METHOD FOR INCREASING VIRIDIFLOROL CONTENT IN TISSUES

Abstract

The present invention relates to the development of high viridiflorol containing variety for enhancing the aroma as well as medicinal properties of Mentha piperita by overexpressing MPTPS4 gene through genetic transformation. This transgenic plant possesses better growth and able to produce essential oil with good quality contains high viridiflorol maximum of about 25% and proportionate decrease in menthone, menthofuran and menthol of Mentha piperita as compared to other control genotype. Mentha piperita (CIM-madhuras) that produces viridiflorol, a molecule of potential demand in perfumery, cosmetics, toiletries, drugs and sanitation products.

Claims

1. A method for increasing viridiflorol content in tissues, said method comprising the steps; a) isolating sesquiterpene synthase gene (MPTPS4) from Mentha piperita having SEQ ID NO: 4; b) cloning of gene MPTPS4 obtained at step (a) into pHANNIBAL vector to obtain a cassette with SEQ ID NO: 8; c) re-cloning the cassette obtained at step (b) into pART27 binary vector system with SEQ ID NO: 9; and d) transforming the vector obtained at step (c) into Agrobacterium tumerfaciens for overexpression of MPTPS4 and further transfecting into tissues to obtain the increased viridiflorol content.

2. The method according to claim 1, wherein the terminator sequence is an octapine synthase (OCS) terminator sequence set forth in SEQ ID NO: 10.

3. The method according to claim 1, wherein the promoter sequence is a tissue specific promoter (Limonene synthase, 797 bp) sequence set forth in SEQ ID NO: 7.

4. A transgenic plant obtained by the method according to claim 1.

5. The plant according to claim 4, wherein the plant has increased viridiflorol content up to 25%.

6. A method of increasing aroma in perfumery, cosmetics, toiletries, drugs and sanitation industries using plant according to claim 4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 is schematic representation of preparation of overexpression construct.

BRIEF DESCRIPTION OF THE TABLES

[0024] Table 1 is viridiflorol analysis in essential oil of transgenic Mentha plants over expressing MPTPS4. Values are represented as mean±SD (n=3 biological replicates), VC: Vector.

[0025] Table 2 is a comparative analysis of other metabolites in MPTPS4 overexpressing lines of Mentha in GC-MS (values are shown in percentage).

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will be illustrated in the following examples, which are in no way intended to limit the scope of the present invention.

Example 1

[0027] Expression of MPTPS4 Gene

[0028] 1. Selection of Genes to be Used

[0029] Comparative Transcriptome Analysis of Mentha piperita and M. spicata

[0030] To investigate the terpene synthesis pathway of Mentha Piperita recently constructed the Mentha piperita transcriptome through RNA sequencing of the GT's rich tissue the transcriptomes are deposited at NCBI under the accession no. SRP056511) (Akhtar, 2017 Physiologia Plantarum, 160:128-141) and compared with transcriptome of the GT's rich tissue of Mentha spicata (the transcriptomes are deposited at NCBI under the accession No. SRP056511) (Jin, 2014, BMC Plant Biology, 14).

[0031] In Silico Analysis of Database Sequences for TPSs Genes

[0032] In order to identify the valuable TPS genes, Plant trichrome EST database of Mentha piperita was mined to retrieve all the TPSs related transcripts/genes. Further, a local BLAST of these ESTs was carried out against the transcriptome database of Mentha piperita. This led to the identification of some uncharacterized TPS related genes. Based on nucleotide and amino acid sequences, partial ESTs were shortlisted and used to obtain full-length genes for further characterization.

[0033] Transcriptome Sequencing and Assembly

[0034] The sequencing of paired end cDNA library of transcriptome from trichomes of Mentha piperita and M. spicata generated a total of 412,458,378 (412.46 million) raw reads for Mentha piperita and 230,383,922 (230.38 million) raw reads for M. spicata. Out of which, number of paired end reads were 143,546,248 (143.5 million) for Mentha piperita and 81,145,014 (81.1 million) for M. spicata. Since, paired end sequencing generates high quality alienable sequence data and is more likely to align to a reference; these reads were used for further processing.

[0035] Assembly of Transcripts Showing Differential Expression

[0036] Transcripts from the libraries of both Mentha piperita and Mentha spicata were clustered using CD-HIT EST tool applying the criterion of sequence identity threshold as 0.8 for obtaining the set of non-redundant transcripts. A total of 52321 transcripts were obtained both from Mentha piperita and M. spicata out of which, 16138 transcripts were exclusive to Mentha piperita while, 896 transcripts were exclusive to Mentha spicata and 35287 transcripts were common in both Mentha piperita and Mentha spicata (456 up-regulated; 438 down-regulated and 34393 neutral with respect to Mentha piperita).

[0037] Functional Annotation and GO Classification

[0038] Annotations of all the unique transcripts (>300 bp) was performed using a BLASTx homology search against TAIR Database. BLAST hits with e-value scores ≤0.001 were considered as annotated homologous proteins. Transcripts were subjected to Gene Ontology (GO) classification in order to categorize them under the three categories of biological process (BP), molecular function (MF) and cellular component (CC). Out of the total 52321 transcripts in both plants, 36793 transcripts got annotated with different GO terms in aforementioned three categories of BP, MF and CC. The order of the GO term assignment was CC (68.61%)>MF (21.43%)>BP (9.94%).

Example 2

[0039] Identification of Transcripts Related to Terpene Synthases

[0040] To explore the array of terpenoids, it was important to identify the transcripts related to the terpene synthases. 89 and 82 TPS transcripts were identified in Mentha piperita and Mentha spicata, respectively when annotated against the TAIR database of Arabidopsis shows number of transcripts for each gene identified in the annotation.

TABLE-US-00001 TABLE 1 SAMPLE MARCH APRIL MAY JUNE JULY MPOT_1  8.66 +/− 0.0049 7.86 +/− 0.0015 8.71 +/− 0.0058 8.75 +/− 0.0025  7.65 +/− 0.0164 MPOT_2 25.98 +/− 0.0043 26.6 +/− 0.0045 25.2 +/− 0.0248 21.7 +/− 0.0341 24.43 +/− 0.0285 MPOT_3 20.40 +/− 0.0029 22.5 +/− 0.0038 19.2 +/− 0.0494 22.7 +/− 0.0107 15.14 +/− 0.0049 MPOT_4 14.53 +/− 0.0013 14.8 +/− 0.0065 14.2 +/− 0.0235 11.3 +/− 0.0198 12.43 +/− 0.0071 VC 0.40 +/− 2.30  0.56 +/− 0.0004 0.53 +/− 0.0001 0.54 +/− 6.67   0.42 +/− 0.0004

TABLE-US-00002 TABLE 2 Compound name MPOT_1 MPOT_2 MPOT_3 MPOT_4 Vector α-Pinene 0.59 0.54 0.58 0.60 0.69 β-Pinene 1.44 1.23 1.32 1.38 1.61 D-Limonene 5.70 4.88 5.23 5.48 6.47 Linolool 0.21 0.18 0.19 0.20 0.23 Menthone 23.7 20.2685 21.95 22.924 27.394 Menthofuran 17.75 15.03 16.14 16.80 20.12 Menthol 18.95 16.14 17.36 18.16 21.71 α-Terpineol 0.66 0.59 0.62 0.65 0.78 Pulegone 3.38 2.89 3.17 3.29 3.97 Menthyl acetate 3.426 2.911 3.154 3.2865 3.93 β-Bourbonene 0.10 0.09 0.09 0.11 0.12 Caryophyllene 3.04 2.59 2.79 2.93 3.47 β-Cubebene 0.13 0.11 0.12 0.12 0.15 Alloaromadendrene 0.28 0.54 0.39 0.28 0.02 Humulene 0.19 0.16 0.17 0.18 0.21 cis-β-Farnesene 0.38 0.34 0.35 0.35 0.38 Germacrene D 0.73 0.63 0.69 0.71 0.86 γ-Muurolene 0.12 0.10 0.10 0.11 0.13 Viridiflorol 7.77 26.03 21.94 14.38 0.585

[0041] Pathway Analysis (Differentially Expressed)

[0042] MVA and MEP Pathway

[0043] Differential expression profile of transcripts annotated as MVEP and MVA pathway genes showed that the transcripts of genes involved in MVEP pathway namely CMK, DXR, DXS, MCT, MCS, HDR and GGPP are upregulated in Mentha spicata and MVA pathway transcripts namely AACT, HMGS, WVK, PMK, PMD and FPP are downregulated compared to the reference gene ACT (Actin).

[0044] Terpene Synthases

[0045] In the differential gene expression analysis, a total of 94 TPS transcripts were found, out of which, 12 were up-regulated, 9 were down-regulated and 44 were neutral in Mentha spicata when compared to Mentha piperita, while 11 TPS transcripts were exclusively expressed in Mentha piperita and 18 were in Mentha spicata.

[0046] Analysis of Available Mentha piperita Trichome EST Database for Terpene Synthase

[0047] ESTs analysis of the Plant trichOME database revealed the presence of about eight different TPS ESTs.

Example 3

[0048] Identification, Isolation and Sequence Characterization of the TPSs in Mentha piperita

[0049] A local BLAST of 45 ESTs from Plant trichOME database was performed against transcriptome database of Mentha piperita. Out of these 11 ESTs which were found less annotated to the database were used to get full-length genes. RACE primers were designed from the available TPS partial sequences to obtain full-length genes. A total of six genes namely MPTPS1 with SEQ ID NO: 1; MPTPS2 with SEQ ID NO: 2; MPTPS3 with SEQ ID NO: 3; MPTPS4 with SEQ ID NO: 4; MPTPS6 with SEQ ID NO: 5 and MPTPS7 with SEQ ID NO: 6 were cloned from Mentha piperita. All the genes were amplified from the cDNA of trichome rich tissue of Mentha piperita (CIM-Madhuras), cloned, sequenced and characterized. The sequences obtained were subjected to BLASTx and BLASTn analysis. The amino acid sequence of Mentha piperita was compared to ones existing in the NCBI database for the identification of signature sequences/motifs and or domains.

[0050] Out of these six terpene synthases, four (MPTPS1, MPTPS2, MPTPS3, and MPTPS4) are sesquiterpenes and two (MPTPS6 and MPTPS7) are monoterpenes. In sesquiterpenes, MPTPS3 and MPTPS4 showed less identity to others from the database after BLASTx analysis, therefore, MPTPS3 and MPTPS4 were considered for further analysis and characterization.

[0051] MPTPS3 and MPTPS4 Gene Expression in Different Tissues.

[0052] Comparison of the deduced amino acid sequences of MPTPS3 and MPTPS4 showed 81% similarity. Tissue-specific expression was analyzed for MPTPS3 and MPTPS4 in different tissues in Mentha piperita for comparison. MPTPS4 showed significantly higher spacial expression than MPTPS3 in the order of trichome>leaf>stem>root. Hence, only MPTPS4 was taken into consideration for the further downstream functional characterization.

[0053] MPTPS4

[0054] EST of MPTPS4 was extracted from the plant trichOME EST database of Mentha piperita annotated as Terpene synthase (Accession no AW255698). The partial sequence thus obtained was 628 bp and which was subjected to 3′ and 5′RACE in order to get the full-length MPTPS4 gene. Full-length cDNA sequence of MPTPS4 gene was found to be 1686 bp, encoding a polypeptide of 561 amino acid residues. The molecular weight of the deduced protein was predicted to be 65.09 kDa and computed isoelectric point (pI) was 4.91. The nucleotide sequence has been submitted to the NCBI database with the accession number (MH790402). Nucleotide sequences of MPTPS4 from Mentha piperita showed 82% similarity with TPS4 of Origanum vulgare, while amino acid sequences showed 77% sequence similarity with TPS4 of Origanum vulgare which is reported to be a Bicyclogermacrene synthase.

Example 4

[0055] Cloning of MPTPS4 Gene in pET28a (+) and Transformation for Bacterial Expression

[0056] The MPTPS4 gene (˜1.6 kb) was sub cloned from pGEM-T Easy cloning vector onto the pET28a (+) expression vector. The positive recombinant plasmid was then transformed 30 into E. coli BL21 (DE3) bacterial expression host cell and both the induced and uninduced host proteins were isolated to analyze on the SDS PAGE. 64 kDa MPTPS4 protein was obtained after purification and desaltation from the induced host cell was confirmed by running SDS PAGE and comparing with the protein molecular weight marker.

[0057] Enzyme Assay

[0058] GC-MS Analysis

[0059] The recombinant MPTPS4 protein was produced in E. coli BL21 (DE3), enzyme assay reactions were set up with crude extracts in which extract was incubated with FPP and the product was analyzed in GC, a unique peak of viridiflorol was observed when compared to the vector control. Similarly, purified MPTPS4 protein was incubated with enzyme substrates FPP, GPP and GGPP and the reaction products were analyzed using GC-MS. The reactions in which GPP and GGPP were used as substrates did not show any product formation. On the other hand, when FPP was used as the substrate of MPTPS4 protein, a product peak was generated. The product was identified to be “Viridiflorol” based on the mass spectra similarity as per the NIST library.

[0060] Enzyme Kinetics

[0061] The purified recombinant MPTPS4 enzyme was characterized for their catalytic activities with FPP. Rate of reaction for different substrate (FPP) concentration (0-350 μM) (was also analyzed). The amount of substrate converted in each reaction was calculated, and the amount of enzyme was standardized to micromole per minute per microgram. The reaction rate per microgram enzyme per second was established, and the reaction rate (1/second) was calculated.

[0062] Product Confirmation of MPTPS4 Through NMR Studies

[0063] Structural Analysis of MPTPS4 Product

[0064] The expression construct pET28a-MPTPS4 was introduced into E. coli BL21-CodonPlus (DE3) cells together with plasmid Add gene Plasmid 35150: pBbA5c-MevT-MBIS, for the utilization of FPP as the substrate, were grown in LB medium and the product extracted was 25.2 mg. This product was subjected to .sup.1H-nuclear magnetic resonance spectrometry, .sup.13C NMR and DEPT 135 spectrum. The .sup.1H and .sup.13C chemical shift of viridiflorol is shown in GC-MS and TLC analysis.

Example 5

[0065] Preparation of Over Expression Construct of MPTPS4

[0066] The MPTPS4 (1686 bp) gene with SEQ ID NO: 4 was cloned in pHANNIBAL vector system by replacing the PDK intron to yield phannibal-MPTPS4, under tissue specific promoter (Limonene synthase, 797 bp) with SEQ ID NO: 7. The whole cassette (˜3.5 kb) SEQ ID NO: 8 was cloned into pART27 binary vector system with SEQ ID NO: 9 and confirmed by restriction analysis. The binary vector with and without the genes were then transformed into GV3103 strain of Agrobacterium tumefaciens separately, positive clones selected on kanamycin antibiotic plates and confirmed with the help of colony PCR.

[0067] Generation of Transgenic Mentha Lines of MPTPS4

[0068] In vitro maintained Mentha plants in the laboratory were used for Agrobacterium mediated internode transformation for generating transgenic plants. All individual transformation experiments were accompanied with vector control. PART-MPTPS4 transformed internode was placed on selective MSB_M media. After 3-4 weeks, direct regeneration was induced as a result of pART-MPTPS4 gene transformation on kanamycin (50 mg L.sup.1) selection medium.

[0069] Analysis of Transgenic Mentha Plants

[0070] PCR analysis was carried out to confirm the transfer of transgene cassette into the transgenic lines. Genomic DNA was extracted from each putative transgenic line and PCR analyzed using primers of npt II gene. Four lines of pART-MPTS4 exhibited an amplification of 750 bp, which was absent in the no template control lane, hence were confirmed to be transgenic lines.

[0071] Overexpression Studies Through Quantitative Expression

[0072] Four independent kanamycin-resistant Mentha piperita transformants of pART-MPTPS4 (MPOT_1, MPOT_2, MPOT_3 and MPOT_4) and pART only (Vector Control, VC) transformants were grown in green house. The expression levels of the transgenic lines were determined by quantitative RT-PCR. Overexpression in four independent transgenic lines was in order of MPOT_2 (18.9-37.7 fold)>MPOT_3 (13.4-26.4-fold)>MPOT_1 (9.4-25 fold)>MPOT_4 (5.4-16.0 fold) compared to the vector control. The range denotes in planta fold expression as estimated from the isolated mRNA during March to July.

[0073] Overexpression Analysis Through GC-MS

[0074] In order to study the effect of increased MPTPS4 expression on the content of plant essential oils, four transgenic (MPOT_1, MPOT_2, MPOT_3 and MPOT_4) plant lines produced through Agrobacterium transformation were grown in a greenhouse and essential oil profiles for all these lines were generated using GC-MS. All the four MPTPS4 over-expressed transgenic lines exhibited significant increase in viridiflorol content compared to the vector control. The range denotes in planta fold expression as estimated from the isolated mRNA during March to July.

[0075] Correlation of Metabolite Synthesis with Gene Expression

[0076] To see the effect of MPTPS4 overexpression on the other metabolites, comparative analysis was performed through GC-MS and values (%). All the four MPTPS4 overexpressing transgenic lines exhibited increase in the viridiflorol content while, other metabolites like menthone, menthofuran and menthol decreased as flux diverted towards increased content of viridiflorol as well as one sesquiterpene, alloaromadendrene also increased compared to control plants.

[0077] Subcellular Localisation of the MPTPS4

[0078] N-terminal in-frame GFP fusions were made with the MPTPS4 full-length coding regions in the p326-SGFP vector between the XbaI and BamH I restriction enzyme sites to create p326-MPTPS4/SGFP. GFP fluorescence for full-length p326-MPTPS4/SGFP coding regions was observed as a diffused signal exclusively in the cytosol. Expression of a control GFP construct was also localized in the cytoplasm. These results confirm the predicted cytosolic localization of the enzyme.

[0079] Procurement Details of all the Biological Materials Used in Invention

[0080] Mentha plants: The germplasm accessions of Mentha piperita cv. ‘Cim-Madhuras’ was obtained from National gene Bank for medicinal and Aromatic Plants (NGBMAP) maintained at Central Institute of medicinal and Aromatic Plants (sponsored by Department of Biotechnology, Regime of India) in Lucknow (26.5° N, 81.010 E), India.

TABLE-US-00003 Bacterial Host Strains Strain Description Reference Escherichia supE44 Δ lacU169 (φ80 lacZ Δ M15) hsd Sambrook et al., 1989 coli R17 rec Al endA1gyrA96 thi-1 rel A1 (DH5α) Escherichia F.sup.−ompT hsdSB(r B.sup.− m B.sup.−) gal dcm lacY1 Novagen coli (DE3) www.novagen.com BL21 Star ™ (DE3) Agrobacterium Resistance for gentamycin and rifampicin Intact Genomics tumefaciens antibiotics www.intactgenomics.com (GV3103)

TABLE-US-00004 Vectors Plasmid vectors Description Source pGEM ®-T 3.0 kb in size, multiple cloning Promega sites having 15 unique www.promega.com restriction sites, LacZ fragment, pUC/M13 forward and reverse priming sites, T7 and SP6 promoter/primer binding site, fl origin, T7 RNA polymerase transcription initiation site and has ampicillin resistance ORFs. pET 28a(+) 5.369 kb, N-terminal His•Tag ® Novagen configuration with an optional www.novagen.com C-terminal His•Tag sequence, f1 origin of replication, T7 promoter, T7 terminator, lacI coding sequence, kan coding sequence, f1 origin replication. pHANNIBAL 5.824 kb in size with bacterial CSIRO ampicillin resistance is www.pi.csiro.au designed for directional insertion of PCR products on either side of the PDK intron. pART 27 Size of 11.667 kb, RK2 CSIRO minimal replicon for www.pi.csiro.au maintenance in Agrobacterium, the ColE1 origin of replication, Tn7 spectinomycin/streptomycin resistance gene as a bacterial selectable marker, nos promoter, nos terminator, LacZ fragment p326-sGFP 4.488 kb in size, CaMV35S Provided by Inhwan Hwang promoter, nos terminator, (POSTEC, Korea) ampicillin resistance, 900 bp sGFP genes cloned between the promoter and terminator. pBbA5c 1.3264 kb in size, lacUV5 Addgene promoter, rrnB terminator, https://www.addgene.org chloroamphinicol resistance, low copy.

[0081] pART-MPTPS4: —The MPTPS4_sense gene (500 bp) and MPTPS4_antisense gene (500 bp) were subcloned from pGEM-T Easy cloning vector onto the pHANNIBAL cloning vector under tissue specific promoter by replacing cauliflower mosaic virus (CaMV) 35S promoter with octopine synthas (ocs) terminator with SEQ TD NO: 10, sequentially on either side of the intron between EcoRI/KpnI and XbaI/HindIII restriction enzyme sites respectively to get the two arms of the hairpin which was confirmed by restriction analysis using the XhoI and XbaI, the first and the last restriction enzyme sites of multiple cloning sites, MCS1 and MCS2 of pHANNIBAL respectively. The hairpin cassette was then cloned into a pART 27 binary vectors in a single step using the Not I restriction sites as there are two NotI restriction enzyme sites in pHANNIBAL and a single site in pART27 binary vector.

[0082] cDNA of trichome rich tissue of Mentha piperita (CIM-Madhuras):—cDNA of trichome rich tissue of Mentha piperita were isolated in Dr. Ajit K. Shasany's Lab, Biotechnology Division, CSIR-CIMAP, Lucknow, India.

[0083] Kanamycin-resistant Mentha piperita transformants of pART-MPTPS4 (MPOT_1, MPOT_2, MPOT_3 and MPOT_4):—To prepare the overexpression construct of the MPTPS4, specific primers with SEQ ID NO: 11 and SEQ ID NO: 12 were designed introducing BamHI with and EcoRI with restriction enzyme sites by replacing the PDK intron into pHANNIBAL vector system to yield pHANNIBAL-MPTPS4 under tissue specific promoter by replacing cauliflower mosaic virus (CMV) 35s promoter with octapine synthase (OCS) terminator. The cloned fragments were confirmed by restriction analysis and sequencing. The whole cassette with promoter, gene and terminator was then cloned into pART27 binary vector system. The cloned fragments were confirmed by restriction analysis. Binary vector with and without the transgene (MPTPS4) was then transformed into GV3103 strain of Agrobacterium separately.

[0084] Generation of transgenic Mentha lines of MPTPS4—In vitro maintained Mentha plants in the laboratory were used for Agrobacterium mediated internode transformation for generating transgenic plant. All individual transformation experiments were accompanied with vector control. PART-MPTPS4 transformed internodes were placed on selective MSB_M media. After 3-4 weeks, direct regeneration was induced as a result of pART-MPTPS4 gene transformation on kanamycin (50 mg L.sup.1) selection medium. Four independent kanamycin-resistant Mentha Piperita transformants of pART-MPTPS4 (MPOT_1, MPOT_2, MPOT_3 and MPOT_4) and pART only (Vector Control, VC) transformants were obtained and grown in green house. Experiments were performed in Dr. Ajit K. Shasany's Lab, Biotechnology Division, CSIR-CIMAP, Lucknow, India.

Advantages of the Invention

[0085] The present invention provides for: [0086] 1.) A method for obtaining transgenic plants with high viridiflorol containing variety for enhancing the aroma, therapeutic properties. [0087] 2.) A method for enhancing the medicinal properties of Mentha piperita by overexpressing MPTPS4 through genetic transformation. [0088] 3.) The method provides better vegetative growth. [0089] 4.) The method produces plants with essential oil with good quality containing high viridiflorol maximum of about 25%. [0090] 5.) The plants obtained with increased viridiflorol content produced by the method of the invention are used in perfumery, cosmetics, toiletries, drugs and other sanitation products. [0091] 6.) The produced viridiflorol adds sweetness to the aroma of plant essential oil. [0092] 7.) The obtained viridiflorol possess anticarcinogenic and antitumorigenic properties.

REFERENCES

[0093] 1. Albert-Puleo M (1980) Fennel and anise as estrogenic agents. J Ethnopharmacol 2:337-344. [0094] 2. Akhtar M Q, Qamar N, Yadav P, et al (2017) Comparative glandular trichome transcriptome-based gene characterization reveals reasons for differential (−)-menthol biosynthesis in Mentha species. Physiologia Plantarum 160:128-141. doi: 10.1111/ppl.12550. [0095] 3. Jin J, Panicker D, Wang Q, et al (2014) Next generation sequencing unravels the biosynthetic ability of Spearmint (Mentha spicata) peltate glandular trichomes through comparative transcriptomics. BMC Plant Biology 14. doi: 10.1186/s12870-014-0292-5.