Preliminary Construction Of The Multi-Gene Expression Vector For Glycyrrhetinic Acid Heterologous Biosynthesis In Tobacco

Glycyrrhiza uralensis is a kind of traditional Chinese medicine with a long history,which is the dried roots and rhizomes of the specific species of Glycyrrhiza in the leguminous family.It has lots of pharmacological functions including anti-inflammatory,antiviral,anti-tumor,expectorant cough,detoxification and so on,as well as replenishing qi,relieving pain,reconciling drugs and other effects.As the most important active component of Glycyrrhiza uralensis,the content of glycyrrhizic acid(GL)is the most important index to measure the quality of Glycyrrhiza uralensis as prescribed in Chinese Pharmacopoeia,the GL in Glycyrrhiza uralensis should not be less than 2.0%.Glycyrrhetinic acid(GA)is the metabolite of GL in human body,and it is hydrolyzed by GL to produce further effects.As a secondary metabolite,the content of GA in plant is pretty low.Scarce resources of wild basal plants,coupled with a wide divergence in GA content in cultivated plants,renders many of these plants inadequate for the ideal standard.In view of the important pharmacological effects and application value,how to efficiently produce GL and GA has become a research focus in the face of demand exceeding supply.Over the past few years,synthetic biology and bioinformatics technologies are continually being developed,the biosynthetic pathway of GA has been thoroughly studied in recent years,and a number of essential genes have been pinpointed.This has provided the theoretical basis for constructing synthetic pathways,as well as the conditions to simulate the metabolic control system of medicinal plants in heterologous hosts.Simultaneously,synthetic biology and bioinformatics technologies have been continually advancing.As a result,it is a practical and feasible method to increase yield by metabolic engineering of the heterologous host as well as transgenic technology.In this study,a multi-gene vector containing several key gene expression boxes was assembled,aiming to construct a new high efficiency GA synthesis system and open up the heterologous biosynthesis pathway,with a further expect of the ultimate efficient synthesis of GA in tobacco.The results of this study can effectively promote the efficient synthesis and industrial production of GA and other terpenoids,as well as the sustainable use of medicinal plant resources.It also provides reference for the synthesis of other triterpenoids in heterologous plant hosts.In addition,the cloned and verified green tissue specific promoters from eight different plant sources can lay a foundation for the balance of metabolic flow in the subsequent synthetic pathway,and provide a reference for other studies of multi-gene transformation.The achievements of this research paper are as follows:1.Eight green tissue specific promoters from six plants were cloned,pCAMBIA2300promoter-green fluorescent protein(GFP)expression vectors were constructed.Verification and comparison of the activity of the eight promoters was accomplished by observing the fluorescence intensity of GFP.Using the constitutive promoter CaMV 35S as control,the order of promoter activity was CaMV 35S>Ibrbcs>PNZIP>Ntrbcs>D540,Cmrbcs,Psak>Rca>PEPC.2.Multi-gene vectors were designed and constructed using TGSII system.The Matrix Attachment Region(MAR)TM2 was cloned from tobacco,and the first donor vector pYL322d1-TM2 was constructed.3.In order to avoid excessive unnecessary metabolic burden in other tissues of the heterologous host,and the transgene silence caused by sequence repetition,five different green tissue specific promoters and one constitutive promoter were constructed in turn with different terminators in the donor vectors pYL322dl and pYL322d2 to form six universal vectors,which include pYL322d2-PNZIP-Tnos;pYL322d1-Ibrbcs-Trbcs;pYL322d2-Ntrbcs-TNtR19;pYL322d1-35S-Tnos;pYL322d2-D540-TGluB4;pYL322d1-Cmrbcs-T35S.4.Six key structural genes involved in GA biosynthesis including tHMGR,FPPS,SQS,β-AS and CYP88D6 were constructed into each vector to form the five intermediate vectors(donor vectors).5.Two donor vectors were constructed into the acceptor vector successively.At last,the multi-gene vector pYLTAC380GW-2G was formed,which possesses TM2 and tHMGR expression frame.