Cloning, Overexpression And Antisense RNA Silencing Of Patchoulol Synthase

Pogostemon cablin(Blanco)Benth.is one of “the top Traditional Chinese medicine(TCM)in Lingnan area”.As a traditional dampness resolving Chinese medicinal herbs,its volatile oil has biological activities such as regulating the gastrointestinal tract,anti-inflammatory and anti-pathogenic microorganisms.It is considered to be an important raw material in the field of medicine.In addition,it is widely used in the food and cosmetics fields.P.cablin oil contains more than 24 kinds of sesquiterpenoids,of which the patchoulol is the main component.Therefore,studying the molecular mechanism of sesquiterpenoids biosynthesis in P.cablin and improving the content of P.cablin oil in medicinal materials,especially the production of patchoulol,is of great significance for the development and application of medicinal resources of P.cablin.Patchoulol synthase(PTS)is a key enzyme for the synthesis of patchoulol in P.cablin.It can catalyze the formation of sesquiterpene by the sesquiterpene precursor-Farnesyl diphosphate(FDP).In this study,the cDNA sequence of PTS gene was obtained from P.cablin planted in Danzhou city HaiNan province by one-step RT-PCR and analyzed by bioinformatics.The result shows that the full-length cDNA of P.cablin PTS gene is 1659 bp,which is a complete open reading frame(ORF)and encodes 552 amino acids.The protein has a molecular weight of 64.1 kD and an isoelectric point of 5.39.It has two sesquiterpene synthase domain,one of which call C-terminal domain include 268 amino acid residue may be involved in the cyclization of linear terpenes,which contain three conserved sequences of DDxxD,RxR and RRx8 W.The obtained PTS gene was constructed into the prokaryotic expression vector pET28 a,pET32b and the plant expression vector pRI101-AN using circular polymerase extension clone(CPEC).In addition,500 bp of the PTS gene was cloned as an antisense sequence,and used the same way to constructed on the pRI101-AN vector.The constructed pET28a-PTS and pET32b-PTS expression vectors were induced to express in BL21(DE2)strain,and it was found that patchoulol synthase had a strong tendency to form inclusion bodies when expressed in E.coli,even fusion with thioredoxin which enhances protein solubility,at lower temperatures and IPTG concentrations(20 ? and 0.25 mM),soluble expression was still not observed.The induction conditions of the pET32b-PTS strain were optimized,but the soluble protein was still invisible.At 25 ° C,0.5 mM IPTG,the expression of the target protein reached the top.Then,a large amount of TRX-PTS protein is expressed under this condition and the inclusion body was renatured.It was dissolved in a refolding solution containing 6 M guanidine hydrochloride,and dialysis was sequentially performed in the order of a refolding liquid containing 3 M,2 M,1 M,0 M,and 0 M guanidine hydrochloride.Finally,it dialysis into 10 mM HEPES buffer.SDS-PAGE results showed that the renaturation was successful and the pure PTS recombinant protein was obtained.The constructed overexpression vector pRI101-PTS(GBD)and the antisense RNA interference expression vector pRI101-PTS(GR)were transformed into LBA4404 Agrobacterium tumefaciens by freeze-thaw method.The results of bacterial liquid PCR showed that both two vectors were successfully introduced into Agrobacterium.Therefore,the leaf disc of P.cablin cultured for 3 days in the preculture medium was used as an explant,and the infection was inactivated with the overexpressing and interfering strains,respectively.After 3~4 days of co-cultivation,the leaf discs were transferred to the screening medium.250 bottles of each of the overexpression group and the antisense RNA silencing group were obtained,and 3 to 4 leaf disks per bottle.During the screening process,the untransformed explants gradually browned and died,and the successfully transformed leaf discs gradually grew callus at the incision.Because P.cablin is more sensitive to kanamycin,it is more difficult to differentiate into budding.After releasing pressure of Kan,the callus began to differentiate into buds.When the buds grow to 1.5-2 cm,they are cut down for rooting culture.A total of 58 over-expressed groups of P.cablin regenerated seedlings and 104 antisense RNA silencing regenerated seedlings were obtained.Finally,3 transgenic PTS overexpressing P.cablin plants and 5 antisense RNA silencing plants were identified by PCR.