Establishment Of Hairy Root Induction System And Genetic Transformation System For Stephania Tetrandra And Corydalis Yanhusuo

China has a long history of using medicinal plants.Medicinal plants are rich in secondary metabolites,which have great medicinal value and economic value.However,medicinal plant resources are under serious threat due to over-exploitation.It is urgent to find solutions to achieve sustainable use of medicinal plant resources.With the development of plant physiology,genetics and plant genetic modification technology,a variety of biotechnology strategies,such as plant tissue culture,genetic transformation,gene editing are gradually applied to medicinal plants,which greatly promotes the mining and development of traditional Chinese medicine resources.As a method of plant tissue culture,hairy root culture has been widely used in the research of secondary metabolites production,synthesis pathway analysis and metabolic regulation of medicinal plants,due to its advantages of stable genetic and biochemical reactions,abundant secondary metabolism and easy to genetic modification."Fangji" and "Yanhusuo" are the traditional Chinese medicine."Fangji" is the dried succulent taproot of Stephania tetrandra S.Moore.Tetrandrine,the main pharmacological active component of Fangji,is disbenzylisoquinoline alkaloid,and is the first choice for Ebola virus infection,silicosis and rheumatic pain."Yanhusuo" is the dry tuber of Corydalis yanhusuo.Its main pharmacological active componentsrotundine and corydaline-are protoberberines alkaloids,and have unique analgesic and sedative mechanism that why them can be used to treat SUD,including the treatment of opiate and other psychotropic drugs misuse."Fangji" and "Yanhusuo"are often in short supply,not only because of the serious shortage of wild resources,but also because of the problems of germplasm confusion,climate change,pests and diseases faced by artificial cultivation.The establishment of their hairy root induction system and genetic transformation system will provide technical support and research platform for the study of disbenzylisoquinoline and protoberberine alkaloids,and will also provide reference for the research of genetic modification and variety improvement of them.Based on this,this paper conducts the following research:1.Establishment of hairy roots induction system in S.tetrandraIn this study,we constructed the hairy root system of S.tetrandra by co-cultivating leaf explants with Agrobacterium rhizogenes C58C1 and determined hairy root growth curves and BIAs accumulations.The qualitative results showed that there were a variety of BIAs in the hairy root,which were similar to the alkaloids in plant roots,but their contents were different.According to the determination of hairy root growth curve and BIAs accumulation,it was found that the S.tetrandra hairy roots were suitable for subculture and expansion culture at 15-10 days,and harvested after 50 days.The highest content of tetrandrine was 8.382±0.160 mg/g DW,which was 59.67%of the content in the cultivated S.tetrandra roots.In addition,0.1 mM MJ and 0.2 g/L YE elicitors were used to treat the hairy roots.It was found that 0.1 mM MJ could significantly increase the content of BIAs alkaloids,and the content of tetrandrine was increased by 85%compared with the control.While,0.2 g/L YE had no significant effect on the growth and accumulation of BIA alkaloids.This study provides a technical support for the factory production of S.tetrandra hairy root and a new way for the production of tetrandrine.2.Establishment of hairy roots genetic transformation system in S.tetrandraThe aim of this study is to establish a genetic transformation system for S.tetrandra hairy roots,so as to realize the genetic transformation of hairy roots.Agrobacterium rhizogenes C58C1 was used to transfer the binary vector pCAMBIA1300 with EGFP gene into the S.tetrandre hairy roots.The transformed lines showed bright green fluorescence,and EGFP bands were successfully amplified by genomic PCR,which proved that the exogenous EGFP gene could be integrated into the hairy root genome and expressed successfully.In addition,we overexpressed CyOMT-7(60MT),which is the key enzyme gene in the BIAs synthesis pathway,in the S.tetrandre hairy root.The quantitative results showed that the contents of coclaurine,N-methycoclaurine and the protoberberine alkaloid scoulerine were increased in CyOMT-7 overexpressing hairy roots,compared with the control.This experiment verified the in vivo function of CyOMT-7,which catalyzes norcoclaurine to coclaurine,and also proved that the content of BIAs could be increased by genetic modification of S.tetrandre haiey roots.The construction of this system not only provides technical support for the genetic improvement,the analysis of BIAs biosynthesis mechanism,the discovery of transcription factors and other studies,but also provides a reference for the construction of genetic transformation system of S.tetrandre plants.3.Establishment of hairy roots induction system and genetic transformation system in C.yanhusuoIn this study,we established the hairy root system of C.yanhusuo by co-cultivating leaf explants with Agrobacterium rhizogenes C58C1.Qualitative results showed that the C.yanhusuo hairy roots contain rotundine and corydaline,and the content of rotundine was 0.449 mg/g DW,which was 35%of the content in the cultivated C.yanhusuo.In addition,in order to solve the problem of short supply time of leaves and achieve sustainable supply of explants,we established the method of callus induction and callus proliferation.The induction system of C.yanhusuo hairy roots was established with callus as explants,and the induction rate was about 10%.At the same time,we established the genetic transformation system of C.yanhusuo hairy roots by transferring the binary vector pCAMBIA1300 carrying the EGFP gene.Hairy roots can show bright green fluorescence.This study provides a new way and new ideas for the study of the production of active components of C.yanhusuo,and provides technical support for the study of the biosynthesis and regulation mechanism of BIAs compounds.In conclusion,this study successfully constructed the hairy root induction system of two species,S.tetrandre and C.yanhuauo,which product benzylisoquinoline alkaloids.BIAs compounds were abundant in both hairy roots,which increased the material source and material basis for BIAs production.The genetic transformation system of hairy roots was established,which provided technical support for subsequent studies on the biosynthesis pathway and metabolic regulation of BIAs using hairy roots.