Screening Of Genetic Elements For Heterologous Synthesis Of Cannabidiol In Plants

ObjectiveCannabis sativa L.is an annual herb that is rich in cannabinoid compounds.Cannabidiol has good pharmacological effects and promising applications.However,the cultivation of cannabis is strictly controlled,and the low cannabidiol content of the existing species has led to its limited source.Based on this,the use of synthetic biology methods to heterologous synthesis cannabidiol from plants is one of the important development directions.The objectives of this study were to(1)screen the promoters of tissue-specific expression genes and cannabidiol synthesis pathway genes,verify their expression patterns,and provide genetic elements for the heterologous synthesis of cannabidiol in plants.(2)verify the functions of genes involved in cannabidiol biosynthesis through heterologous expression of a tobacco transient expression system,and to explore the possibility of heterologous synthesis of cannabidiol in plants.Methods(1)Verify the expression pattern of cannabis promoter and screen out heterologous expression promoters.The cannabis genome data and transcriptome data were used to screen tissue-specific expressed genes,and the cannabidiol synthesis pathway genes were identified through literature review.The above genes were used as candidate genes.The expression intensity of candidate genes was visualized using TBtools software.Meanwhile,the promoter regions of the candidate genes were extracted,and the cisacting elements were analyzed using the online website Plant Care to predict the factors affecting the regulation of promoters.The promoters of tissue-specific expression genes and cannabidiol synthesis pathway genes were cloned from cannabis genomic DNA by polymerase chain reaction(PCR).The cloned promoter was ligated to the upstream of GUS gene of p CAMBIA1305.1 by seamless ligation technology,and the recombinant vectors were introduced into Agrobacterium tumefaciens EHA105.Then,Arabidopsis was transformed by flowers dipping method,and the transgenic Arabidopsis was obtained by hygromycin resistance screening and PCR detection.Finally,GUS staining was performed on different tissues of transgenic Arabidopsis to verify the expression pattern of the promoters.(2)The functions of genes involved in cannabidiol synthesis pathway were verified by transient expression in tobacco.First,cannabis genomic RNA was extracted and reverse transcribed to obtain c DNA.PCR cloning was performed to obtain cannabidiol synthesis pathway genes,acyl-activating enzyme1(AAE1),olivetol synthase(OLS),olivetolic acid cyclase(OAC),aromatic prenyltransferase 4(PT4)and cannabidiolic acid synthase(CBDAS).The expression vectors were constructed by seamless cloning technology and in vitro multiple gene stacking technology,and then transfected into Agrobacterium tumefaciens EHA105.Cannabidiol synthesis pathway genes were hetero-expressed in different combinations by transient expression in tobacco leaves.Finally,the control substance and ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-QTOF-MS)were used to detect the compounds in tobacco leaves to verify the function of heterologous genes expression.Results(1)Validation of cannabis promoter expression pattern.Through transcriptome analysis and literature review,a total of 21 candidate promoters were obtained.Among them,6 were seed-specific expression promoters(S1-S6),5 were glandular hairspecific expression promoters(G1-G5),5 were root-specific expression promoters(R1-R5),and 5 were cannabidiol synthesis pathway gene promoters(p AAE1,p OLS,p OAC,p PT4,p CBDAS).14 promoters(p AAE1,p OLS,p OAC,p PT4,p CBDAS,S1,S2,S3,S4,S6,G2,G5,R2,R4)were obtained by PCR cloning,and all of them were constructed and transformed into Arabidopsis.Among them,9 promoters(p AAE1,p OLS,p OAC,p PT4,p CBDAS,S2,S4,G2,R4)obtained transgenic Arabidopsis.GUS staining in different tissues of transgenic Arabidopsis showed that p AAE1,p OLS,and p OAC were expressed in the trichome,flowers,and roots,but not in the stems and seeds.p PT4 showed a strong constitutive expression pattern.p CBDAS were expressed in the trichome,stigma,seeds and roots,but not in the stems and leaves.S2 is expressed in inflorescences and seedlings.S4 was weakly expressed in inflorescences and trichome.G2 was only weakly expressed in inflorescences.R4 was strongly expressed in all tissues except seeds.(2)Functional verification of cannabidiol synthesis pathway genes.5 cannabidiol synthesis pathway genes(AAE1,OLS,OAC,PT4 and CBDAS)were successfully cloned from the cannabis genome c DNA,and the single gene vector p EAQ-HT was constructed.Meanwhile,p AAE1-OLS-OAC-HT and p AAE1-OLS-OAC-PT4-HT multigene vectors were constructed.The results of Agrobacterium-mediated transient expression in tobacco leaves showed that in group A: co-injected with hexanoic acid and p EAQ-AAE1-HT,p EAQ-OLS-HT,p EAQ-OAC-HT vectors,olivetolic acid and olivetolic acid glucose were detected.Group B: no cannabigerolic acid was detected by co-injection of olivetolic acid and p EAQ-PT4-HT vector.Group C: Co-injection of cannabigerolic acid and p EAQ-CBDAS-HT vector produced cannabidiolic acid,but cannabidiol was not detected.Conclusion(1)Cannabis promoter expression pattern validation.The cloned cannabis promoter could be expressed in heterologous plant chassis.The p PT4 showed constitutive expression and the rest of promoters showed specific expression in flowers,trichomes,roots and seeds.Although the expression results differed from transcriptome data,all could be used as genetic elements for the subsequent heterologous synthesis of cannabidiol in plants.(2)Functional validation of cannabidiol synthesis pathway genes.AAE1,OLS and OAC can catalyze the production of olivetolic acid from hexanoic acid in tobacco.CBDAS can catalyze the production of cannabidiolic acid from cannabigerolic acid in tobacco.All of the above four genes were able to perform their functions correctly in tobacco.However,the function of PT4 has not been verified in tobacco and needs further confirmation.