CN116694673A - Method for blocking metabolic pathway of tomato endogenous monoterpene compounds - Google Patents

Abstract

The invention belongs to the technical fields of plant molecular biology and plant genetic engineering, and particularly relates to a method for blocking metabolic pathways of endogenous monoterpene compounds of tomatoes. The method knocks out the pentenyltransferase CPT1 and the terpene synthase TPS20 by CRISPR/Cas9 technology, thereby blocking the synthesis and accumulation of terpenoid compounds such as beta-Phellandrene. The method can not only block the anabolism of terpenoid in tomato and reduce the interference of endogenous secondary metabolism to heterologous artificial metabolic pathway, but also provide sufficient precursor for anabolism of heterologous terpenoid. The method and the tomato chassis material prepared by the method are suitable for heterologous production of high-value terpenoid, have important value for development of plant synthesis biology research, and lay a foundation for further development of new varieties and industrialized application.

Description

Method for blocking metabolic pathway of tomato endogenous monoterpene compounds

Technical Field

The invention belongs to the technical fields of plant molecular biology and plant genetic engineering, and particularly relates to a method for blocking metabolic pathways of endogenous monoterpene compounds of tomatoes.

Background

Terpenoids (also known as isoprenoids) are a class of natural products widely occurring in nature and having a variety of structures composed of isoprene units. The terpenoid has unique biological activity and plays important biological functions in the processes of plant growth and development, insect resistance, disease resistance and the like. Some terpenoids, such as artemisinin, paclitaxel, menthol, etc., are important pharmaceutical, fragrance and industrial raw materials, and have important values in human health and national economy.

According to the isoprene unit (C) in the chemical structure ₅ ) The number of terpenoids is different and the terpenoids are divided into monoterpenes (C ₁₀ ) Sesquiterpenes (C) ₁₅ ) Diterpene (C) ₂₀ ) Triterpenes (C) ₃₀ ) Etc. In plant cells, the precursor compounds isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are synthesized from the mevalonate pathway in the cytoplasm (MVA pathway) and the methyl erythritol-4-phosphate pathway in the plastid (MEP pathway), and geranyl pyrophosphate (GPP, C) is synthesized by the catalytic action of different amounts of IPP and DMAPP by the Pentenyltransferase (PT) ₁₀ ) Farnesyl pyrophosphate (FPS, C) ₁₅ ) And geranylgeranyl pyrophosphate (GGPP, C) ₂₀ ) Then, terpene synthase (TPS) catalyzes and generates monoterpenes, sesquiterpenes and diterpenoid compounds with different structures. These terpenoids may be released directly into the environment or stored in specific organs, tissues or cells, or oxidized, reduced, glycosidated modified by cytochrome P450, alcohol dehydrogenase, glycosidase, etc., to form structurally diverse derivatives. In plants, the cytoplasmic MVA pathway and the plastid MEP pathway are conserved, and in different tissues, different hairsPT and TPS expressed during the incubation period together determine the structural diversity, distribution characteristics and environmental response pattern of plant terpenoids. Therefore, the metabolism of plant terpenoid can be changed by modifying PT and TPS in the plant genome.

As a secondary metabolite, terpenoid content in plant tissues is usually low, and medicinal plants tend to grow slowly, so that it is difficult to obtain a large amount of terpenoid having important value directly through medicinal plants. The synthetic biology is based on the principle of understanding and engineering the operation rule of biological systems, designs and reforms the existing living system in nature or constructs an artificial living device or system which is not in nature from scratch, and opens up a brand new and efficient way for researching and developing natural products of plants and sustainable utilization and development of plant resources. Tomato (Solanum lycopersicum) is an important cash crop, grows rapidly, has large biomass, is well metabolized secondarily, and leaves and stems are rich in glandular hairs on the surfaces, wherein a large amount of terpenoid compounds are synthesized, stored and secreted, and is an ideal plant bioreactor. The high-value terpenoid synthesis pathway genes specific to medicinal plants are recombined and optimized through a synthesis biological technology, and then introduced into tomato genome, so that tomatoes can be used as natural active substances required by heterologous synthesis of host plants, and the novel recombinant strain has important academic, economic and social values.

The surfaces of stems, leaves, flowers and young fruits of tomato distribute a large number of multicellular glandular hairs in which a large amount of beta-phellandene is synthesized and accumulated, and the metabolic pathways thereof have been resolved: pentenyltransferase CPT1 Synthesis of Neryl Diphosphate Using IPP and DMAPP precursors (NPP, C ₁₀ Units) and then transformed by TPS20. Beta. -Phellandene for storage in cells. When tomato is taken as a receptor plant and an artificial metabolism way is constructed to heterologously synthesize the high-value terpenoid, the endogenous beta-Phellandene metabolic way not only consumes most of IPP and DMAPP precursor substances in plant cells, so that the heterometabolism way precursor is not supplied enough, the yield of the target compound is obviously influenced, a large amount of beta-Phellandene is mixed in the extract, the difficulty of separating, extracting and purifying the target metabolite is increased, and the production cost is increased.

According to the analyzed biosynthesis pathway information of the beta-Phellandrene, CPT1 and TPS20 genes are edited through a CRISPR/Cas9 system, anabolism of the beta-Phellandrene is blocked, a chassis material suitable for heterologous production of high-value terpenoid is created, and the method has important value for developing plant synthesis biology research and lays a foundation for further developing new varieties and industrialized application.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for blocking the metabolic pathway of endogenous monoterpene compounds of tomato, which is realized by reducing the expression quantity of pentenyltransferase CPT1 and monoterpene synthase TPS20 or blocking the production of pentenyltransferase CPT1 and monoterpene synthase TPS20.

Further, the sequences of the pentenyltransferase CPT1 gene and the monoterpene synthase TPS2 gene are sequentially shown as SEQ ID NO. 1-2.

Furthermore, the pentenyltransferase CPT1 gene and the monoterpene synthase TPS2 gene are mutated to block the tomato endogenous monoterpene compound metabolic pathway.

Furthermore, the mutation is to add, substitute or delete one or more bases from the nucleotide sequence shown in SEQ ID NO. 1-2.

Still further, the pentenyltransferase CPT1 gene and the monoterpene synthase TPS2 gene are mutated using CRISPR/Cas9 technology.

Furthermore, a target sequence based on a CRISPR/Cas9 system is designed aiming at a pentenyltransferase CPT1 gene and a monoterpene synthase TPS2 gene, a primer is designed aiming at the target sequence, a target joint is obtained after primer denaturation annealing, the target joint is connected into a carrier carrying CRISPR/Cas9, tomatoes are transformed, and the blocking of the metabolic pathway of endogenous monoterpene compounds of tomatoes is realized.

Furthermore, the target point of the pentenyltransferase CPT1 gene is shown as SEQ ID NO.3-4, and the target point of the monoterpene synthase TPS2 gene is shown as SEQ ID NO. 5-6.

Furthermore, the primer of the target point shown in SEQ ID NO.3 is shown as SEQ ID NO.7-8, the primer of the target point shown in SEQ ID NO.4 is shown as SEQ ID NO.9-10, the primer of the target point shown in SEQ ID NO.5 is shown as SEQ ID NO.11-12, and the primer of the target point shown in SEQ ID NO.6 is shown as SEQ ID NO. 13-14.

Still further, the endogenous monoterpene compounds include α -Pinene, (+) -2-care, and β -Phellandene.

The invention has the following beneficial effects:

the invention uses CRISPR/Cas9 technology to knock out tomato pentenyltransferase CPT1 and monoterpene synthase TPS20 genes, blocks endogenous terpenoid and synthetic pathway, and the obtained transgenic tomato plant can not synthesize and accumulate monoterpenoid, thus obviously reducing the influence on heterologous metabolic pathway. Meanwhile, the precursor substances of the isoprene metabolic pathway in the material are accumulated in a large quantity, so that the material has the potential of heterologously producing high-value terpenoid, has important value for plant metabolic engineering and synthetic biology research, and also has wide industrial application prospect and large-scale development potential.

Drawings

FIG. 1 shows the plasmid map of pCPT 1-1.

FIG. 2 shows the plasmid map of pCPT 1-2.

FIG. 3 is a map of pTPS20-1 plasmid.

FIG. 4 is a map of pTPS20-2 plasmid.

FIG. 5 shows a plasmid map of pY 359.

FIG. 6 shows that the CPT1 gene of transgenic tomato (pY 359-13 and pY 359-14) was edited.

FIG. 7 shows that TPS20 gene of transgenic tomato (pY 359-13 and pY 359-14) was edited.

FIG. 8 shows that the content of terpenoid compounds such as beta-Phellandrene in transgenic tomatoes (pY 359-13 and pY 359-14) is significantly reduced.

Detailed Description

The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.

Example 1: construction, transformation and plasmid extraction of recombinant vectors

2 targets 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) respectively, crCPT-1, according to the tomato genome reference sequence: 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'-AGATGCAAGGGATAGAATAA-3' (SEQ ID NO. 6). Forward and reverse primers were designed according to each target. The nucleotide sequences of the forward and reverse primers of the crCPT-1 are shown as SEQ ID NO.7 and SEQ ID NO.8, the nucleotide sequences of the forward and reverse primers of the crCPT1-2 are shown as SEQ ID NO.9 and SEQ ID NO.10, the nucleotide sequences of the forward and reverse primers of the crTPS20-1 are shown as SEQ ID NO.11 and SEQ ID NO.12, and the nucleotide sequences of the forward and reverse primers of the crTPS20-2 are shown as SEQ ID NO.13 and SEQ ID NO. 14.

Dissolving the forward and reverse primers into 20 mu M solution respectively by using 10mM Tris (pH 8.0), mixing 20 mu L of the forward and reverse primers respectively, denaturing at 95 ℃ for 5 minutes, and naturally cooling to room temperature to obtain the target joint. Each target linker was digested and ligated with pICSL01009: atU6p (Addgene Plasmid # 46968) Plasmid. The reaction system is as follows: 2. Mu.L of 10 XT 4 DNA Ligase buffer, 1. Mu. L T4Ligase, 1. Mu.L of BsaI, 1. Mu.L of pICSL01009: atU6p, 1. Mu.L of target linker. The reaction conditions were 37℃for 1 hour and 16℃for 24 hours. The reaction product was transformed into E.coli DH 5. Alpha. Clone under the following conditions: mu.L of the ligation product was added to 100. Mu.L of competent cells, and the mixture was gently mixed and then ice-bathed for 30 minutes; rapidly placing into a water bath at 42 ℃ for heat shock for 90 seconds, and immediately placing on ice for 2 minutes; 800. Mu.L of LB liquid medium was added thereto, and the mixture was incubated at 37℃for 1 hour with slow shaking. The bacterial liquid was centrifuged at 6000rpm for 1 minute, 700. Mu.L of the supernatant was discarded, and the bacterial cells were suspended and plated on LB plates containing spectinomycin (Spec, 100 mg/L), and were subjected to inverted dark culture at 37℃for 16 hours. And (3) adopting colony PCR to carry out positive clone screening, selecting positive monoclonal colonies, extracting plasmids, and then delivering to sequencing verification. The constructed correct plasmids were named pCPT1-1, pCPT1-2, pTPS20-1 and pTPS20-2, respectively, and the vector structures are shown in FIGS. 1-4.

The pCPT1-1, pCPT1-2, pTPS20-1 and pTPS20-2 plasmids were constructed as CRISPR/Cas9 gene editing vectors. The reaction system is as follows: mu.L of 10 XT 4 DNA Ligase buffer, 1. Mu. L T4Ligase, 1. Mu.L of BsaI, 1. Mu.L of pICH86966 (Addgene Plasmid # 48075), 1. Mu.L of pNPTII (Addgene Plasmid # 165836), 1. Mu.L of pEPOR1CB0002 (Addgene Plasmid # 117543), 1. Mu.L of pCPT1-1, pCPT1-2, pTPS20-1 and pTPS20-2 plasmids, respectively. The reaction conditions were 37℃for 1 hour and 16℃for 24 hours. The reaction product was transformed into E.coli DH 5. Alpha. Clone under the following conditions: mu.L of the ligation product was added to 100. Mu.L of competent cells, and the mixture was gently mixed and then ice-bathed for 30 minutes; rapidly placing into a water bath at 42 ℃ for heat shock for 90 seconds, and immediately placing on ice for 2 minutes; 800. Mu.L of LB liquid medium was added thereto, and the mixture was incubated at 37℃for 1 hour with slow shaking. The bacterial liquid was centrifuged at 6000rpm for 1 minute, 700. Mu.L of the supernatant was discarded, and the bacterial cells were suspended and plated on LB plates containing kanamycin (50 mg/L), and were subjected to inverted dark culture at 37℃for 16 hours. And (3) adopting colony PCR to carry out positive clone screening, selecting positive monoclonal colonies, extracting plasmids, and then delivering to sequencing verification. The correct plasmid was constructed and named pY359 (fig. 5).

Example 2: transgenic tomato cultivation

mu.L of pY359 plasmid was added to 100. Mu.L of Agrobacterium LBA4404 competent cells, quick-frozen with liquid nitrogen for 2 minutes, left at 37℃for 30 minutes, added to 1mL of LB liquid medium, incubated at 28℃for 3 hours, plated on LB plates containing 25mg/L rifampicin, 25mg/L streptomycin, 50mg/L kanamycin, and incubated at 28℃for 3 days. Agrobacterium was selected and inoculated into 50ml of LB medium (containing 25mg/L rifampicin, 25mg/L streptomycin, 50mg/L kanamycin) and cultured overnight at 28℃and 220 rpm. Centrifuging the bacterial liquid at 8000rpm for 2 min, collecting precipitate, re-suspending in 1/2MS liquid culture medium, and adjusting OD ₆₀₀ =0.6, as an infectious agent.

Tomato (Solanum lycopersicum) Ailsa Craig dry seeds were soaked in 75% ethanol for 2 min, soaked in 20% hydrogen peroxide solution for 10min, washed 4 times with sterile water, and sown on 1/2MS (containing 15g/L sucrose) solid medium for 1 week at 25℃under 16h light/8 h dark conditions. The cotyledons of the seedlings are cut into 0.5 cm fragments, soaked in an agrobacterium infection solution for 15 minutes, transferred to a co-culture medium (MS medium+30 g/L sucrose+2 mg/L ZT+8g/L Agar) for culturing for 48 hours in a dark place, transferred to a screening medium (MS medium+30 g/L sucrose+2 mg/L ZT+50mg/L kanamycin+8 g/L Agar) for culturing for 4-8 weeks at 25 ℃ under 16h light/8 h dark conditions. The resistant shoots were excised and subcultured into rooting medium (MS medium+30 g/L sucrose+8 g/L Agar) for 2 weeks. Transplanting the regenerated seedlings into a flowerpot, and placing the flowerpot in a climatic chamber for growth (at 25 ℃ for 16h light/8 h dark).

Example 3: pentenyltransferase CPT1 and monoterpene synthase TPS20 double mutant tomato screening

100mg of tomato leaf material was thoroughly ground in liquid nitrogen, transferred to a 1.5mL centrifuge tube, added with 1mL of DNA extract (100mM Tris,2M NaCl,2% CTAB,2% PVP) and mixed well, placed at 65℃for 15min, centrifuged at 12000rpm for 10min, and the pellet was discarded. 0.5ml of chloroform was added to the supernatant, and the mixture was homogenized, centrifuged at 12000rpm for 10 minutes, and 0.5ml of isopropanol was added to the supernatant to precipitate DNA. Centrifuge at 12000rpm for 10min and the pellet was dissolved in 100. Mu.L water.

PCR detection was performed using a 2 XTaq Master Mix (Vazyme). Using primer pair seqCPT1-F:5'-GAGAGTGGCGAGCGAGATTG-3' (SEQ ID NO. 15) and seqCPT1-R:5'-CAGTAGAGAATGCAAAAGCAGTG-3' (SEQ ID NO. 16) amplifying the pentenyltransferase CPT1, using primer pair seqTPS20-F:5'-GATGGAGAGAATGCTAGCGAG-3' (SEQ ID NO. 17) and seqTPS20-R:5'-ACCTCTTTTGATTTGCTCATCTC-3' (SEQ ID NO. 18) amplified the monoterpene synthase TPS20. The PCR reaction system is as follows: 2 XTaq Master Mix 25. Mu.L, 2. Mu.L forward primer, 2. Mu.L reverse primer, 1. Mu.L DNA. The PCR conditions were: 95 ℃ for 5min;95 ℃ 30s,55 ℃ 30s,72 ℃ 60s,35 cycles; extending at 72℃for 5min. The PCR products were detected by electrophoresis on a 1% agarose gel. And (5) recovering the PCR product for sequencing and sequence alignment analysis.

As shown in FIG. 6, the CPT1 gene loci of pY359-13 and pY359-14 transgenic tomatoes were edited.

As shown in FIG. 7, the TPS20 gene loci of pY359-13 and pY359-14 transgenic tomato were edited.

Example 4 tomato monoterpene detection

100mg of tomato leaf material was thoroughly ground in liquid nitrogen, transferred to a 1.5mL centrifuge tube, 1mL pentane was added, shaken for 2 minutes, centrifuged at 13000rpm for 1 minute, and the supernatant was collected for GC-MS detection. Gas Chromatography (GC) instrument model: thermo Trace1300 (HP-5 ms:30 m.times.0.25 mm.times.0.25 μm). Mass spectrometry instrument model Thermo ITQ 900 (EI ion source; ion trap detector). Sample injection amount: 1 μl. Chromatographic conditions: keeping the temperature at 50 ℃ for 3min, heating to 280 ℃ at 10 ℃/min, and keeping the temperature for 5min, wherein the helium flow rate is 1ml/min. Mass spectrometry conditions: the ion source temperature is 250 ℃, the interface temperature is 250 ℃, and the scan mode acquisition m/z is 50-500.

As shown in FIG. 8, the content of terpenoid compounds such as beta-Phellandrene in transgenic tomatoes (pY 359-13 and pY 359-14) is remarkably reduced.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (9)

1. A method of blocking the metabolic pathway of endogenous monoterpene compounds of tomato, characterized in that it is achieved by reducing the expression level of pentenyltransferase CPT1 and monoterpene synthase TPS20 or blocking the production of pentenyltransferase CPT1 and monoterpene synthase TPS20.

2. The method according to claim 1, wherein the sequence of the pentenyltransferase CPT1 gene and the monoterpene synthase TPS2 gene is shown in SEQ ID NO. 1-2.

3. The method according to claim 2, characterized in that the pentenyltransferase CPT1 gene and the monoterpene synthase TPS2 gene are mutated to block the tomato endogenous monoterpene compound metabolic pathway.

4. A method according to claim 3, wherein the mutation is the addition, substitution or deletion of one or more bases to the nucleotide sequence shown in SEQ ID No. 1-2.

5. The method of claim 4, wherein the pentenyltransferase CPT1 gene and monoterpene synthase TPS2 gene are mutated using CRISPR/Cas9 technology.

6. The method according to claim 5, wherein a target sequence based on a CRISPR/Cas9 system is designed for the CPT1 gene of the pentenyltransferase and the TPS2 gene of the monoterpene synthase, a primer is designed for the target sequence, a target joint is obtained after primer denaturation annealing, the target joint is connected into a carrier carrying the CRISPR/Cas9, tomatoes are transformed, and the blocking of the metabolic pathway of the endogenous monoterpene compounds of the tomatoes is realized.

7. The method according to claim 6, wherein the target of the pentenyltransferase CPT1 gene is shown in SEQ ID NO.3-4 and the target of the monoterpene synthase TPS2 gene is shown in SEQ ID NO. 5-6.

8. The method of claim 7, wherein the primer of the target shown in SEQ ID NO.3 is shown in SEQ ID NO.7-8, the primer of the target shown in SEQ ID NO.4 is shown in SEQ ID NO.9-10, the primer of the target shown in SEQ ID NO.5 is shown in SEQ ID NO.11-12, and the primer of the target shown in SEQ ID NO.6 is shown in SEQ ID NO. 13-14.

9. The method of claim 8, wherein the endogenous monoterpene compounds comprise α -Pinene, (+) -2-Carene, and β -phellandene.