Mangifera indica terpene synthase gene tps4 and application thereof
By cloning and validating the mango terpene synthase gene TPS4, constructing a recombinant expression vector and engineered bacteria, the synthesis of mango terpenoid compounds was achieved, solving the problem of aroma loss in traditional breeding and promoting the improvement of mango aroma quality and the cultivation of high-quality varieties.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI
- Filing Date
- 2026-03-17
- Publication Date
- 2026-06-09
AI Technical Summary
Traditional mango breeding focuses on phenotypic traits such as disease resistance and fruit size, while neglecting the genetic improvement of volatile aroma components. This leads to the loss of fruit aroma, which affects the development of the mango industry. Furthermore, the loss of function of terpene synthase genes results in significant differences in aroma.
The mango terpene synthase gene TPS4 was cloned and verified. A recombinant expression vector and recombinant engineered bacteria were constructed. Genetic engineering technology was used to regulate the synthesis of terpenoid compounds by GPP, NPP and FPP, thereby realizing the functional expression of terpene synthase and aroma regulation.
The application of the mango terpene synthase gene TPS4 was provided, which improved the aroma quality of mangoes through genetic engineering, cultivated high-quality aroma varieties, solved the problem of aroma loss, and provided a molecular biological basis for mango breeding.
Smart Images
Figure CN122168646A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a mango terpene synthase gene TPS4 and its application, particularly to a mango terpene synthase gene (Terpenesynthase 4, TPS4) that catalyzes the synthesis of terpene compounds in mangoes, its encoded protein, and its application. Background Technology
[0002] Mango (Mangifera indica L.), belonging to the genus Mango in the family Anacardiaceae, is known as the "King of Tropical Fruits" and is an important economic fruit tree in tropical regions worldwide. Fruit flavor is the core indicator for evaluating the commercial quality of mangoes, and aroma characteristics are the primary dimension of flavor perception. To date, approximately 400 volatile components have been identified in mangoes, covering chemical categories such as terpenes, esters, alcohols, aldehydes, ketones, and lactones. Among these, terpenes have the highest content and the most diverse composition, constituting their characteristic aroma. Different cultivars exhibit significant aroma differentiation, and this variety-dependent aroma variation provides an important target for targeted breeding.
[0003] However, traditional mango breeding has long focused on phenotypic traits such as disease resistance, fruit size, appearance, color, and sugar-acid ratio, with insufficient attention paid to the genetic improvement of volatile aroma components. Furthermore, during breeding and cultivation, it has been observed that mango fruits gradually lose their aroma, posing new challenges to the mango industry. This loss of aroma may be due to functional changes in terpene synthase (TPS) genes during long-term production and breeding, or it may be due to the lack of expression or very low expression levels of many TPS molecules in the fruit. In addition, mango aroma is also affected by factors such as harvest time, origin, cultivation management practices, storage conditions, and varietal differences.
[0004] Terpenes are the most structurally complex class of natural products in plant secondary metabolism, with over 30,000 different structures discovered to date. They are widely involved in the regulation of plant growth and development, as well as environmental adaptation. Based on the number of isoprene units in their carbon skeleton, terpenes can be classified into monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), and triterpenes (C30), among which volatile monoterpenes and sesquiterpenes are the main contributors to the floral and fruity aroma characteristics. These compounds not only endow plants with unique allelopathic effects but also exhibit multiple pharmacological activities in human medicine and health, including anti-inflammatory, antitumor, and antibacterial properties. Terpenoid biosynthesis in plants relies on two independent metabolic pathways: the mevalonate (MVA) pathway, located in the cytoplasm, and the methyl erythritol phosphate (MEP) pathway, occurring within the plastid. These two pathways synthesize isopentenyl diphosphate (IPP) and its isomer, dimethyl allyl diphosphate (DMAPP), respectively. These two C5 units are the basic precursors for building all terpenoid skeletons. Under the catalysis of pentenyltransferase, IPP and DMAPP undergo head-to-tail condensation to successively generate geraniyl diphosphate (GPP, C10), nerolidinyl diphosphate (NPP, C10), farnesyl diphosphate (FPP, C15), and geraniylgeraniyl diphosphate (GGPP, C20), which provide direct substrates for the synthesis of monoterpenes, sesquiterpenes, and diterpenes, respectively.
[0005] TPS (terpene synthases) are key enzyme nodes in the terpene metabolic network, determining the cyclization, rearrangement, and functional group modification of the carbon skeleton, thus endowing terpene products with extremely high structural diversity. This enzyme family has numerous members with varying substrate specificity and catalytic mechanisms, capable of generating hundreds of structurally diverse terpene end products using GPP, NPP, FPP, or GGPP. In mangoes, the functional differentiation of the terpene synthase gene family directly leads to differences in aroma types among different varieties. Therefore, identifying and characterizing mango-specific terpene synthase genes, elucidating their catalytic properties and expression patterns, is a crucial breakthrough for revealing the molecular formation mechanism of highly aromatic mangoes and guiding genetic engineering improvements. Summary of the Invention
[0006] The purpose of this invention is to provide a mango terpene synthase gene TPS4, its encoded protein, and a method for its preparation.
[0007] The present invention also aims to provide a recombinant expression vector containing the mango terpene synthase gene TPS4, a recombinant expression engineered bacterium, and recombinant mango terpene synthase TPS4.
[0008] The final objective of this invention is to provide the mango terpene synthase gene TPS4, the protein encoded by the mango terpene synthase gene TPS4, a recombinant expression vector containing the mango terpene synthase gene TPS4, and the application of recombinant engineered bacteria or recombinant mango terpene synthase TPS4 in regulating the synthesis of terpenoid compounds from geraniol diphosphate (GPP), neroli diphosphate (NPP), and farnesyl diphosphate (FPP).
[0009] The first objective of the present invention can be achieved by the following technical solution: a mango terpene synthase gene TPS4, the nucleotide sequence of which is shown in SEQ ID NO:1.
[0010] The amino acid sequence of the protein encoded by the mango terpene synthase gene TPS4 is shown in SEQ ID NO:2.
[0011] The method for preparing the mango terpene synthase gene TPS4 includes the following steps: reverse transcription of total RNA from Sacred Heart mango pulp into cDNA, and PCR amplification using the cDNA as a template with primer pairs shown in SEQ ID NO:3 and SEQ ID NO:4.
[0012] Specifically, this invention designs a pair of specific primers (PF forward primer: 5'-cgcggcagccatATGTCTCTTCAAGTTTCAGC-3', as shown in SEQ ID NO:3, PR reverse primer: 5'-gtggtggtgctcgagCTATATTGACACAGGGTCAA-3', as shown in SEQ ID NO:4) to amplify cDNA from mango pulp samples by PCR, obtaining the functional gene CDS sequence (full length 1677bp, as shown in SEQ ID NO:1) that catalyzes the synthesis of terpenoid compounds from GPP, NPP and FPP in mango pulp.
[0013] The second objective of the present invention can be achieved by the following technical solution: a recombinant expression vector comprising the above-mentioned mango terpene synthase gene TPS4 and the expression vector.
[0014] Preferably, the expression vector is PSJ8.
[0015] In some embodiments of the present invention, the recombinant expression vector is PSJ8-TPS4.
[0016] The present invention also provides a recombinant expression engineered bacterium, wherein the mango terpene synthase gene TPS4 is constructed into the expression vector PSJ8, the obtained recombinant expression vector is used to transform BL21, positive strains are screened to construct an engineered Escherichia coli bacterium, and the recombinant expression engineered bacterium is obtained.
[0017] In some embodiments of the present invention, the recombinant expression engineered bacteria of the present invention first integrates a maltose-binding protein (MBP) tag into the pET21a(Novagen) prokaryotic expression vector to construct a PSJ8 prokaryotic expression vector; then, the mango terpene synthase gene TPS4 is constructed into the PSJ8 vector, and the obtained recombinant expression vector PSJ8-TPS4 is used to transform BL21(DE3), and positive strains are screened to construct engineered Escherichia coli bacteria to obtain recombinant expression engineered bacteria.
[0018] The present invention also provides a recombinant mango terpene synthase TPS4, comprising transforming host cells with the recombinant expression vector, culturing the transformants, and obtaining recombinant mango terpene synthase TPS4 from the culture.
[0019] Preferably, the host cell is Escherichia coli.
[0020] In addition to recombinant expression vectors, recombinant engineered bacteria, and recombinant mango terpene synthase TPS4, it can also include expression cassettes containing the mango terpene synthase gene TPS4, transgenic cell lines, etc.
[0021] The last objective of the present invention can be achieved by the following technical solution: the mango terpene synthase gene TPS4, the protein encoded by the mango terpene synthase gene TPS4, a recombinant expression vector containing the mango terpene synthase gene TPS4, the recombinant expression engineered bacteria, or the application of the recombinant mango terpene synthase TPS4 in regulating the synthesis of terpenoid compounds from geraniol diphosphate (GPP), neroli diphosphate (NPP), and farnesyl diphosphate (FPP).
[0022] This invention enables the application of the mango terpene synthase gene TPS4, the protein encoded by the mango terpene synthase gene TPS4 (mango terpene synthase TPS4), an expression cassette containing the mango terpene synthase gene TPS4, a recombinant expression vector, a recombinant engineered expression bacterium, and the recombinant mango terpene synthase gene TPS4 in regulating the synthesis of terpenoid compounds by TPS. Furthermore, it can also be applied in biosynthetic technologies such as genetic engineering and cell engineering. Moreover, genetic engineering technology can be used to improve mango varieties in order to obtain varieties with special or intense aromas.
[0023] This invention has the following advantages: Addressing the current lack of comprehensive and systematic research on the terpene synthase gene family in mangoes, this invention is the first to clone and verify the terpene synthase gene TPS4 from the Sacred Heart mango genome. TPS4 is capable of simultaneously catalyzing the synthesis of monoterpenes and sesquiterpenes from three substrates: GPP, NPP, and FPP. This gene is a key gene in the final step of the biosynthetic pathway of mango terpenes, providing an important molecular biological basis and theoretical foundation for future use of genetic engineering or metabolic engineering techniques to improve the aroma quality of mangoes and cultivate high-quality aromatic varieties. It has broad application prospects and significant economic value. Attached Figure Description
[0024] Figure 1 This is a TIC diagram of the GPP reaction product catalyzed by TPS4 protease in Example 3;
[0025] Figure 2 This is a TIC diagram of the NPP reaction product catalyzed by TPS4 protease in Example 3;
[0026] Figure 3 This is a TIC diagram of the product of the TPS4 protease-catalyzed FPP reaction in Example 3;
[0027] Figure 4 This is a TIC diagram of the enzymatic GPP reaction product of the negative control in Example 3;
[0028] Figure 5 This is a TIC diagram of the enzymatic NPP reaction product of the negative control in Example 3;
[0029] Figure 6 This is a TIC diagram of the enzymatically catalyzed FPP reaction product of the negative control in Example 3. Detailed Implementation
[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.
[0031] Example 1: Cloning of the mango terpene synthase gene TPS4
[0032] Based on the previous method of combining PacBio SMRT Iso-seq sequencing on the PacBio Sequel II platform with Illumina NovaSeq sequencing on the Illumina NovaSeq 6000 platform, a full-length transcriptome of a mixture of pulp, peel, and leaves of *Saccharum semperflorens* was constructed. A pair of specific primers (PF forward primer: 5'-cgcggcagccatATGTCTCTTCAAGTTTCAGC-3' and PR reverse primer: 5'-gtggtggtgctcgagCTATATTGACACAGGGTCAA-3', as shown in SEQ ID NO:3 (without the linker cgcggcagccat) and SEQ ID NO:4 (without the linker gtggtggtgctcgag), respectively) were designed according to the splicing sequence. Total RNA was extracted from the pulp of *Saccharum semperflorens* using the Tiangen RNAprep Pure Kit, and cDNA was synthesized by reverse transcription. The full-length transcriptome of the sample obtained by reverse transcription was amplified from the cDNA using the aforementioned primers PF and PR, as shown in SEQ ID NO:3 (without the linker cgcggcagccat) and SEQ ID NO:4 (without the linker gtggtggtgctcgag), respectively. IDNO:1 shows the CDS sequence of the mango terpene synthase gene TPS4, which catalyzes the formation of terpenoid compounds in mangoes. The full-length CDS sequence of the mango terpene synthase gene TPS4 is 1677 bp.
[0033] The nucleotide sequence of the mango terpene synthase gene TPS4 is shown below:
[0034] TAG As shown in SEQ ID NO:1;
[0035] The amino acid sequence of the protein encoded by the mango terpene synthase gene TPS4:
[0036] *, as shown in SEQ ID NO:2.
[0037] Note: The underlined codon is the stop codon.
[0038] The specific steps for obtaining the mango terpene synthase gene TPS4 are as follows:
[0039] (1) Cool and grind fresh Sacred Heart Mango pulp with liquid nitrogen, weigh 200mg of powder and add it to a 2mL centrifuge tube pre-cooled with liquid nitrogen, add the lysis buffer from the Tiangen RNAprep Pure Kit total RNA reagent kit, vortex mix and let stand for 10min.
[0040] (2) Extract total RNA from the pulp according to the instructions of the Tiangen RNAprep Pure Kit;
[0041] (3) Using total RNA extracted from the pulp of Sacred Heart Mango as a template, the first strand of cDNA was synthesized by reverse transcription using the PrimeScript™ RT Master Mix reverse transcriptase kit (purchased from Takara Bio Engineering Dalian Co., Ltd.). The reaction conditions were carried out according to the kit instructions.
[0042] (4) The plant CDS sequence of the mango terpene synthase gene TPS4 was amplified from the cDNA obtained by reverse transcription of RNA using the above primers PF and PR.
[0043] (5) Reaction conditions: 95°C pre-denaturation for 3 min; 95°C for 30 sec, 55°C for 30 sec, 72°C for 2 min, 35 cycles; 72°C extension for 10 min, the solution system is added according to the manufacturer's instructions;
[0044] (6) The PCR products obtained by amplification are extracted by gel extraction. For specific gel extraction methods, please refer to the manufacturer's instructions for the GelExtraction Kit (Omega).
[0045] Example 2: Construction and transformation of mango TPS4 expression vector
[0046] Following the construction strategy of Nadler C et al. (2012): the base vector was pET21a (Novagen, catalog number: 69770-3), the insert fragment was MBP-8His-TeV protease cleavage site, the MBP gene (malE) was from Escherichia coli, the 8×His tag was used, the TeV protease cleavage site sequence was ENLYFQ / G, and the cloning site was BamHI / EcoRI, etc., to first construct the PSJ8 prokaryotic expression vector; then, it was linearized by enzyme digestion, the enzyme digestion sites were NdeI and XhoI (purchased from NEB), and the enzyme digestion reaction system was (50 μL system), as shown in Table 1 below.
[0047] Table 1 Enzyme digestion reaction system
[0048]
[0049] The enzyme digestion reaction products were subjected to gel extraction at 37°C for 1 hour and 65°C for 20 minutes. The specific method for gel extraction was as described in the manufacturer's instructions for the Gel Extraction Kit (Omega).
[0050] The recovered target product was ligated into the linearized expression vector PSJ8 using In-Fusion cloning (Clontech), transformed into E. coli competent cells BL21(DE3), positive clones were screened and sequenced to obtain the required full-length gene. The recombinant expression vector containing the target gene was named PSJ8-TPS4. An equal volume of 50% sterilized glycerol was added to the obtained positive strain to obtain a bacterial culture, which was then mixed thoroughly and stored at -80°C.
[0051] Example 3: Functional Verification of the TPS4 Gene in Mango
[0052] To analyze the catalytic function of the mango gene TPS4, the recombinant strain obtained in Example 2 was induced to express the crude enzyme. The gene function was verified by feeding the gene with the precursor compounds GPP, NPP, and FPP, as well as by detecting the synthesis of the product GGPP.
[0053] The specific steps are as follows:
[0054] The recombinant strain obtained in Example 2 was induced to express:
[0055] (1) The bacterial culture preserved in Example 2 was inoculated into 50 mL LB medium at 1% by volume (with 0.1% by volume of 50 mg / mL kana working solution added), and cultured overnight at 37°C with shaking at 200 rpm.
[0056] (2) The overnight culture was inoculated into 1 L LB medium at 1% volume (with 0.1% volume of 50 mg / ml Kana working solution added), and cultured in a shaker at 200 rpm at 37°C for 2-4 h until the bacterial culture OD was reached. 600 =0.6~1.0;
[0057] (3) Cool the bacterial culture to room temperature, add IPTG inducer to a final concentration of 1 μM, and culture at 20°C and 200 rpm for 10-12 h with shaking.
[0058] (4) Centrifuge the bacterial culture at 4°C and 14,000 rpm for 10 min in an ultra-high speed low temperature refrigerated centrifuge and collect the bacterial cells;
[0059] (5) Add 10 mL of pre-cooled Lysis Buffer to resuspend and wash the cells, centrifuge at 4°C and 14,000 rpm for 10 min in an ultra-high speed low temperature refrigerated centrifuge, and collect the cells;
[0060] (6) Add 100 mL of pre-cooled Lysis Buffer to resuspend the bacterial cells;
[0061] (7) Place the resuspended bacterial cells in an ice water bath and sonicate them (40Hz, sonication for 5s, interval for 2s, for 10min).
[0062] (8) Centrifuge at 4°C and 14,000 rpm for 30 min in an ultra-high speed low temperature refrigerated centrifuge, repeat the centrifugation once, and take the supernatant as crude enzyme;
[0063] (9) The blank vector PSJ8 was transformed into the BL21(DE3) strain and the same induction treatment was performed. The crude enzyme obtained was used as a negative control.
[0064] The steps for the in vitro functional verification of the crude enzyme are as follows: The total reaction volume is 5 mL, to which 10 μM·L⁻¹ substrate (GPP, NPP, FPP and GGPP were added respectively, purchased from Echelon Biosciences) and 2 mL of mango TPS4 crude enzyme were added, and the reaction buffer was added to the total volume of 5 mL; the blank vector PSJ8 was transformed into BL21(DE3) strain and the same induction treatment was performed. The resulting crude enzyme was used as a negative control. 2 mL of this crude enzyme was added to the negative control, and the other components were the same as above.
[0065] All reagents were added to gas chromatography-mass spectrometry vials, sealed, and placed in a shaker at 28°C and 150 rpm for 40 minutes.
[0066] The reaction buffer consisted of 15 mM MOPSO, 13% (v / v) glycerol, 1 mM ascorbic acid, 1 μL·mL⁻¹ Tween 20, 1 mM magnesium chloride, and 2 mM DTT, adjusted to pH 7.5.
[0067] After the reaction was complete, the solid-phase microextraction fiber (Supelco 50 / 30μm; CAR / PDMS / DVB) was inserted into a gas chromatography-mass spectrometry (GC-MS) vial, and extraction was performed at room temperature for 30 min. The products were then detected by GC-MS.
[0068] GC-MS conditions were as follows: Gas chromatography (Agilent GC7890B; Agilent MSD5977A) using an HP-5ms (Agilent) capillary column (30m × 0.25mm inner diameter with 0.25μm film thickness). Helium was used as the carrier gas at a flow rate of 1mL / min and a split ratio of 20:1. The injection port temperature was 250°C, the ion source temperature was 230°C, and the quadrupole temperature was 150°C. The temperature program was as follows: initial temperature 60°C, hold for 1 minute, ramp to 120°C at a rate of 4°C / min; then ramp to 200°C at a rate of 5°C / min and hold for 3 minutes. Ionization was performed using EI with an electron energy of 70 eV. The mass scan range was 35–500 m / z.
[0069] The GC-MS results were analyzed using Masshunter software, and the gaseous products were matched against the NIST14.L library. Substances with a matching score of 85 or higher and a relative content of more than 1% were selected and listed in Table 1.
[0070] TIC diagrams of the products of TPS4 protease-catalyzed GPP, NPP, and FPP reactions are shown below. Figure 1-3 As shown, the TIC diagrams of the enzyme-catalyzed GPP, NPP, and FPP reaction products of the negative control are as follows. Figure 4-6 As shown.
[0071] From Table 2 and Figure 1-3 It can be seen that in heterologous expression analysis:
[0072] TPS4 protein can catalyze the production of three monoterpenes from GPP: γ-Terpinene, β-Phellandrene, and α-Phellandrene. The major product is γ-Terpinene, while the minor products are β-Phellandrene and α-Phellandrene.
[0073] TPS4 protein can catalyze the formation of five monoterpenes from NPP: Sabinene, α-Phellandrene, γ-Terpinene, D-Limonene, and Terpinolene, with D-Limonene and γ-Terpinene being the main products.
[0074] TPS4 protein can catalyze the formation of three monoterpenes from FPP: γ-Terpinene, α-Terpinolene, and Terpinolene, and five sesquiterpenes: Cadinene, Aromandendrene, Alloaromadendrene, Guaiene, and Himachalene.
[0075] TPS4 protein cannot catalyze the formation of corresponding diterpenes from GGPP.
[0076] Table 2. Products of TPS4 protease reaction
[0077]
[0078] The above embodiments are only used to illustrate the present invention, and the scope of protection of the present invention is not limited to the above embodiments. Those skilled in the art can achieve the purpose of the present invention based on the above disclosure. Any improvements and modifications made based on the concept of the present invention fall within the scope of protection of the present invention, and the specific scope of protection is determined by the claims.
Claims
1. A mango terpene synthase gene TPS4, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:
1.
2. The mango terpene synthase gene TPS4 according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the mango terpene synthase gene TPS4 is shown in SEQ ID NO:
2.
3. The method for preparing the mango terpene synthase gene TPS4 according to claim 1, characterized in that, Includes the following steps: Total RNA from Sacred Heart Mango pulp was reverse transcribed into cDNA, and cDNA was used as a template for PCR amplification with primer pairs shown in SEQ ID NO:3 and SEQ ID NO:
4.
4. A recombinant expression vector, characterized in that, Includes the mango terpene synthase gene TPS4 and expression vector as described in claim 1.
5. The recombinant expression vector according to claim 4, characterized in that, The expression vector is PSJ8.
6. A recombinant expression engineered bacterium, characterized in that, The mango terpene synthase gene TPS4 described in claim 1 was constructed into the expression vector PSJ8, and BL21 was transformed with the obtained recombinant expression vector. Positive strains were screened to construct engineered Escherichia coli, and recombinant engineered expression bacteria were obtained.
7. A recombinant mango terpene synthase TPS4, characterized in that: This includes transforming host cells with the recombinant expression vector of claim 5, culturing the transformants, and obtaining mango recombinant terpene synthase TPS4 from the culture.
8. The application of the mango terpene synthase gene TPS4 of claim 1, the mango terpene synthase TPS4 of claim 2, the recombinant expression vector of claim 4 or 5, the recombinant expression engineered bacteria of claim 6, or the recombinant mango terpene synthase TPS4 of claim 7 in regulating the synthesis of terpenoid compounds from geraniol diphosphate (GPP), neroli diphosphate (NPP), and farnesyl diphosphate (FPP).