Prokaryotic Expression Of AvTPS1 From Amomum Villosum Lour. In Escherichia Coli And Its Genetic Transformation In Tobacco

Purpose:The monoterpenoid compounds are the most widely distributed and have a wide range of pharmacological effects in the terpene compounds of plant secondary metabolites.Amomum villosum Lour.is a wetland of traditional Chinese medicine,Amomum villosum Lour.is the main source of Fructus Amomi.The main components of the pharmacological action are the volatile oil,containing rich monoterpene compounds,among which the representative monoterpene compounds are the bomyl acetate,the borneol and the camphor.Although the upstream route of the plant terpenoid biosynthetic pathway has been researched,the downstream pathway of the monoterpene compounds synthesis,namely,from the terpenoid compound precursors to the specific terpenoid compounds is still unclear.In this study,the transcriptions of the transcriptome and methyl jasmonate-induced transcriptome were studied in this paper.Several monoterpene synthase genes related to the downstream pathway of the synthesis of Monoterpenoids were identified,and AvTPS1 might participate in the synthesis of monoterpene compounds from geranyl pyrophosphate to borneol,bomyl acetate and camphor.Therefore,on the basis of cloning AvTPS1,prokaryotic expression vector and eukaryotic expression vector of AvTPSl were constructed and expressed in N.tabacum,which laid the foundation for the functional identification of this gene.Method:1.Construciotion of recombinant expression vectors using In-Fusion and Gateway systems methodsUsing the recombinant clone vector pLB-AvTPS1 plasmid as template,Primer Premier5.0 software was used to design specific In-Fusion primers and Gateway primers to amplify the corresponding gene fragments.The expression vector pET-3 2a(?)with 6× HIS tag was linearized with NcoI and then ligated with the fragment(truncation of chloroplast Transit Peptide)(-tp)of AvTPS1 to construct.The recombinant expression vector pET-32(+)-(-tp)AvTPS1 was successfully constructed.Similarly,the pBI121 vector was linearized by XbaI and SacI digestion,and then ligated with the complete coding region of AvTPS1 to construct eukaryotic recombinant expression vector pBI-AvTPS1.The amplified of AvTPSl was ligated into the entry cloning vector pENTR using the Gateway method and then ligated to the pDEST17 containing the 6xHIS tag via the LR exchange reaction to construct the recombinant expression vector pDEST17-AvTPSl.The correctness of the vector construction is verified by comparing with the existing sequences.2.AvTPS1 prokaryotic expressionThe potent pET-32a(+)-(-tp)AvTPSl and pDEST17-AvTPSl were transformed into E.coli BL21DE3,BL21-CodonPlus,BL21DE3(Rosetta)and BL21(DE3)pLyS by heat shock method.Positive recombinants E.coli strains BL21DE3 and BL21-CodonPlus containing the expression vector pET-32a(+)-(-tp)AvTPSl respectively were cultured at 37? for 2 h with 0.5 mM IPTG,and the cells were collected by centrifugation.The recombinant E.coli engineering strain BL21(DE3)pLyS wih vector pDEST17-AvTPS1,was induced with 1 mM IPTG and incubated at 37? for 4 h.The cells were collected by centrifugation.The cells were disrupted by sonication,the protein was obtained,followed by SDS-PAGE.Positive recombinants E.coli strains containing pET-32a(+)-(-tp)AvTPSl and pDEST17-AvTPSl were harvested at 37? for 6 h,and the cells were treated with the same method.Then Western blot was used to detect the expression of HIS and incubated overnight with anti-rabbit secondary antibody.After transfection,the results were compared with infrared fluorescence.3.Genetic transformation of AvTPSl gene in tobaccoThe eukaryotic expression vector was successfully transformed into Agrobacterium tumefaciens EHA105 competent cells by freeze-thawing method,and the positive recombinants were screened by bacterial liquid PCR.In this paper,tobacco(labeled DH)was co-transformed with AvDXR transgenic tobacco(labeled D),AvDXR and AvHMGR(the key enzyme gene of the upstream of the terpenoid synthesis MVA pathway),which was used as the key enzyme gene of the above-mentioned terpenoid synthesis of the terpenoid,corresponding wild-type tobacco(labeled C)for genetic transformation.The tobacco leaf dics were firstly cultured with MS1 solid medium for 3 d,and then were infected by activated recombination Agrobacterium tumefaciens for 15 min,and then transferred to MS2 solid medium for co-culture for 2 days,.After that the tobacco leaf discs were transferred to MS3 differentiation culture medium for buds growth,which was subcultured once every 20 days,in the end,transferred to MS4 medium for rooting.The genomic DNA of tobacco leaves was extracted using the plant genomic DNA kit.The target genes AvTPSl,AvDXR and AvHMGR were detected by PCR and transgenic tobacco was screened.Results:1.Vector ConstructionPCR was used to detect the positive recombination of pET-32a(+)-(-tp)AvTPS1,pDEST17-AvTPSl and pBI-AvTPSl.The sequencing results showed that the coding region of pDEST17-AvTPS1 and pBI-AvTPS1 was identical with the coding region of pLB-AvTPSl.And pET-32a(+)-(-tp)AvTPSl was consistent with the sequence after truncation of signal peptide.The prokaryotic expression vector pET-32a(+)-(-tp)AvTPSl and the prokaryotic expression vector pDEST17-AvTPS1 with the complete coding region sequence were constructed,as well as the transgenic plants eukaryotic expression vector pBI-AvTPS1.2.Prokaryotic expressionThe recombinant expressio vector pET-32a(+)-(-tp)AvTPSl and pDEST 17-AvTPSl were successfully transferred into different Escherichia coli strains by hot shocksee Method 2 above).SDS-PAGE showed that the expression of pET-32a(+)-(-tp)AvTPSl in E.coli BL21DE3 and BL21-CodonPlus,and pDEST17-AvTPSl in BL21(DE3)pLyS were not observed clearly band.However,after Western blotting,it showed that pET-32a(+)-(-tp)AvTPSl and pDEST17-AvTPS1 were expressed in E.coli BL21DE3(Rosetta),and then the corresponding protein size without transtit peptide protein has less molecular weight than intact coding region.3 AvTPS1 transgenic tobacco was obtainedAgrobacterium tumefaciens EHA105 containing recombinant expression vector(pBI-AvTPS1/EHA105)was successfully screened by PCR.Fifty-five transformed tobacco plants wered obtained by Agrobacterium tumefaciens leaf disc method.Among them,there were 30 transformation plants of pBI-AvTPS1 transformed D strains and 25 transformation plants of pBI-AvTPS1 transformed DH strains.Twenty-two transgenic tobacco plants with good growth were selected and total DNA of them was extracted.Eleven plants of AvTPSl transgenic tobacco were confirmed by PCR.Among them,five co-transformed tobacco plants with AvTPS1 and AvDXR was screened,and three tobacco plants with AvTPS1 and AvDXR and AvHMGR were screened,three transgenic tobacco plants with only AvTPS1 was screened.Conclusion:Three expression vectors pET-3 2a(+)-(-tp)-AvTPS1?pDEST17-AvTPS1 and pBI-AvTPS1 were constructed.The two expression vectors in BL21DE3(Rosetta)in the large volume expression is detected by Western blotting.The expression of the corresponding protein provided the foundation for the subsequent optimization of the expression conditions to obtain purified protein for functional identification Another vector pBI-AvTPS1 was genetically transformed in tobacco to obtain AvTPSl single-transformed and co-transformed tobacco with AvHMGR and AvHMGR,which provided the foundation to study the effect of AvTPSl on terpenoid synthesis and its interaction with the key enzyme genes in the different upstream pathways in model plant tobacco.