CN116694679A - Method for blocking metabolic pathway of tobacco endogenous cembratriene diol compound - 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 tobacco endogenous cembrane triene diol compounds. According to the method, a CRISPR/Cas9 technology is adopted to knock out a tobacco cembratrienol synthase (CBTS) gene, so that synthesis and accumulation of terpenoid compounds such as cembratriene diol, cembratriene monool and the like are blocked. The method can not only block the anabolism of the terpenoid in the tobacco and reduce the interference of endogenous secondary metabolism on the heterologous artificial metabolic pathway, but also provide sufficient precursor substances for the anabolism of the heterologous terpenoid. The method and the tobacco 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 tobacco endogenous cembratriene diol compound

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 tobacco endogenous cembrane triene diol compounds.

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, whereas PT and TPS expressed in different tissues, different developmental stages 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. Tobacco (Nicotiana tabacum) 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 a tobacco genome, so that tobacco can be used as a natural active substance required by the heterologous synthesis of host plants, and the novel high-value terpenoid synthesis pathway genes have important academic, economic and social values.

The stem, leaf, flower surface of tobacco is distributed with a large number of multicellular glandular hairs, in which a large amount of cembratriene diol (cembratriene-diol) is synthesized and accumulated, and its metabolic pathway has been resolved: geranylgeranyl pyrophosphate (GGPP) synthesized by MEP metabolic pathway in plastid synthesizes cembratrienol under cembratrienol synthase (CBTS) catalysis, and then cytochrome P450 monooxygenase catalyzes to generate cembratrienol. When tobacco is used as a receptor plant and high-value terpenoids are synthesized in a heterologous way through constructing an artificial metabolism way, the metabolism way of endogenous cembrane diterpenoid compounds consumes most of IPP and DMAPP precursor substances in plant cells, so that the supply of the precursor of the heterologous metabolism way is insufficient, the yield of target compounds is obviously influenced, a large amount of cembrane triene diol and cembrane triene monool are mixed in the extract, the difficulty of separating, extracting and purifying the target metabolites is increased, and the production cost is increased.

According to the analyzed cembratriene diol biosynthesis pathway information, the CRISPR/Cas9 system is used for editing the metabolic pathway key enzyme cembratrienol synthase gene to block the anabolism of the cembratriene diol compound, so that a chassis material suitable for the heterologous production of high-value terpenoid compounds is created, the important value is provided for developing plant synthesis biology research, and a foundation is laid 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 tobacco endogenous cembratriene diol compounds, which is realized by mutating cembratrienol synthase genes, wherein the cembratrienol synthase genes have sequences shown in any one of SEQ ID NO. 1-3.

Further, the mutation is to add, substitute or delete one or more bases from the nucleotide sequence shown in SEQ ID NO. 1-3.

Still further, the cembratrienol synthase gene was mutated using CRISPR/Cas9 technology.

Furthermore, a target sequence based on a CRISPR/Cas9 system is designed aiming at a cembratrienol synthase 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, and the carrier is converted into tobacco to block the metabolic pathway of the endogenous cembrane triene diol compound of the tobacco.

Furthermore, the target sequence is shown as SEQ ID NO. 4-5.

Furthermore, the primer sequence of the target point shown in SEQ ID NO.4 is shown in SEQ ID NO.6-7, and the primer sequence of the target point shown in SEQ ID NO.5 is shown in SEQ ID NO. 8-9.

Further, transferring the vector into escherichia coli for amplification and propagation to obtain positive recombinant bacteria, extracting recombinant plasmids in the positive recombinant bacteria, transferring the recombinant plasmids into agrobacterium to obtain agrobacterium containing the recombinant plasmids, and introducing the recombinant plasmids into tobacco plants through an agrobacterium-mediated genetic transformation system.

Still further, the step of introducing the recombinant plasmid into a tobacco plant by an agrobacterium-mediated genetic transformation system comprises: and infecting tobacco leaves by using the agrobacterium, co-culturing, screening resistant buds on a kanamycin-containing screening medium, and transferring the resistant buds to a rooting medium to induce the growth of complete transgenic plants.

Still further, the E.coli is E.coli strain DH 5. Alpha.

Still further, the agrobacterium is agrobacterium strain LBA4404.

The invention has the following beneficial effects:

the invention provides a method for creating a tobacco chassis material suitable for heterologous synthesis of terpenoid, which uses CRISPR/Cas9 technology to knock out tobacco cembratrienol synthase gene and blocks the synthesis way of endogenous cembrane triene diol. The obtained transgenic tobacco plants can not synthesize and accumulate compounds such as cembratriene glycol, cembratriene monool and the like, and can obviously reduce the influence on heterologous metabolic pathways. 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 is a nucleotide sequence alignment of tobacco CBTS1, CBTS2, CBTS 3.

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

FIG. 3 shows the plasmid map of pCBTS-2.

FIG. 4 shows a pY182 plasmid map.

FIG. 5 is the CBTS gene of transgenic tobacco (pY 182-3 and pY 182-4) was edited.

FIG. 6 shows that the sibutrene diol content in transgenic tobacco (pY 182-3 and pY 182-4) was 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

A total of 3 cembratrienol synthase (CBTS) genes, designated CBTS1, CBTS2 and CBTS3, were found in the tobacco genome, with nucleotide sequence similarity of about 95% (FIG. 1). According to the sequence comparison result, 2 targets are designed in exons of a conserved region, namely crCBTS-1:5'-ATGGTCACCCCAAATTGTTG-3' (SEQ ID NO. 4) and crCBTS-2:5'-AAGAAGAATGAATCGAGCAA-3' (SEQ ID NO. 5). Forward and reverse primers were designed according to each target. Wherein the nucleotide sequences of the forward and reverse primers of the crCBTS-1 are shown as SEQ ID NO.6 and SEQ ID NO.7, and the nucleotide sequences of the forward and reverse primers of the crCBTS-2 are shown as SEQ ID NO.8 and SEQ ID NO. 9.

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 pCBTS-1 and pCBTS-2, respectively. The carrier structure is shown in fig. 2-3.

The pCBTS-1 and pCBTS-2 plasmids were constructed as CRISPR/Cas9 gene editing vectors. 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 pICH86966 (Addgene Plasmid # 48075), 1. Mu.L of pNPTII (Addgene Plasmid # 165836), 1. Mu.L of pEPOR1CB0002 (Addgene Plasmid # 117543), pCBTS-1 1. Mu.L, pCBTS-2 1. Mu.L. 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 pY182 (FIG. 4).

Example 2: transgenic tobacco cultivation

mu.L of pY182 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.

Tobacco (Nicotiana tabacum) variety K326 dry seeds were soaked in 75% ethanol for 2 minutes, added with 20% hydrogen peroxide solution for 10 minutes, washed 4 times with sterile water, and sown onto 1/2MS (containing 15g/L sucrose) solid medium for 2 months under dark conditions of 25℃and 16h light/8 h. The leaves of the seedlings are cut into small pieces of 0.5X0.5 cm, soaked in an agrobacterium infection solution for 15 minutes, transferred to a co-culture medium (MS medium+30 g/L sucrose+2 mg/L6-BA+8 g/L Agar) for culturing for 48 hours in the absence of light, transferred to a screening medium (MS medium+30 g/L sucrose+2 mg/L6-BA+50 mg/L kanamycin+8 g/L Agar) for culturing for 4-8 weeks under dark conditions at 25 ℃ for 16 hours. 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: CBTS gene mutation tobacco material screening

100mg of tobacco leaf material is fully ground in liquid nitrogen, transferred into a 1.5mL centrifuge tube, added with 1mL of DNA extract (100mM Tris,2M NaCl,2% CTAB,2% PVP) and mixed evenly, placed at 65 ℃ for 15min, centrifuged at 12000rpm for 10min, and the precipitate is 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 seqCBTS-F:5'-ATGAGTCAATCAATTTCTCCA-3' (SEQ ID NO. 10) and seqCBTS-R:5'-ATTGAAATGATATGCTAATCCCA-3' (SEQ ID NO. 11) amplified CBTS gene fragment. 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. 5, the CBTS gene loci of transgenic tobacco lines pY182-3 and pY182-4 were subject to editing events.

Example 4 tobacco Selaginella three ene glycol Compound detection

100mg of tobacco leaf material was thoroughly ground in liquid nitrogen, transferred to a 1.5mL centrifuge tube, 1mL of n-hexane 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: the temperature is kept at 60 ℃ for 3min, the temperature is increased to 280 ℃ at 10 ℃/min, the temperature is kept for 5min, and 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. 6, the content of cembratriene diol in transgenic tobacco pY182-3 and pY182-4 was significantly 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 (10)

1. A method for blocking the metabolic pathway of an endogenous cembratrienediol compound of tobacco, characterized in that it is carried out by mutating cembratrienol synthase gene, said cembratrienol synthase gene having the sequence as shown in any one of SEQ ID nos. 1-3.

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

3. The method of claim 2, wherein the cembratrienol synthase gene is mutated using CRISPR/Cas9 technology.

4. A method according to claim 3, characterized in that a target sequence based on a CRISPR/Cas9 system is designed for a cembratrienol synthase gene, 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 CRISPR/Cas9, the carrier is transformed into tobacco, and the blocking of the metabolic pathway of the tobacco endogenous cembratriene diol compound is realized.

5. The method of claim 4, wherein the target sequence is set forth in SEQ ID No. 4-5.

6. The method of claim 5, wherein the primer sequence of the target represented by SEQ ID NO.4 is shown as SEQ ID NO.6-7 and the primer sequence of the target represented by SEQ ID NO.5 is shown as SEQ ID NO. 8-9.

7. The method according to claim 6, wherein the vector is transferred into escherichia coli for amplification and propagation to obtain a positive recombinant bacterium, a recombinant plasmid in the positive recombinant bacterium is extracted, the positive recombinant bacterium is transferred into agrobacterium to obtain agrobacterium containing the recombinant plasmid, and the recombinant plasmid is introduced into a tobacco plant through an agrobacterium-mediated genetic transformation system.

8. The method of claim 7, wherein the step of introducing the recombinant plasmid into a tobacco plant by an agrobacterium-mediated genetic transformation system comprises: and infecting tobacco leaves by using the agrobacterium, co-culturing, screening resistant buds on a kanamycin-containing screening medium, and transferring the resistant buds to a rooting medium to induce the growth of complete transgenic plants.

9. The method of claim 8, wherein the escherichia coli is escherichia coli strain DH5 a.

10. The method of claim 9, wherein the agrobacterium is agrobacterium strain LBA4404.