A method for blocking the metabolic pathway of endogenous monoterpenes in tomato

Abstract

The application belongs to the technical field of plant molecular biology and plant genetic engineering, and particularly relates to a method for blocking an endogenous monoterpene compound metabolic pathway of tomato. The method knocks out pentenyltransferase CPT1 and terpene synthase TPS20 through CRISPR / Cas9 technology, so as to block the synthesis and accumulation of terpenoids such as β-Phellandrene. The method can not only block the synthesis metabolism of terpenoids in tomato, reduce the interference of endogenous secondary metabolism on the heterologous artificial metabolic pathway, but also provide sufficient precursor substances for the synthesis metabolism of heterologous terpenoids. The method and the created tomato chassis material are suitable for the heterologous production of high-value terpenoids, have important value for carrying out plant synthetic biology research, and lay a foundation for further development of new varieties and industrial application.

Description

Technical Field

[0001] This invention belongs to the fields of plant molecular biology and plant genetic engineering technology, and specifically relates to a method for blocking the metabolic pathway of endogenous monoterpenoid compounds in tomatoes. Background Technology

[0002] Terpenes (also known as isoprene compounds) are a class of naturally occurring products with diverse structures composed of isoprene units, widely found in nature. Most terpenes possess unique biological activities, playing important biological functions in plant growth and development, and in resistance to insects and diseases. Some terpenes, such as artemisinin, paclitaxel, and menthol, are important pharmaceuticals, fragrances, and industrial raw materials, holding significant value for human health and the national economy.

[0003] Based on the number of isoprene units (C5) in their chemical structure, terpenoids are classified into monoterpenes (C5, C6, C7, C8, C9 ... 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), triterpenes (C 30 In plant cells, the precursor compounds isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are synthesized via the mevalonate pathway (MVA pathway) in the cytoplasm and the methyl erythritol-4-phosphate pathway (MEP pathway) in the plastids. Pentenyltransferase (PT) catalyzes the synthesis of geranyl pyrophosphate (GPP, C...) from varying amounts of IPP and DMAPP. 10 ), farnesyl pyrophosphate (FPS, C 15 ) and geraniol geraniol pyrophosphate (GGPP, C 20 Terpenoids, terpene synthase (TPS), catalyze the formation of monoterpenes, sesquiterpenes, and diterpenes with diverse structures. These terpenoids can be directly released into the environment, stored in specific organs, tissues, or cells, or subjected to oxidation, reduction, and glycosylation modifications by cytochrome P450, alcohol dehydrogenases, and glycoside transferases to form structurally diverse derivatives. In plants, the cytoplasmic MVA pathway and the plastid MEP pathway are relatively conserved, while the expression of PT and TPS in different tissues and developmental stages jointly determines the structural diversity, distribution characteristics, and environmental response patterns of plant terpenoids. Therefore, modifying PT and TPS in the plant genome can alter the metabolism of plant terpenoids.

[0004] As secondary metabolites, terpenoids are typically present in low concentrations in plant tissues. Medicinal plants often grow slowly, making it difficult to directly obtain large quantities of valuable terpenoids from them. Synthetic biology, based on the understanding of the operating principles of biological systems and engineering principles, designs and modifies existing life systems in nature, or constructs artificial life devices or systems that do not exist in nature from scratch. This opens up a new and efficient pathway for the research, development, and sustainable utilization and development of plant natural products and plant resources. Tomato ( Solanum lycopersicum Tomato is an important economic crop, characterized by rapid growth, large biomass, and vigorous secondary metabolism. Its leaves and stems are rich in glandular trichomes, which synthesize, store, and secrete large amounts of terpenoids, making it an ideal plant bioreactor. By recombinating and optimizing genes specific to medicinal plants that synthesize high-value terpenoids using synthetic biology techniques, and then introducing them into the tomato genome, tomatoes can be used as a host plant for heterologous synthesis of the necessary natural active substances, possessing significant academic, economic, and social value.

[0005] The stems, leaves, flowers, and young fruits of tomatoes are covered with numerous multicellular glandular trichomes, which synthesize and accumulate large amounts of β-Phellandrene. The metabolic pathway of this process has been elucidated: the pentenyltransferase CPT1 utilizes IPP and DMAPP precursors to synthesize Neryl Diphosphate (NPP, C 10 The endogenous β-Phellandrene (a precursor of terpenoids) is converted by TPS20 and stored in the cell as β-Phellandrene. When tomato is used as the recipient plant and high-value terpenoids are synthesized heterologously through artificial metabolic pathways, the endogenous β-Phellandrene metabolic pathway not only consumes most of the IPP and DMAPP precursors in the plant cells, resulting in insufficient supply of precursors for heterologous metabolic pathways and significantly affecting the yield of target compounds, but also a large amount of β-Phellandrene is mixed in the extract, increasing the difficulty of separating, extracting and purifying the target metabolites and increasing production costs.

[0006] Based on the resolved information on the biosynthetic pathway of β-Phellandrene, this invention uses the CRISPR / Cas9 system to edit the CPT1 and TPS20 genes, blocking the biosynthetic metabolism of β-Phellandrene and creating a chassis material suitable for heterologous production of high-value terpenoids. This is of great value for conducting research in plant synthetic biology and lays the foundation for further development of new varieties and industrial applications. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for blocking the metabolic pathway of endogenous monoterpenoid compounds in tomatoes, which is achieved by reducing the expression levels of pentenyltransferase CPT1 and monoterpenoid synthase TPS20 or blocking the production of pentenyltransferase CPT1 and monoterpenoid synthase TPS20.

[0008] Furthermore, the sequences of the pentenyltransferase CPT1 gene and the monoterpene synthase TPS20 gene are shown in SEQ ID NO.1-2, respectively.

[0009] Furthermore, the CPT1 gene of pentenyltransferase and the TPS20 gene of monoterpene synthase were mutated to block the metabolic pathway of endogenous monoterpenoid compounds in tomatoes.

[0010] Furthermore, the mutation is the addition, substitution, or deletion of one or more bases in the nucleotide sequence shown in SEQ ID NO.1-2.

[0011] Furthermore, the CRISPR / Cas9 technology was used to mutate the pentenyltransferase CPT1 gene and the monoterpene synthase TPS20 gene.

[0012] Furthermore, target sequences based on the CRISPR / Cas9 system were designed for the pentenyltransferase CPT1 gene and the monoterpene synthase TPS20 gene. Primers were designed for the target sequences, and target adapters were obtained after primer denaturation and annealing. The target adapters were then ligated into a vector carrying CRISPR / Cas9 and transformed into tomatoes to block the metabolic pathways of endogenous monoterpenes in tomatoes.

[0013] Furthermore, the target sites of the pentenyltransferase CPT1 gene are shown in SEQ ID NO.3-4, and the target sites of the monoterpene synthase TPS20 gene are shown in SEQ ID NO.5-6.

[0014] Furthermore, the primers for the target shown in SEQ ID NO.3 are shown in SEQ ID NO.7-8, the primers for the target shown in SEQ ID NO.4 are shown in SEQ ID NO.9-10, the primers for the target shown in SEQ ID NO.5 are shown in SEQ ID NO.11-12, and the primers for the target shown in SEQ ID NO.6 are shown in SEQ ID NO.13-14.

[0015] Furthermore, the endogenous monoterpenoid compounds include α-Pinene, (+)-2-Carene, and β-Phellandrene.

[0016] The present invention has the following beneficial effects: This invention utilizes CRISPR / Cas9 technology to knock out the tomato pentenyltransferase CPT1 and monoterpene synthase TPS20 genes, blocking endogenous terpenoid compounds and their synthesis pathways. The resulting transgenic tomato plants cannot synthesize or accumulate monoterpenoid compounds, significantly reducing the impact on heterologous metabolic pathways. Simultaneously, this material accumulates a large amount of precursors for the isoprene metabolic pathway, demonstrating the potential for heterologous production of high-value terpenoid compounds. This has significant value for plant metabolic engineering and synthetic biology research, and also possesses broad industrial application prospects and the potential for large-scale development. Attached Figure Description

[0017] Figure 1 This is a map of the pCPT1-1 plasmid.

[0018] Figure 2 This is a map of the pCPT1-2 plasmid.

[0019] Figure 3 This is a map of the pTPS20-1 plasmid.

[0020] Figure 4 This is a map of the pTPS20-2 plasmid.

[0021] Figure 5 This is a map of the pY359 plasmid.

[0022] Figure 6 The CPT1 gene was edited in transgenic tomatoes (pY359-13 and pY359-14).

[0023] Figure 7 The TPS20 gene was edited in transgenic tomatoes (pY359-13 and pY359-14).

[0024] Figure 8 The content of terpenoids such as β-Phellandrene was significantly reduced in transgenic tomatoes (pY359-13 and pY359-14). Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0026] Example 1: Construction, transformation and plasmid extraction of recombinant vectors Based on the tomato genome reference sequence, two target sites were designed in the exons of the pentenyltransferase CPT1 (Solyc08g005680.2.1, SEQ ID NO. 1) and the monoterpene synthase TPS20 (NM_001247700, SEQ ID NO. 2), namely crCPT1-1: 5′-GTTCTTTGGTTCTTCAATGT-3′ (SEQ ID NO. 3), crCPT1-2: 5′-CCTCTGACAGTGTCTGCTCG-3′ (SEQ ID NO. 4), crTPS20-1: 5′-TGCCTAGCTTAGGATGAGAA-3′ (SEQ ID NO. 5) and crTPS20-2: 5′-AGATGACAAGGGATAGAATAA-3′ (SEQ ID NO. 6). Forward and reverse primers were designed for each target site. The nucleotide sequences of the forward and reverse primers for crCPT1-1 are shown in SEQ ID NO. 7 and SEQ ID NO. 8, the nucleotide sequences of the forward and reverse primers for crCPT1-2 are shown in SEQ ID NO. 9 and SEQ ID NO. 10, the nucleotide sequences of the forward and reverse primers for crTPS20-1 are shown in SEQ ID NO. 11 and SEQ ID NO. 12, and the nucleotide sequences of the forward and reverse primers for crTPS20-2 are shown in SEQ ID NO. 13 and SEQ ID NO. 14.

[0027] Dissolve the forward and reverse primers separately in 10 mM Tris (pH 8.0) solution to prepare 20 μM solutions. Mix 20 μL of each primer and denature at 95°C for 5 minutes, then allow to cool naturally to room temperature. The resulting product is the target adapter. Perform enzyme digestion-ligation reactions with the pICSL01009::AtU6p (Addgene Plasmid #46968) plasmid. The reaction mixture consisted of 2 μL 10×T4 DNA Ligase buffer, 1 μL T4 ligase, 1 μL BsaI, 1 μL pICSL01009::AtU6p, and 1 μL target adapter. The reaction conditions were 37°C for 1 hour and 16°C for 24 hours. The reaction product was transformed into *E. coli* DH5α clone strain under the following conditions: 20 μL of ligation product was added to 100 μL of competent cells, gently mixed, and incubated on ice for 30 minutes; the cells were then rapidly heat-shocked in a 42 °C water bath for 90 seconds, immediately placed on ice for 2 minutes; 800 μL of LB broth was added, and the cells were incubated at 37 °C with gentle shaking for 1 hour. The bacterial suspension was centrifuged at 6000 rpm for 1 minute, 700 μL of supernatant was discarded, and the bacterial cells were resuspended and plated on LB agar plates containing spectinomycin (Spec, 100 mg / L). The plates were incubated upside down in the dark for 16 hours. Positive clones were screened using colony PCR. Positive single colonies were selected, and plasmids were extracted and sent for sequencing verification. The correctly constructed plasmids were named pCPT1-1, pCPT1-2, pTPS20-1, and pTPS20-2, respectively. The vector structures are shown below. Figure 1-4 As shown.

[0028] CRISPR / Cas9 gene editing vectors were constructed using pCPT1-1, pCPT1-2, pTPS20-1, and pTPS20-2 plasmids. The reaction mixture consisted of: 2 μL 10×T4 DNA Ligase buffer, 1 μL T4 ligase, 1 μL BsaI, 1 μL pICH86966 (Addgene Plasmid #48075), 1 μL pNPTII (Addgene Plasmid #165836), 1 μL pEPOR1CB0002 (Addgene Plasmid #117543), and 1 μL each of pCPT1-1, pCPT1-2, pTPS20-1, and pTPS20-2 plasmids. The reaction conditions were 37°C for 1 hour followed by 16°C for 24 hours. The reaction product was transformed into *E. coli* DH5α clone strain under the following conditions: 20 μL of ligation product was added to 100 μL of competent cells, gently mixed, and incubated on ice for 30 minutes; the cells were then rapidly heat-shocked in a 42 ℃ water bath for 90 seconds, immediately placed on ice for 2 minutes; 800 μL of LB broth was added, and the cells were incubated at 37 ℃ with gentle shaking for 1 hour. The bacterial suspension was centrifuged at 6000 rpm for 1 minute, 700 μL of supernatant was discarded, the bacterial cells were resuspended, and plated on LB agar plates containing kanamycin (50 mg / L). The plates were incubated upside down in the dark for 16 hours. Positive clones were screened using colony PCR. Positive single colonies were selected, and plasmids were extracted and sent for sequencing verification. The correctly constructed plasmid was named pY359. Figure 5 ).

[0029] Example 2: Transgenic Tomato Breeding Add 5 μL of pY359 plasmid to 100 μL of Agrobacterium LBA4404 competent cells, flash freeze in liquid nitrogen for 2 minutes, incubate at 37°C for 30 minutes, add 1 mL of LB liquid medium, and incubate at 28°C for 3 hours. Spread the culture onto LB agar plates containing 25 mg / L rifampin, 25 mg / L streptomycin, and 50 mg / L kanamycin, and incubate at 28°C for 3 days. Pick a single Agrobacterium colony and inoculate it into 50 mL of LB medium (containing 25 mg / L rifampin, 25 mg / L streptomycin, and 50 mg / L kanamycin) and incubate overnight at 28°C and 220 rpm. Centrifuge the bacterial culture at 8000 rpm for 2 minutes, collect the precipitate, resuspend it in 1 / 2 MS liquid medium, and adjust the OD. 600 =0.6, reserved as infection fluid.

[0030] tomato( Solanum lycopersicumAilsa Craig seeds were soaked in 75% ethanol for 2 minutes, then in 20% hydrogen peroxide solution for 10 minutes, and aseptically washed four times. They were then sown on 1 / 2 MS (containing 15 g / L sucrose) solid medium and cultured at 25 ℃ under 16 h light / 8 h dark conditions for one week. Seedling cotyledons were cut into 0.5 cm segments, soaked in Agrobacterium infection solution for 15 minutes, and transferred to co-culture medium (MS medium + 30 g / L sucrose + 2 mg / L ZT + 8 g / L Agar) and cultured in the dark for 48 hours. They were then transferred to selection medium (MS medium + 30 g / L sucrose + 2 mg / L ZT + 50 mg / L kanamycin + 8 g / L Agar) and cultured at 25 ℃ under 16 h light / 8 h dark conditions for 4-8 weeks. Resistant shoots were subcultured on rooting medium (MS medium + 30 g / L sucrose + 8 g / L Agar) and cultured for 2 weeks. The regenerated seedlings were transplanted into flowerpots and placed in an artificial climate chamber for growth (25 ℃, 16h light / 8h darkness).

[0031] Example 3: Screening of double mutant tomatoes containing pentenyltransferase CPT1 and monoterpene synthase TPS20 100 mg of tomato leaf material was thoroughly ground in liquid nitrogen and transferred to a 1.5 ml centrifuge tube. 1 mL of DNA extraction buffer (100 mM Tris, 2 M NaCl, 2% CTAB, 2% PVP) was added and mixed well. The mixture was incubated at 65°C for 15 min, then centrifuged at 12000 rpm for 10 min, and the precipitate was discarded. 0.5 ml of chloroform was added to the supernatant, mixed well, and centrifuged at 12000 rpm for 10 min. 0.5 ml of isopropanol was added to the supernatant to precipitate the DNA. The mixture was centrifuged at 12000 rpm for 10 min, and the precipitate was dissolved in 100 μL of water.

[0032] PCR detection was performed using 2 × Taq Master Mix (Vazyme). Pentenyltransferase CPT1 was amplified using primer pairs seqCPT1-F: 5'-GAGAGTGGCGAGCGAGATTG-3' (SEQ ID NO. 15) and seqCPT1-R: 5'-CAGTAGAGAATGCAAAAGCAGTG-3' (SEQ ID NO. 16), and monoterpene synthase TPS20 was amplified using primer pairs seqTPS20-F: 5'-GATGGAGAGAATGCTAGCGAG-3' (SEQ ID NO. 17) and seqTPS20-R: 5'-ACCTCTTTTGATTTGCTCATCTC-3' (SEQ ID NO. 18). The PCR reaction mixture consisted of 25 μL of 2 × Taq Master Mix, 2 μL of forward primer, 2 μL of reverse primer, and 1 μL of DNA. PCR conditions were as follows: 95 ℃ for 5 min; 35 cycles of 95 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 60 s; extension at 72 ℃ for 5 min. PCR products were detected by 1% agarose gel electrophoresis. PCR products were recovered for sequencing and sequence alignment analysis.

[0033] like Figure 6 As shown, the CPT1 gene locus in pY359-13 and pY359-14 transgenic tomatoes was edited.

[0034] like Figure 7 As shown, the TPS20 gene locus in pY359-13 and pY359-14 transgenic tomatoes was edited.

[0035] Example 4: Detection of Monoterpenoids in Tomato 100 mg of tomato leaf material was thoroughly ground in liquid nitrogen, transferred to a 1.5 mL centrifuge tube, 1 mL of pentane was added, the mixture was shaken for 2 minutes, centrifuged at 13000 rpm for 1 minute, and the supernatant was collected for GC-MS analysis. Gas chromatography (GC) instrument model: Thermo Trace1300 (HP-5ms: 30m×0.25mm×0.25µm). Mass spectrometry instrument model: Thermo ITQ 900 (EI ion source; ion trap detector). Injection volume: 1 µl. Chromatographic conditions: 50 ℃ for 3 min, ramped to 280 ℃ at 10 ℃ / min, held for 5 min, helium flow rate 1 ml / min. Mass spectrometry conditions: ion source temperature 250 ℃, interface temperature 250 ℃, scan mode acquisition m / z 50-500.

[0036] like Figure 8As shown, the content of terpenoids such as β-Phellandrene was significantly reduced in transgenic tomatoes (pY359-13 and pY359-14).

[0037] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0038] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for blocking the metabolic pathway of endogenous monoterpenoid compounds in tomatoes, characterized in that, This was achieved by reducing the expression levels of pentenyltransferase CPT1 and monoterpene synthase TPS20. The sequences of the pentenyltransferase CPT1 gene and the monoterpene synthase TPS20 gene are shown in SEQ ID NO.1-2, respectively. The endogenous monoterpene compounds include α-Pinene, (+)-2-Carene, and β-Phellandrene. The expression levels of pentenyltransferase CPT1 and monoterpene synthase TPS20 were reduced by mutating the pentenyltransferase CPT1 gene and the monoterpene synthase TPS20 gene using CRISPR / Cas9 technology. The method specifically includes: Target sequences based on the CRISPR / Cas9 system were designed for the pentenyltransferase CPT1 gene and the monoterpene synthase TPS20 gene. Primers were designed for the target sequences, and target adapters were obtained after primer denaturation and annealing. The target adapters were then ligated into a vector carrying CRISPR / Cas9 and transformed into tomatoes to block the metabolic pathway of endogenous monoterpenoid compounds in tomatoes. The target sites of the pentenyltransferase CPT1 gene are shown in SEQ ID NO. 3-4, and the target sites of the monoterpene synthase TPS20 gene are shown in SEQ ID NO. 5-6; the primers for the target site shown in SEQ ID NO. 3 are shown in SEQ ID NO. 7-8, the primers for the target site shown in SEQ ID NO. 4 are shown in SEQ ID NO. 9-10, the primers for the target site shown in SEQ ID NO. 5 are shown in SEQ ID NO. 11-12, and the primers for the target site shown in SEQ ID NO. 6 are shown in SEQ ID NO. 13-14.

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