Functional Identification Of Monoterpene Synthases And Cloning,Prokaryotic Expression Of Transcription Factors MYC In Amomum Villosum

1.ObjectiveThe dried fruit of Amomum villosum is called Fructus Amomi,has much medicinal value and is one of the best genuine medicinal materials in Guangdong province,China.The fruit Fructus Amomi has the function of appetizer with dampness,warming the spleen and stopping diarrhea,regulating qi and tocolysis.Our group has already conducted the transcriptome analysis on two transcriptomes of A.villosum and screened out several important terpene synthases and transcription factors.Here,the functional characterization of the monoterpene synthases AvTPS1 and AvTPS3,which have already obtained the full-length genes,and the cloning and functional characterization of two transcription factors AvMYC2 and AvMYC4b were carried out to further understand the regulatory mechanism of the secondary metabolism of A.villosum.2.Method2.1 Functional identification of monoterpene synthases AvTPS1 and AvTPS32.1.1 Enzyme assayBased on the cloning of AvTPS1 and AvTPS3,the ORF excluding the N-terminal transit peptide was ligated into the pET32a expression vector using the In-fusion cloning Kit.The positive constructs were transformed into the competent Rosetta cells to do prokaryotic expression.The recombination proteins were purified with NI-NTA resin.Finally,enzyme assay and product analysis by GC-MS was performed.2.1.2 Subcellular localizationThe complete ORF of AvTPS1 and AvTPS3 were ligated into constructs pAN580 for subcellular localization using the In-fusion cloning Kit to get pAN580-AvTPS1 and pAN580-AvTPS3 vectors.The protoplast of Nicotiana tubacum was isolated by enzymolysis,and then the vectors transformed into protoplast by the PEG-mediated method.Finally,laser confocal was used for subcellular localization.2.1.3 Quantitative Real-time PCR and volatile terpenoid extraction and analysisOn the one hand,the total RNA of different tissues in A.villosum was extracted and transcripted reversely into cDNA.The gene-specific primer for AvTPS1 and AvTPS3were tested for amplification kinetics and linearity with a standard curve by quantitative Real-time PCR.Then target gene transcript levels were monitored with double reference genes as control and calculated using 2~-??Cq??Cq method.On the other hand,volatile terpenoid was extracted by the method of ultrasonic extraction and detected by GC-MS.Finally,our research documented a correlation between the gene transcript levels and the related volatile terpenoid in A.villosum.2.1.4 Cloning and activity analysis of AvTPS1 promoterAvTPS1 and AvTPS3 were cloned from genomic DNA(gDNA)of A.villosum leaves.Furthermore,the promoter of AvTPS1 was cloned by FPNI-PCR and the sequence was analyzed.The recombinant vector pCAMBIA-AvTPS1p was constructed.The activity of AvTPS1 promoter was verified by the transient expression that Agrobacterium-mediated infiltration was carried on using the leaves of Nicotiana benthamiana.2.2 Cloning and prokaryotic expression of transcription factors AvMYC2 and AvMYC4bBased on the transcriptome data of A.villosum,the whole cDNA sequence of AvMYC2 was obtained by normal PCR amplification method and the whole cDNA sequence of AvMYC4b was obtained by RACE method.And then the GATEWAY TOPO cloning vector,and the expression vector pMAL-C5X-AvMYC2 and pET32a-AvMYC4b were constructed by the In-fusion cloning Kit.The positive constructs were transformed into the competent Rosetta cells to do prokaryotic expression.The recombinant proteins were purified with MBP and Ni-NTA resin.3 Result3.1 Functional identification of monoterpene synthase AvTPS1 and AvTPS3 in A. villosum.3.1.1 Protein function of AvTPS1 and AvTPS3 in vitroThrough enzyme assay,we found that the recombinant protein AvTPS1 can catalyze GPP to?-pinene(36.69%)and?-pinene(63.31%).So it was named as pinene synthase(AvPS).The recombinant protein AvTPS3,with GPP as its substrate,after dephosphorylation,produced borneol diphosphate(65.91%)as the major product with camphene(17.31%),limonene(13.14%)and?-myrcene(3.63%)as a minor product.So we named AvTPS3 as bornyl diphosphate synthase(AvBPPS).3.1.2 Subcellular localization of AvPS and AvBPPSAccording to subcellular localization,AvPS and AvBPPS were both localized in plastic and were similar to the biological information prediction and the characterization of monoterpene synthases.AvPS and AvBPPS were the plastic protein,which work in the chloroplast.3.1.3 Correlation between gene expression levels of AvPS and AvBPPS with metabolite accumulationqRT-PCR of AvPS and AvBPPS in seven tissues of A.villosum was performed,including 45 DAF(Days after flowers)pericarp,45 DAF seeds,75 DAF pericarp,75DAF seeds,roots,creeping stems and leaves.AvPS was highly expressed in the pericarp of both stages(PYF and PF)with the highest expression level in the pericarp of young fruit,but not expressed in the seeds.AvBPPS was highly expressed in the seeds of both stages(SYF and SF).The results of the contents of volatile terpenes indicate that the fruits are rich in volatile terpenes,especially monoterpenes.A total of 15 monoterpene compounds and nine sesquiterpene compounds were detected,among which,bornly acetate was significantly enriched in seeds(relative content>50%),the medicinal part.Alpha-pinene is present in all tissues,while?-pinene is not present in the seeds,indicating that AvPS is involved in the synthesis of terpenes in the pericarp,roots,creeping stems and leaves.As the pinene is a terpenoid related to biotic defense,it is speculated that AvPS may participate in the biotic defense of A.villosum.AvBPPS were highly expressed in 45 DAF and 75 DAF seeds of A.villosum.Since the main product of bornyl diphosphate in AvBPPS is a key precursor of borneol,camphor,and bornyl acetate.The direct and indirect associations of AvBPPS with terpenes,including camphene,limonene,?-myrcene,borneol,camphor and bornyl acetate were analyzed in this research.The high expression of AvBPPS in the seeds was found to be associated with these 6 volatile terpenoids,particularly borneol acetate,The enrichment in the seed is related to the hypothesis that AvBPPS is the key enzyme in the biosynthetic pathway of bornyl acetate,the main bioactive components in the plant.3.1.4 Cloning of AvPS and AvBPPS gDNA,cloning and characteristic identification of AvPS promoterThe gene sequence of AvPS and AvBPPS were 2 444 bp and 2 374 bp,both including seven exons and six introns.The 568 bp AvPS promoter was successfully cloned using FPNI-PCR.Furthermore,the sequence has cis-elements including conserved elements TATA-box,CAAT-box,MYC related element G-box and other elements.The results predicted that the AvPS promoter was regulated by the transcription factors MYC.Finally,the GUS staining showed the tobacco leaves infiltrated by the pCAMBIA-AvTPS1p were blue.The result confirms that the AvPS promoter can drive the transcription of GUS gene and then was verified to have the promoter activity.3.2 Cloning and prokaryotic expression of transcription factors AvMYC2 and AvMYC4bAvMYC2 cDNA gene had 2 057 bp,including 171bp 5'UTR,1644 bp ORF,and 1 644bp ORF,which encoded a deduced protein of 547 amino acid with a calculated molecular weight of 59 kDa.And AvMYC4b cDNA gene had 2 579 bp,including 165bp 5'UTR,1 995 bp ORF,and 389 bp 3'UTR,which encoded a deduced protein of644 amino acid with a calculated molecular weight of 72 211.Bioinformatics analysis indicated that AvMYC2 and AvMYC4b had the conserved domain of transcription factor MYC family and predicted that AvMYC2 and AvMYC4b could be located in the nucleus.We constructed the expression vectors pMAL-C5X-AvMYC2 and pET32a-AvMYC4b successfully and transformed into Rosetta(DE3)cell.The AvMYC2 and AvMYC4b protein was expressed in E.coil and purified successfully with a molecular mass of about 100 kDa and 90 kDa,which was consistent with the predicted molecular weight.4.ConclusionThis research identified the function of AvPS and AvBPPS,found that AvPS catalyzed GPP as the substrate to?-pinene and?-pinene;AvBPPS,with GPP as its substrate,was treated with alkaline phosphatase to produce borneol diphosphate as the major product and had camphene,limonene and?-myrcene as an additional product.So we named AvTPS3 as bornyl diphosphate synthase(AvBPPS).Furthermore,we found AvPS and AvBPPS were localized in plastic.Metabolite accumulation and gene expression pattern combined with AvPS biochemical characterization suggest that AvPS might play a role in the biosynthesis of?-pinene and?-pinene in pericarps,roots,creeping stemsand leaves.On the other hand,the most active ingredient,bornyl acetate was highly accumulated in seeds,consistent with the high expression of AvBPPS,indicating that AvBPPS is responsible for the biosynthesis of bornyl acetate,the final metabolite of bornyl diphosphate in A.villosum.AvMYC2 and AvMYC4b were also cloned and established prokaryotic expression system.This study will benefit the improvement of the quality of A.villosum through metabolic engineering.