Effects Of Exogenous Lanthanum Chloride,ethephon And Methyl Jasmonate On Accumulation Of Active Components In Hairy Roots Of Astragalus Membranaceus(Fisch.)

Astragalus membranaceus is a major medicinal material in northern my country.Astragalus saponins,flavonoids and phenolic acids are its main active components.The field production of Astragalus has a series of problems,such as low content,long cycle and unstable quality due to environmental influence.The use of the hairy root system of Astragalus can achieve continuous and controllable production.In addition,the hairy root system,as a cell culture system,is easily stimulated by exogenous elicitors to cause changes in metabolites.And as a cell culture system,it is easily stimulated by exogenous elicitors to cause changes in metabolites.Lanthanum is an antagonist of cellular calcium transport,and Ethylene and Jasmonic acid are signal substances that play an important role in the regulation of secondary metabolism in medicinal plants.Accordingly,in this study,LaCl₃,Ethephon（Eth）and Methyl Jasmonate（MeJA）were selected as exogenous signal substances to be added to the hairy roots of Astragalus capsulata that were cultured for 21days.Samples were taken at 7 time points and the contents of astragaloside Ⅳ,total phenolic acid and total flavonoids in hairy roots were detected by high performance liquid chromatography and spectrophotometry.At the same time,real-time quantitative PCR was used to detect the responses of key genes（AACT,HMGS,HMGR,IDI,FPS,SS,SE,CAS,AmCYP88D6 and AmCYP93E3）in the biosynthesis of astragaloside Ⅳ.Furthermore,the effects of exogenous elicitors LaCl₃,Eth and MeJA on the metabolic regulation of astragaloside Ⅳ in hairy roots of A.membranaceus,and the accumulation rule of active components were described.The results of the study are as follows:（1）100μM La Cl3 and 5 mM Eth had significant inhibitory effects on the accumulation of astragaloside Ⅳ in hairy roots of A.membranaceus（P<0.05）.75μM MeJA significantly promoted the accumulation of astragaloside Ⅳ in the hairy roots of A.membranaceus（P<0.05）.（2）The responses of FPS,SS,SE,CAS,AmCYP88D6 and AmCYP93E3 were significantly down-regulated（P<0.05）after 100μM La Cl3 treatment of hairy roots of A.membranaceus for 2 h.After 12 h of treatment,the responses of AACT,HMGR,IDI,FPS,SS,CAS,AmCYP88D6 and AmCYP93E3 were significantly up-regulated（P<0.05）.（2）The responses of FPS,SS,SE,CAS,AmCYP88D6 and AmCYP93E3 were significantly down-regulated（P<0.05）after 100μM LaCl₃ treatment of hairy roots of A.membranaceus for 2 h.After 12 h of treatment,the responses of AACT,HMGR,IDI,FPS,SS,CAS,AmCYP88D6 and AmCYP93E3 were significantly up-regulated（P<0.05）.However,LaCl₃had inhibitory effect on the content of astragaloside Ⅳ in the hairy roots of A.membranaceus.And 100μM LaCl₃ has the strongest inhibitory effect.（3）In this study,5 mM Eth and 75μM MeJA were selected to treat the hairy roots of A.membranaceus.After adding Eth for 3 days and then adding MeJA,MeJA may alleviate the inhibitory effect of Eth on astragaloside biosynthesis in hairy roots of A.membranaceus by up-regulating the relative expression of AACT、HMGS、IDI、FPS、SS、SE、CAS和AmCYP93E3.When MeJA was added for 3 days and then induced by Eth,the addition of Eth could up-regulate the responses of AACT,HMGS,IDI and AmCYP93E3 down-regulated by MeJA after 2h and 12h.After adding MeJA for 3 days and then adding Eth for 4 days,the content of astragaloside Ⅳ in the roots of MeJA,Eth and JAE groups was similar and significantly lower than that of the control group.（4）The content of total phenolic acids and total flavonoids in hairy roots of A.membranaceus treated with exogenous elicitors showed that La CL₃ at a concentration of100μM significantly promoted the accumulation of total phenolic acids in hairy roots of A.membranaceus.The coexistence of Ca CL₂ and La CL₃ could promote the synthesis of total phenolic acids in the hairy roots of A.membranaceus.There was no significant effect on the synthesis of total flavonoids.The interaction of Eth and MeJA had similar effects on the content of total phenolic acids and total flavonoids in hairy roots of Astragalus.That is,Eth has a significant promoting effect on the accumulation of total phenolic acids and total flavonoids in the hairy roots of A.membranaceus,and MeJA has no effect on it.At the same time,the addition of Eth in JAE also significantly increased the content of total phenolic acids and total flavonoids in the roots.The presence of MeJA had no effect on the induction of total phenolic acid and total flavonoid synthesis in roots by Eth.In conclusion,LaCl₃ can significantly inhibit the accumulation of astragaloside Ⅳ.Eth and MeJA could inhibit and promote the accumulation of astragaloside Ⅳ,respectively.The key genes in the biosynthesis of astragaloside Ⅳ can significantly respond to exogenous LaCl₃,Eth and MeJA signals.The accumulation of total phenolic acids,total flavonoids and astragaloside Ⅳ in hairy roots of A.membranaceus is homeostatically regulated.This study provides a theoretical basis for further elucidating the metabolic regulation mechanism of the active components of Astragalus.It also provides strong support for the subsequent use of plant genetic engineering strategies to enrich traditional Chinese medicine resources.