O₂and NO content of the test group compared with the blank control,and when the Aspergillus niger elicitor was 50mg·L^-1,the content of both reached the maximum.It is speculated that H₂O₂and NO may participate in the regulation of the Aspergillus niger elicitor on the TAs synthesis pathway.4.In order to clarify the regulation mechanism of the Aspergillus niger elicitor on the TAs metabolic pathway of Atropa belladonna L.hairy roots,the 12 d treated Atropa belladonna L.hairy root was used to study the effect of different concentrations of Aspergillus niger elicitor on TAs synthesis precursor substances putrescine（Put）,bird The influence of the activity of amine decarboxylase（ODC）and arginine decarboxylase（ADC）and the relative expression of key enzyme genes PMT,TR I,PPAR and H6H on the TAs biosynthetic pathway.The results show that the treatment of Atropa belladonna L.hairy roots with Aspergillus niger elicitor can significantly increase the content of putrescine（Put）,the precursor substance in the TAs metabolic pathway,and promote the increase of ODC and ADC activities;stimulate the key enzyme gene PMT in the TAs biosynthetic pathway,TR I,PPAR and H6H,among which the expression of PMT and H6H is the most significant,which improves the synthesis of scopolamine and promotes the conversion of hyosyamine to scopolamine.In addition,they all showed a trend of first rising and then falling with the increase of the elicitor concentration of Aspergillus niger,reaching the highest at 50 mg·L^-1,which was consistent with the changes in the content of hyosyamine and scopolamine.In summary,the Aspergillus niger elicitor can increase the content of soluble total sugar,reduce antioxidant enzyme activity,enhance the ability of hairy roots to absorb and assimilate nitrogen,promote the growth of Atropa belladonna L.hairy roots,cause the burst of H₂O₂and NO,and enhance the synthesis of polyamines.The key synthase activity in the pathway can increase the content of putrescine,which is a substrate required for the synthesis of TAs,to stimulate the expression of key enzyme genes on the tropane alkaloid synthesis pathway,and finally realize the accumulation of tropane alkaloid production.