Functional Characterization Of Monoterpene And Sesquiterpene Synthases In Amomum Villosum And Function Comparison Of BPPSs

1.ObjectiveChinese herbal medicine Fructus Amomi is mainly derived from the dry and mature fruits of Amomum villosum Lour.It has the functions of moistening appetizer,regulating qi and placating fetus,warming spleen and stopping diarrhea.The main medicinal components of Amomum villosum are terpenoids,such as borneol acetate,camphor,borneol,etc.Terpene synthases(TPSs)are the key enzymes in the downstream synthesis pathway of terpenoids.Previous research has selected 10 TPS from transcriptome,and identified the pinene synthase and bomyl diphosphate synthase.Terpenoids are abundant in Amomum villosum,especially in the medical parts of the fruits with abundant monoterpenoids and sesquiterpenoids.Therefore,TPS family involved in the synthesis of these terpenoids remains to be explored.In this study,more TPS genes were screened out from fruit-based transcriptome,and then were cloned and functionally identified,in order to establish better understanding of the biosynthesis of terpenoids and TPS family in Amomum villosum fruits and provide more candidate genes for the metabolic engineering of volatile terpenes.On the other hand,bomyl diphosphate synthase(AvBPPS),the most critical TPS in the synthesis of terpenoids in medical effect of Amomum villosum,is special not only in the diversity of its products,but also in the fact that its main product,bomyl diphosphate(BPP),is the direct or indirect precursor of bomyl acetate,camphor and bomeol,which are the main terpenoids in the medicinal effect of Amomum villosum.Amomum longiligular T.L.Wu is related species of Amomum villosum and a source plant of Fructus Amomi.In this study,the AlBPPS was cloned from Amomum longiligular and LdBPPS derived from Lippia dulcis was synthesized.Through functional comparison and amino acid sequence comparison of AvBPPS and these two BPPS,the key amino acid sites were found in order to provide the basis for optimizing the function of AvBPPS.2.Method2.1 Transcriptome sequencing and volatile terpenoids of the fruit of Amomum villosum2.1.1 Transcriptome data analysis and AvTPS miningSequencing quality and annotation information were analyzed by performing transcriptome sequencing using the pericarp,seed cluster and rhizome of two mature fruits(45 DAF and 75 DAF)of Amomum villosum(three biological replicates in each tissue).Local blast was conducted with the sequences of AvTPS 1-10 candidate genes to find the corresponding unigene,and all unigene predicted as TPS were screened out according to the prediction results of Pfam protein domain.Then,unigene with a sequence less than 500bp was filtered out and unigene which may be of the same TPS were merged to finally obtain the candidate AvTPS.At last,the tissue expression pattern of candidate AvTPS and its correlation with volatile terpenes content were preliminarily analyzed according to RPKM value of transcriptome sequencing2.1.2 Extraction and determination of terpenoids from Amomum villosumExtraction of volatile terpenoids from 45 DAF(Days after flowers)pericarp(PYF),45 DAF seeds(SYF),75 DAF pericarp(PF),75 DAF seeds(SF)and rhizome(CS)by ultrasonic extraction with hexane.The terpenoids in these tissues were determined by GC-MS,and the standard substance was used to make standard curves for quantitative analysis.2.2 Cloning and functional identification of candidate TPS genes2.2.1 Cloning and prokaryotic expression of TPS genesThe candidate TPS genes were cloned using the tissue cDNA with high expression as the template,and the recombinant cloning vector was constructed.The recombinant cloning vector plasmid was used as the template.Construction of recombinant prokaryotic expression vector pET 32a by In-fusion method with double His tag,and the Rosetta(DE3)expression strain was used for prokaryotic expression.2.2.2 Protein purification and functional identification of TPS proteinThe expression of fusion TPS protein was induced by IPTG,the fusion protein was purified by NI-NTA column,and the TPS catalyzed product was detected by GC-MS using GPP or FPP as substrate for in vitro enzymatic reaction.2.2.3 Gene expression pattern analysisExtracting RNA from 75 DAF seeds(SF),75 DAF pericarp(PF),flower(F),Leaves(L)and rhizome(CS)of Amomum villosum and transcribing it reversely as cDNA.The expressions of AvTPSs in different tissues were detected by real-time fluorescence quantitative method.2.2.4 Cloning of TPS gDNA and promoterThe genome of TPS was cloned using the genome of Amomum villosum leaves as a template.Designed three specific primers based on gDNA fragments.The promoter of TPS was cloned from the genome of Amomum villosum leaf by FPNI-PCR,and the transcription initiation site of the promoter and the cis-expression element were analyzed.2.3 Comparison of BPPS functions2.3.1 Cloning and expression vector construction of Amomum longiligular BPPSThe transcriptome was sequenced from the immature seed mass of Amomum longiligular,and the candidate TPS genes were screened from the transcriptome data for sequence alignment and phylogenetic tree analysis.The sequence design primers with the highest similarity to AvBPPS were selected,and the coding region of it was amplified with nested PCR method.In addition,the method of In-fusion was adopted to construct AvBPPS-T47 and AlBPPS into the pET-32a expression vector.2.3.2 Synthesis of LdBPPS from Lippia dulcisThe nucleotide sequence(MF133333.1)of LdBPPS from GenBank was downloaded to synthesize the coding region of its prokaryotic expression,and the expression vector pET32a was constructed.2.3.3 Comparison of enzyme functions of different BPPSProkaryotic expression and protein purification methods were the same as 2.2.1 and2.2.2 In order to detect the product bomeol,alkaline phosphatase is added after the substrate reaction,the product was dephosphorylated,and GC-MS detection was performed again.2.3.4 Expression pattern analysis of BPPS in pericarp and seeds of Amomum villosum and Amomum longiligular and correlation with corresponding terpenoidsAmomum villosum 45 DAF pericarp(AvP),Amomum villosum 45 DAF seeds(AvS),Amomum longiligular 45 DAF pericarp(AlP),Amomum longiligular 45 DAF seeds(AlS)were used to detect the relative gene expression by Real-time fluorescence quantitative method.The volatile terpenoids from the corresponding tissues of Amomum longiligular were determined,and the correlation between gene expression and the content of corresponding terpenoids was analyzed.3.Results1.Fruit transcriptome and volatile terpenoids from Amomum villosumIn order to have a better understanding of TPS family and volatile terpenoids in the fruit of Amomum villosum,the transcriptiome data of 45 DAF and 75 DAF were analyzed,and the unigene sequence obtained from transcriptome with better assembly quality was generally longer,and the annotation rate in the Nr database was up to 99.48%.In the KEGG metabolic pathway,unigene was the most annotated biosynthesis pathway of secondary metabolism;in the terpenoids skeleton synthesis pathway,the unigene was annotated more by MEP than MVA.The detection results of terpenoids indicated that the Amomum villosum was rich in volatile terpenoids,and a total of 45 terpenoids were detected,including 17 monoterpenoids,27 sesquiterpenoids and one diterpenoids.Bomyl acetate,camphor,and bomeol were detected in the seeds of Amomum villosum.The total content of volatile terpenoids in SF is higher than that in SYF,and the total content of volatile terpenoids in PF is also higher than that in PYF,which indicates that the volatile terpenoids in the fruits are in the accumulation process,ranging from 45 DAF to 75 DAF.The species of monoterpenoids in the seeds is richer than in the pericarp and rhizome,and the eontent of terpenoids is also higher than the pericarp and rhizome,but a diterpenoids compound detected in the pericarp and rhizome cannot be detected in the seeds.Correlation analysis between RPKM values of 22 AvTPS at five different tissue and 45 species volatile terpenoids showed that TPS predicted to be monoterpene synthase had a higher correlation with some monoterpenoids compounds.Sesquiterpene synthase is not only highly correlated with monoterpenoids,but also has a high correlation with sesquiterpenoids.2.Cloning and functional identification of AvTPS genePhylogenetic trees clustered 22 AvTPS into 7 different subfamilies,among which monoterpene synthases were clustered into the TPS-b subfamily,sesquiterpene synthases were mainly clustered into the TPS-a subfamily,and diterpene synthases were mainly clustered into the TPS-c and d subfamily.Nine AvTPSs were successfully cloned,including monoterpene synthase AvTPS2,sesquiterpene synthases--AvTPS6,AvTPS15,AvTPS16,AvTPS17,AvTPS18 and three diterpene synthases-AvTPS5,AvTPS9 and AvTPS19.Prokaryotic expression vectors were constructed and protein was purified.Soluble proteins were obtained from AvTPS2,AvTPS5,AvTPS6,AvTPS 15 and AvTPS 18.The proteins induced by AvTPS17 and AvTPS 19 were inclusion bodies.AvTPS9 and AvTPS 16 genes did not induce the target protein.The purified proteins were reacted with substrates GPP or FPP,respectively.The main product of AvTPS2 and GPP reaction was Linalool,and the by-products were ?-myrcene,limonene,trans-?-ocimene,?-cis-ocimene and carene.AvTPS2,which could be named as linalool synthase,was highly expressed in PF and flowers,but hardly expressed in SF and CS.Linalool,the main product of volatile terpenoids,was detected in PF and floweres,but not in SF or CS.The main reaction products of AvTPS6 with GPP were(2E)-1-methoxy-3,7-dimethylocta-2,6-diene,and the by-products were ?-myrcene,limonene,a-terpinene,a-terpineol,cis-geraniol;The main product of AvTPS6 with FPP was humulene,and the by-product was caryophyllene,and AvTPS6 could be named as humulene synthase.The expression level of AvTPS6 in SF was higher than that in PF.The content of humulene in SF was higher than PF,corresponding to the expression pattern.The reaction products of AvTPS15 with GPP were ?-myrcene,limonene,cis-?-terpineol,?-terpinene,linalool,linalool methyl ether,?-terpineol,(2E)-1-methoxy-3,7-dimethylocta-2,6-diene,geraniol;The reaction products of AvTPS 15 with FPP were a-santalene and cis-a-bergamotene,by-products were(Z)-?-bisabolenes,?-sesquiphellandrene,therefore AvTPS 15 could be named as a-santalene/a-bergamene synthase.AvTPS 15 was highly expressed in SF,and a-santalene and a-bergamotene are detected in the seeds;The reaction product of AvTPS 18 with GPP was(2E)-1-Methoxy-3,7-dimethylocta-2,6-diene,and the reaction product of AvTPS 18 withFPP was ?-elemene.AvTPS18 could be named as ?-elemene synthase.AvTPS 18 was highlyexpressed in PF and CS,also expressed in the SF,but ?-elemene was detected only in SF.In addition,the genomes containing complete coding regions of AvTPS2 and AvTPS6 were cloned,and both containing 7 exons and 6 introns.The genome structure was similar to other monoterpene or sesquiterpene synthase genes reported.Furthermore,the promoter fragment of AvTPS6 was cloned and found to have cis-regulatory elements such as TATA-box and G-box.3.Comparison of the function of bornyl diphosphosynthasesThe AIBPPS,which is most similar to AvBPPS,was screened out from the transcriptome of immature seeds form Amomum longiligular.The 1791 bp coding region sequence was obtained from the seed cDNA,and the prokaryotic expression vector was constructed and induced to obtain purified protein.Phylogenetic evolution tree showed that it was a branch of AvBPPS and belonged to the TPS-b subfamily.The results of multiple sequence alignment indicated that the sequence consistency of AIBPPS with other species was 60.06%,and that of AvBPPS was 82.55%.The sequence of conservative domain is almost identical.Only one amino acid difference site was found in "DTE",AlBPPS(A496)was alanine(A),and AvBPPS(G495)was glycine(G).AvBPPS,AvBPPS-T26(AvBPPS with N-terminal truncation of 26 aa),AvBPPS-T47(AvBPPS with N-terminal truncation of 47 aa),and LdBPPS(synthesized according to sequences submitted on GenBank)were also subjected to protein induction and purification.When the reaction product was dephosphorylated with alkaline phosphatase,all proteins produced bomeol.The production of bomeol in the catalytic product of AvBPPS-T26 accounted for 57.55%of the total production,and camphene,limonene and ?-mycrene accounted for 21%,19%and 6%of the total production,respectively;The contents of bomeol,campene and limonene in the catalytic products of AvBPPS-T47 decreased,and a new compound a-pinene was produced.In the catalytic product of AIBPPS,camphene as the main product,accounted for 59.92%of the total production,bomeol and limonene accounted for 21.16%and 6.21%,respectively,and the total of other by-products accounted for 12.70%;LdBPPS produced six compounds,of which camphene was the main product,and it accounted for 45.31%.Borneol,limonene,a-pinene,terpinolene,and ?-mycrene accounted for 24.72%,11.36%,8.47%,6.06%,and 4.08%of the total production,respectively.The production of bomeol in the AvBPPS-T26 catalytic product is the highest,and the LdBPPS catalytic product is the most abundant.Semi-quantitative results indicated the main expression of AvBPPS and AlBPPS in the seeds of Amomum villosum and Amomum longiligular.Real-time fluorescence quantification showed that BPPS has higher expression in Amomum villosum 45 DAF seeds(AvZ)than in Amomum longiligular 45 DAF seeds(AlZ),and it has low expression in Amomum longiligular 45 DAF pericarp(AlP)while no expression in Amomum villosum 45 DAF pericarp(AvP).This is basically consistent with the trend of the total content of direct or indirect catalytic products detected in the corresponding tissue parts.4.ConclusionIn this paper,22 candidate AvTPSs were screened out from the fruit-based transcriptome of Amomum villosum.One of the monoterpene synthase(AvTPS2)and three sesquiterpene synthase(AvTPS6,AvTPS15,AvTPS 18)genes were cloned and identified,which were named as linalool synthase,humulene synthase,a-santalene/cis-?-bergamene synthase,and ?-elemene synthase,respectively.Their expression patterns in different tissue were basically eonsistent with those of terpenoids.They were all multi-product TPSs,in which,AvTPS6,AvTPS15 and AvTP518 are bifunctional synthase,which can both catalyze GPP and FPP to formvarious terpenoids products.The results of this study enrich the understanding of the synthetic mechanism of volatile terpenes in Amomum villosum.The comparison of catalytic and dephosphorylation ratios of bomeol in the products of AvBPPS-T26,AvBPPS-T47,AlBPPS and LdBPPS provided a basis for the subsequent optimization of AvBPPS functions.