Study On Terpenoid Synthases And The Promoters Involved In Volatile Terpenes Synthesis In Amomum Villosum

1.ObjectiveFructus Amomi is the dried and mature fruit of the genus zingiberaceae of Amomum villosum Lour.It is one of the famous "four southern medicines" in China.The essential oil is rich in terpenoids such as camphor,borneol,bomyl acetate and so on.Terpene synthase(TPS)is a key enzyme in the downstream biosynthesis pathway of terpenoids.In the previous study,twenty-two TPS genes were screened from the transcriptome of A.villosum and some of them were identified as monoterpene synthase and sesquiterpene synthase.The terpenoids are abundant in the A.villosum,especially in the fruits,its medicinal tissues,which contain monoterpenoids,sesquiterpenoids and a few diterpenoids,and as the fruit ripens,its terpenoid content also changes accordingly.However,the TPS responsible for the synthesis of these monoterpenoids,sesquiterpenoids and diterpenoids has yet to be fully explored.Therefore,in this study,more TPS genes were mined based on the second generation transcriptome and full-length transcriptome data,and their cloning and functional identification were carried out,so as to more comprehensively understand the diversity of volatile terpenoids and the mechanism of their accumulation in fruits.The promoter plays an important role in the regulation of plant gene expression,and the promoter of the terpenoid synthase gene regulates the expression of the gene,thus affecting the synthesis of plant terpenoids.In this paper,the promoter of some terpene synthase gene of A.villosum was cloned,and its cis-regulating elements were analyzed,so as to understand the mechanism of regulating the synthesis of terpenoids.2.Method2.1 Full-length transcriptome sequencing and TPS excavation of A.villosumIn spring leaves,flowers,roots and rhizomes,45 DAF seed,75 DAF(Days after flowering)of seed and pericarp as material of A.villosum,extraction of RNA,respectively,full-length transcriptome sequencing was performed after equal mixing,and the sequencing quality and annotation information were analyzed.According to the known sequences of candidate AvTPS1-22 genes,local blast was conducted in transcriptome data,and then Pfam protein domains were combined to screen out candidate AvTPS for bioinformatics analysis.2.2 Determination of volatile terpenoids in fruits at different developmental stagesVolatile terpenoids from 30,45,60,75 and 90 DAF pericarp and seed were extracted by organic solvent ultrasonic extraction method with n-hexane.Terpenoids in different development stages were determined by GC-MS and quantified by standard samples.2.3 Cloning and functional identification of TPS genesThe TPS gene was cloned from the cDNA of the tissue with high expression of the target gene,and the recombinant cloning vector was constructed.The recombinant cloning vector was used as the template,and the In fusion method was used to construct the recombinant prokaryotic expression vector pET32a with double his tag or pMAL-c5x with MBP tag.The prokaryotic expression was transferred to the Rosetta(DE3)expression strain.IPTG was used to induce the protein expression of fusion TPS,the fusion protein was purified by Ni-NTA column or MBP column,and the substrate GPP,FPP or GGPP were used for in vitro enzymatic reaction,and the catalytic products were detected by GC-MS.2.4 Fluorescent quantitative PCRPrimers of identified functional target genes for qPCR were designed using primer premier 5.0,and fluorescent quantitative PCR was performed using the cDNA of pericarp,seed,leaf,flower and rhizome in different stages of development of A.villosum.Each sample contained two biologic.al replicates,two technical replicates,and the gene expression level was normalized by 2-ΔΔCT.2.5 AvBPPS and AvLIS/NES promoter cloning and analysisThe genomic DNA(gDNA)of the leaves was extracted and the gDNA sequences of AvBPPS and AvLIS/NES were cloned.Three specific primers with the same direction were designed on the basis of the gDNA sequences obtained,and their promoter sequences were amplified by fusion primer and nested polymerase integrated PCR(FPNI-PCR),and the transcriptional starting sites and cis-acting regulatory elements of the promoters were analyzed.3.Result3.1 Full-length transcriptome data analysis and AvTPS mining of A.villosumThe assembly quality of full-length transcriptome was better,and the obtained isoform sequences were generally longer,and the annotation rate in Nr database was as high as 93.98%.In KEGG metabolic pathway,the most isoform was annotated in secondary metabolic biosynthesis pathway.In the synthesis pathway of terpenoid skeletons,more isoform was annotated by MEP pathway than by MVA pathway.A total of 24 candidate AvTPS were selected based on multiple transcriptome data from the early stage of A.villosum.The twenty-four candidate genes were constructed into phylogenetic tree and clustered into seven different subfamilies,among which the monopterpenes synthase mainly clustered into the TPS-b subfamily,the sesiterpenes synthase mainly clustered into the TPS-a subfamily,and the diterpenes synthase mainly clustered into the TPS-c and d subfamilies.3.2 Cloning and functional identification of AvTPS gene3.2.1 Seven AvTPS genes were cloned and eleven AvTPS purified proteins were successfully obtainedIn this study,seven AvTPS were successfully cloned,including three monoterpene synthase genes,AvTPS12,AvTPS13,AvTPS23,two sesquipene synthase genes,AvTPS14,AvTPS’24 and two diterpene synthase genes,AvTPS20 and AvTPST21.Genes that had been cloned but their protein failed to be purified(AvTPS4,AvTPS8,AvTPS16,and AvTPS17)were replaced with expression vectors to express proteins.Inclusion bodies appeared in the AvTPS4,AvTPS8,AvTPS 16,AvTPS17 and AvTPS23 proteins which were expressed in the prokaryotic expression vector with double His tag,so they were constructed into the prokaryotic expression vector containing MBP tag to increase protein solubility.Finally,all eleven AvTPS obtained purified proteins.3.2.2 The enyme function of nine AvTPS was successfullly identifiedThe main products of reactions between mono-terpene synthases AvTPS 13 and AvTPS23 and GPP were both β-ocimene,which were named as β-ocimene synthase 1(AvOCS1)and P-ocimene synthase 2(AvOCS2)respectively.AvTPS4 reacts with FPP to produce a single product,trans-nerolidol,named trans-nerolidol synthase(AvtNES).AvTPS 12,AvTPS 14,AvTPS 16 and AvTPS24 were all identified as bifunctional enzymes,which could react with GPP to produce monoterpenoid products and FPP to produce sesquiterpenoid products in vitro.Linalool and some by-products were the main products of AvTPS 12 reaction with GPP.The reaction with FPP produced a single product nerolidol,named linalool/nerolidol synthase(AvLIS/NES).The main products of AvTPS 14 reaction with GPP and FPP were geranyl methyl ether and copaene respectively,both of which contained several by-products and were named copaene synthase(AvCOS).The main products of AvTPS 16 reaction with GPP and FPP were geranyl methyl ether and aristolochene respectively,and several by-products were also generated,which were named aristolochene synthase(AvARS).The main products of AvTPS24 reaction with GPP and FPP were geranyl methyl ether and bicyclogermacrene,respectively,which produced several by-products named bicyclogermacrene(AvBES).AvTPS20 and AvTPS21 reacted with GGPP to produce a single product,copalyl diphosphate,which was dephosphorylated to produce copalol,which was named copalol synthase 1(AvCPS1)and copalol synthase 2(AvCPS2).AvTPS8 and AvTPS 17 reacted with GPP and FPP and no products were detected.In this paper,it was found that the catalytic products of 4 pairs of AvTPS were similar,and their sequence alignment had a high similarity,namely AvTPS2 and AvTPS12,AvTPS13 and AvTPS23,AvTPS14 and AvTPS24,and AvTPS20 and AvTPS21,respectively.In addition,all bifunctional synthases reacted with GPP to produce geranyl methyl ether,and AvTPS 12 also produced linalool methyl ether,both of which are alcohol methyl ether.3.3 There are abundant and diverse volatile terpenoids in the fruit of A.villosumThe types and contents of volatile terpenoids detected in the fruits of different development stages were abundant,and a total of forty nine kinds of terpenoids were detected.There were nineteen kinds of monoterpenoids,nine kinds of sesquiterpenoids and three kinds of diterpenoids in the pericarp.There were twenty-four kinds of monoterpenoids,twenty-two kinds of sesquiterpenoids and two kinds of diterpenoids in seeds,which were more abundant than in the pericarp.For the first time,this paper compares the volatile terpenoids in five fruits at different developmental stages.The content of volatile terpenoids in most seeds is higher than that in the pericarp.With the development of fruit,the volatile terpenoids in the pericarp did not change much,while the volatile terpenoids in the seeds gradually accumμlated,and reached the highest accumulation at 75 DAF.Bornyl acetate,the main pharmacodynamic substance,was enriched in seed,and began to be enriched from 45 DAF.3.4 Correlation analysis between AvTPS expression pattern and terpenoidsCorrelation analysis was conducted between the expression levels of fifteen identified AvTPS and their terpenoids in fruits and tissues at different development stages.All fifteen AvTPS were expressed in seeds,among them,AvBPPS showed seed expression specificity,and the highest expression was found in 45 DAF seeds,mainly involved in the synthesis of camphene,borneol,bornyl acetate and camphor in seeds.AvC.OS and AvBIS have similar functions and are expressed in all tissues,and the highest expression is found in 45 DAF seeds.mainly responsible for the synthesis of copaene and bicyclogermacrene in 45 DAF seeds.AvLIS and AvLIS/NES have similar functions,but different expression patterns.AvLIS is mainly expressed in pericarps,and the highest expression level is found in 45 DAF pericarps,which is mainly responsible for the synthesis of linalool in pericarps,while AvLIS/NES is the highest expression level in flowers and mainly involved in the synthesis of linalool in flowers.AvOCS1 and AvOCS2 products are similar,but the expression patterns are different--AvOCS1 has the highest expression in 60 DAF pericarps,and is mainly involved in the synthesis of β-ocimene in the pericarps,AvOCS2 has the highest expression in the leaves and is mainly involved in the synthesis of β-ocimene in the leaves.AvCPS1 and AvCPS2 were expressed in all tissues,with the highest expression in 45 DAF pericarps,and the expression of AvCPS1 was higher than that of AvCPS2.AvPIS is expressed in most tissues(except the rhizome),and the highest expression is found in 60 DAF pericarps,which is mainly involved in the synthesis of pinene in pericarps,and pinene exists in all tissues,indicating that the expression of AvPIS is related to the accumulation of pinene.3.5 Obtain the promoter of AvBPPS and AvLIS/NESThe sequence length of gDNA fragments of AvBPPS and AvLIS/NES cloned was 2374 bp and 2295 bp,respectively,both containing 7 exons and 6 introns and the complete open reading frame(ORF)of the corresponding gene.gDNA sequences of AvBPPS and AvLIS/NES were submitted to GenBank,and the serial Numbers MG763230 and MN829545 were obtained respectively.The promoter was cloned by FPNI-PCR,and the promoter was further amplified by AvBPPS and AvLIS/NES on the basis of the obtained genome of the leaves of their leaves.The sequencing resμlts showed that AvBPPS and AvLIS/NES had 264 bp and 156 bp overlapping sequences at the 5 ’end of their gDNA,and the promoter sequences of AvBPPS and ALIS/NES were preliminarily confirmed.3.6 The AvBPPS and AvLIS/NES promoters contain cis-acting regμlatory elements involved in endosperm expression and MeJA reaction,respectivelyBioinformatics analysis of the obtained AvBPPS and AvLIS/NES promoter sequences revealed that the AvBPPS promoter contained GCN4-motif(TGAGTCA),a cis-acting regulatory element involved in the expression of endosperm,and G-box,a component involved in the optical response.In addition,it also contains cis-acting regμlatory elements involved in abscisic acid reaction,low temperature response and MYB binding.In this paper,it was found that AvBPPS promoter contained GCN4-motif,and the results of fluorescence quantitative PCR and transcriptome data analysis showed that AvBPPS was specifically expressed in seeds,and the corresponding terpene products such as camphor,camphor,etc.,were enriched in seeds.The promoter of AvLIS/NES contains CGTCA-motif/TGACG-motif,a cis-acting regulatory element involved in the reaction of methyl jasmonate.There are also AAGAA-motif and ABRE involved in abscisic acid reaction and G-box involved in light response.Combined with the MeJA-treated of A.villosum,it was found that AvLIS/NES responded to the induction of MeJA and the content of corresponding terpenoid linalalol was also regulated by MeJA.4.ConclusionIn this paper,a total of twenty-four candidate AvTPS were screened,and nine AvTPS were successfully cloned and identified.By analyzing all the AvTPS with identified functions in A.villosum,it was found that most of AvTPS were multi-product enzymes,and the predicted sesquiterpene synthase genes were bifunctional synthase genes.All the identified AvTPS were analyzed for expression levels in fruits and tissues at different stages of development,and these genes were found to be expressed in seeds.AvBPPS and AvCOS are specifically expressed in the seeds.AvBPPS is a key enzyme responsible for the synthesis of major pharmacodynamics components such as bornyl and bornyl acetate,etc.AvCOS is an enzyme with the highest diversity of terpenoids.The diversity of AvTPS products and their expression in seeds are the basis of the enrichment and diversity of major pharmacoterpenoids in seeds.Among them,three groups of AvTPS showed similarity in sequence and catalytic products,and their expression patterns were different,revealing that similar genes in the TPS gene family of A.villosum play a role in different tissues.In this paper,the cis-acting regulatory elements involved in the expression of endosperm were first discovered from the promoter of AvBPPS from the medicinal plant A.villosum,which provided the basis for in-depth study on the specific mechanism of seed expression of key terpenoid synthesis enzymes and an operating element for the regulation of terpenoid metabolism.