Gene Function Of Farnesyl Pyrophosphate Synthase And Sesquiterpene Synthase From Pogostemon Cablin

Pogostemon cablin(Blanco)Benth is a plant belonging to the Pogostemon of labiatae,which has the functions of aromatic turbidity,appetizers and antiemetics,and is mainly used to treat gastrointestinal diseases.Its volatile oil is widely used in the perfume industry.Patchouli alcohol,as the main active ingredient in patchouli oil,has anti-inflammatory,anti-tumor,antioxidant and other biological activities.Therefore,the synthesis mechanism of patchouli alcohol was studied at the gene level,and the key enzymes of the patchouli alcohol synthesis pathway were regulated in order to obtain a large amount of patchouli alcohol by fermentation engineering in vitro.At the same time,the expression of patchouli alcohol was increased from plant sources through transgenic pathways,laying a foundation for further utilization of patchouli medicinal resources.Farnesyl pyrophosphate synthase is a key enzyme for the synthesis of intermediate farnesyl pyrophosphate in the patchouli sesquiterpene synthesis pathway.It can catalyze isopentenyl pyrophosphate(IPP)and dimethylallyl pyrophosphate(DMAPP)produces farnesyl pyrophosphate and other substances.The research team performed 2 + 3 generation sequencing on patchouli leaves to obtain patchouli transcriptome data.The cDNA sequence of patchouli FPPS gene was obtained by reverse transcription and analyzed by bioinformatics.It was found that the cDNA of patchouli FPPS gene was 1 050 bp in length,encoding 349 amino acids.The molecular weight of the protein is 40 KD and the isoelectric point is 5.43.The catalytic site of the protein is composed of a large central cavity,which is mainly composed of anti-parallel ?-helix.There are two opposite walls in the central cavity,which are rich in aspartic acid-rich regions(DDXXD).Prokaryotic expression vectorspET-28a-FPPS and pET-32b-FPPS were constructed using seamless cloning and introduced into protein expression strain BL21(DE3).Protein expression was induced by isopropylthio-?-D-galactoside(IPTG)at different temperatures and concentrations.The results showed that the protein expressed by the p ET-28a-FPPS vector was truncated by about 10 KD.The protein expressed by the pET-32b-FPPS vector was significantly regulated by temperature.Most of the proteins were expressed in the supernatant at 20 and 25 ?,while were expressed in the precipitate at 30 and 37 ?.A large amount of protein was induced under the conditions of25 ?,130 rmp,and 1 mM IPTG.The protein was purified using an agarose Ni column,and then the protein concentration was measured by BCA method.Enzymatic reactions were performed in the buffers of IPP and DMAPP.After the reaction,the products were detected by HPLC-MS.It was found that the results of the protease-promoted reaction were in line with expectations,producing monoterpene compounds named geranyl pyrophosphate and sesquiterpene compounds named ?-hydroxy farnesyl phosphonic acid.Sesquiterpene synthase(PTS)is a key enzyme for the synthesis of patchouli alcohol in patchouli and catalyzes farnesyl pyrophosphate to produce sesquiterpene compounds such as patchouli alcohol.In this study,self-induction was used instead of IPTG to induce BL21(DE3)strain with pET-32b-PTS vector.Soluble PTS protein was obtained and purified for concentration detection,followed by enzymatic reaction with farnesyl pyrophosphate as a substrate.It was found that the purified protein was active and could catalyze the production of patchouli alcohol and other by-products from the substrate.The research team obtained Pogostemon cablin that were over-expressed and antisense RNA silenced in the previous work,but did not detect the content of them.In this study,the content of patchouli was determined in these two groups.The results showed that the patchouli alcohol content of the over-expressed plants increased by 87.6 %,47.1 % and 35.7 %(dry weight),and the pogostone content decreased by 59.2 %,-20.4 % and 60.7 %,respectively.Patchouli alcohol content in the interference group decreased by 18.0%(dry weight),and pogostone content increased by 314.1%.Therefore,it isspeculated that patchouli alcohol and pogostone may have a common precursor.At the same time,the PTS gene was introduced into tobacco,and the PTS gene-transformed tobacco was obtained,which provides material for further verification of the function of the PTS gene in the later stage.Many key genes in the sesquiterpene synthesis pathway are regulated by jasmonic acid.In this study,methyl jasmonate was applied to the patchouli leaves,and the leaves were harvested at 7 different time periods.After the RNA was extracted,the FPPS and PTS genes were quantitatively analyzed by fluorescence.It was found that FPPS and PTS genes were regulated by methyl jasmonate.Compared with FPPS,the expression of PTS gene was more significantly regulated by methyl jasmonate.The expression of the two genes at 48 h was most significantly different from that of the control group.Compared with the control group,the expression of FPPS gene was up-regulated by 2.53 times and that of PTS gene was up-regulated by 4.81 times when induced by 0.1 mM methyl jasmonate at 48 h.Compared with the control group,the expression of FPPS gene was increased by 2.20 times,and PTS gene was increased by 4.88 times when induced by 0.25 mM methyl jasmonate at 48 h.And from the expression trend of FPPS gene,it can be inferred that high concentration of methyl jasmonate can inhibit the expression of FPPS gene,and low concentration of methyl jasmonate can promote the expression of FPPS gene.