Construction method of multi-gene synergistic expression vector and application of multi-gene synergistic expression vector in improving synthesis of squalene of camellia oleifera

Abstract

The invention provides a construction method of a multi-gene synergistic expression vector and application of the multi-gene synergistic expression vector in improvement of oil tea squalene synthesis, and belongs to the technical field of plant genetic engineering and metabolic engineering. MAPK, PP1 and HMGR gene segments are obtained through an RT-PCR technology, the MAPK, PP1 and HMGR gene segments are cloned to expression cassettes containing independent promoters and terminators respectively, and after Insulator isolation elements are inserted, the expression cassettes are integrated to a pCAMBIA1300 vector through a modular assembly technology. Wherein the HMGR gene contains S47A and T208A mutation sites so as to enhance the enzyme activity. The vector constructed by the invention can cooperatively express a target gene in plant cells, so that accurate regulation and control on the phosphorylation state of HMGR are realized. When the method is applied to tobacco or camellia oleifera callus, the squalene content is effectively increased, the method has the advantages of multi-target regulation and control, efficient and flexible construction, high universality and the like, and an efficient tool is provided for anabolism enhancement of natural products.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering and metabolic engineering technology, specifically to a method for constructing a multi-gene co-expression vector and its application in enhancing the synthesis of squalene in Camellia oleifera. Background Technology

[0002] Squalene, a triterpenoid natural product with important physiological activities, is an unsaturated triterpenoid olefin widely found in animals, plants, and microorganisms. In the pharmaceutical field, squalene, due to its excellent antioxidant properties, can scavenge free radicals and inhibit lipid peroxidation, and is used to prevent cardiovascular disease, improve liver function, and assist in anti-tumor treatment. In the health supplement industry, as a natural immunomodulator, it can enhance the body's resistance and is often used as a nutritional supplement for sub-healthy individuals. In the high-end cosmetics field, squalene has excellent skin affinity and permeability, can repair the skin barrier, and relieve dryness and sensitivity, making it a core ingredient in anti-aging and moisturizing products. With consumers' increasing preference for natural products, the market demand for squalene continues to grow.

[0003] Currently, the main commercial sources of squalene include extraction from deep-sea shark liver and plant-based extraction. Traditional shark liver extraction methods are increasingly restricted by ethical and environmental policies due to limitations related to shark resource conservation, high extraction costs, and the risk of heavy metal residues. In contrast, plant-based squalene offers advantages such as sustainable sources, high safety, and purity, making it a core alternative to shark-derived squalene. Among these, Camellia oleifera Abel, a unique woody oilseed crop in my country, has squalene content of 0.5-2.0 mg / g in its seeds and fruits. Furthermore, Camellia oleifera has a long history of cultivation and a wide range of suitable growing areas, making it a plant-based squalene raw material with significant development potential.

[0004] The biosynthetic pathway of squalene belongs to the isoprene metabolic pathway in plants, and its core regulatory mechanism has been extensively studied. In plant cells, acetyl-CoA is converted into isopentenyl pyrophosphate (IPP) and dimethylpropene pyrophosphate (DMAPP) via the mevalonate pathway (MVA pathway). These two molecules are then catalyzed by farnesyl pyrophosphate synthase (FPS) to form farnesyl pyrophosphate (FPP), which is ultimately condensed by squalene synthase (SQS) to form squalene. In this pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is recognized as the rate-limiting enzyme, and its activity directly determines the metabolic flux of the MVA pathway, thus affecting the accumulation of downstream squalene. The regulation of HMGR activity is multi-layered, including transcriptional expression regulation, post-translational modification, and feedback inhibition. Among these, phosphorylation modification, due to its rapid response, is a key mechanism for short-term regulation.

[0005] Recent studies have revealed that the phosphorylation state of HMGR is synergistically regulated by mitogen-activated protein kinase (MAPK) and protein phosphatase (PP1). MAPK family kinases can inhibit HMGR activity by recognizing specific serine or threonine residues at these sites and catalyzing their phosphorylation; conversely, PP1 phosphatase can relieve this inhibition and restore HMGR activity by removing the phosphate groups at these sites. For example, in Arabidopsis thaliana, AtMAPK3 reduces the activity of AtHMGR1 by phosphorylating the T177 site, while AtPP1 reverses this process through dephosphorylation, forming a dynamic regulatory network. However, the phosphorylation regulation mechanism of HMGR in Camellia oleifera is not fully elucidated, and its interaction patterns with MAPK and PP1, as well as key sites, require further verification, posing a challenge to the precise regulation of squalene synthesis in Camellia oleifera.

[0006] Currently, research on enhancing plant squalene content through genetic engineering primarily focuses on single-gene overexpression strategies, such as individually introducing the SQS or HMGR genes. However, practice has shown that single-gene regulation has significant limitations: on the one hand, overexpression of a single gene easily leads to metabolic imbalance, causing the accumulation of intermediate products with toxicity or feedback inhibition; on the other hand, single-gene expression cannot simulate the synergistic regulatory relationships in natural metabolic networks, making it difficult to overcome the constraints of complex signaling pathways. Furthermore, existing multi-gene expression tools suffer from low construction efficiency and poor expression coordination. Traditional enzyme digestion and ligation methods require multiple stepwise cloning steps, which is not only cumbersome but may also lead to gene silencing due to interference between expression cassettes, severely limiting the application efficiency of metabolic engineering. More critically, there is a severe lack of specific tools for regulating HMGR phosphorylation. Existing technologies mostly rely on exogenous hormones or environmental stress to induce HMGR dephosphorylation, but these methods suffer from drawbacks such as delayed response, strong regulatory nonspecificity, and susceptibility to plant stress damage, making it difficult to achieve precise and efficient regulation of squalene synthesis. Therefore, developing a vector system capable of synergistic expression has become a core requirement for overcoming existing technological bottlenecks. Summary of the Invention

[0007] To address the problems of low efficiency of plant-derived raw materials, unclear regulatory mechanisms, and lack of genetic engineering tools in existing squalene production, this invention provides a method for constructing a multi-gene co-expression vector and its application in improving squalene synthesis in Camellia oleifera. By constructing a multi-gene co-expression vector, the regulatory networks of MAPK, PP1, and mutant HMGR are integrated to achieve precise regulation of the squalene synthesis pathway.

[0008] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0009] A method for constructing a multi-gene co-expression vector includes the following steps:

[0010] (1) MAPK, PP1, and HMGR gene fragments were extracted from Camellia oleifera tissue using RT-PCR technology;

[0011] (2) The extracted MAPK, PP1, and HMGR gene fragments are cloned into their respective expression cassettes, which include a promoter, a gene coding sequence, and a terminator.

[0012] (3) Insert an Insulator isolation element between the three expression boxes;

[0013] (4) The expression cassette with inserted isolation elements is integrated into the pCAMBIA1300 vector backbone using modular assembly technology to obtain a multi-gene co-expression vector.

[0014] Preferably, in step (1), MAPK is MAPK3 from Camellia oleifera, PP1 is the protein phosphatase 1C subtype from Camellia oleifera, and HMGR is the Camellia oleifera HMGR encoding gene with the number TRINITY_DN57861_c3_g1.

[0015] Preferably, the MAPK expression cassette in step (2) is a 35S promoter-MAPK gene-NOS terminator, the PP1 expression cassette is a Ubi promoter-PP1 gene-NOS terminator, and the HMGR expression cassette is a dual CaMV 35S promoter-HMGR gene-NOS terminator. The construction of the MAPK expression cassette, PP1 expression cassette, and HMGR expression cassette includes one or two structures, either tandem or parallel, inserted into a single vector backbone.

[0016] Preferably, the HMGR gene carries at least one phosphorylation site mutation, and the phosphorylation site mutation of the HMGR gene includes one or both of S47A and T208A.

[0017] Preferably, during modular assembly in step (4), the splicing operation is performed using the BsaI restriction site.

[0018] Preferably, the modular assembly in step (4) includes one or both of Golden Gate and Gibson assembly technologies.

[0019] The application of a method for constructing a multi-gene co-expression vector in improving the synthesis of squalene in Camellia oleifera involves introducing the multi-gene co-expression vector into plant cells.

[0020] Preferably, the plant includes one or both of tobacco and camellia oleifera.

[0021] Preferably, the multi-gene co-expression vector is introduced into plant cells using one or both of Agrobacterium-mediated transient transformation and stable transformation.

[0022] Preferably, the Agrobacterium is GV3101.

[0023] The mechanisms of action of each preparation step are as follows:

[0024] I. Molecular Mechanism of Gene Fragment Extraction by RT-PCR

[0025] RT-PCR technology, through the coupling of reverse transcription and PCR amplification, enables the specific extraction of functional fragments of the MAPK, PP1, and HMGR genes from total RNA in Camellia oleifera tissue. Its core principle is as follows: First, using RNA as a template, cDNA is synthesized under the catalysis of reverse transcriptase, converting unstable RNA into a stable, amplifiable DNA form and avoiding interference from non-coding sequences such as introns in genomic DNA. Then, using gene-specific primers, the coding regions of the target genes are exponentially amplified through the extension action of DNA polymerase in the PCR reaction. This step ensures that the obtained gene fragment sequences are faithful to the original transcripts, providing a precise functional template for subsequent expression cassette construction, and is a prerequisite for ensuring the correct protein encoding by the target genes.

[0026] II. The Regulatory Logic of Expression Box Construction

[0027] Expression cassettes, as functional units of gene expression, achieve spatiotemporally specific regulation through the combination of promoters, gene coding sequences, and terminators. In this invention, the MAPK expression cassette uses the 35S promoter, whose broad-spectrum and highly efficient transcriptional activation characteristics drive the sustained expression of MAPK kinase; the PP1 expression cassette uses the Ubi promoter, leveraging its constitutive expression advantage in plant cells to maintain the basal activity level of the phosphatase; and the HMGR expression cassette enhances the transcriptional efficiency of the rate-limiting enzyme through the synergistic effect of two CaMV 35S promoters. The NOS terminator, by recognizing transcription termination signals, ensures correct mRNA cleavage and poly(A) tail addition, improving transcript stability. The independent regulatory modules of the three expression cassettes avoid transcriptional interference between genes, laying the foundation for multi-gene synergistic expression.

[0028] III. Barrier Mechanism of Insulator Isolation Element

[0029] Insulators are DNA sequences that function as chromatin boundaries, their core role being to block the diffusion of regulatory signals between adjacent expression cassettes. In this invention, the inserter inserted between three expression cassettes maintains expression stability through two mechanisms: first, it physically isolates enhancer effects, preventing non-specific activation of adjacent promoters by strong enhancers such as the 35S promoter; second, it inhibits heterochromatin silencing by recruiting chromatin remodeling complexes to maintain open chromatin in the expression cassette region, avoiding gene expression silencing caused by vector integration location effects. The introduction of this element solves the transcriptional interference problem in multi-expression cassette tandem, ensuring that MAPK, PP1, and HMGR are co-expressed in the expected proportions.

[0030] IV. Precise splicing mechanism of modular assembly

[0031] Modular assembly technologies (GoldenGate and Gibson) achieve efficient integration of expression cassettes through sequence-specific recombination. GoldenGate technology relies on the misaligned cleavage characteristics of Type IIS restriction enzymes (such as BsaI), where the recognition sequence separates from the cleavage site, generating custom sticky ends. This allows for the directional splicing of the expression cassette with the pCAMBIA1300 vector backbone, and the final product contains no residual restriction enzyme sites. Gibson assembly, on the other hand, generates single-stranded overhangs using exonucleases, fills the gaps with DNA polymerase, and seals with ligases, achieving seamless splicing. Both technologies support one-step assembly of multiple fragments. Combined with precise design of BsaI cleavage sites, they can efficiently achieve tandem or parallel integration of expression cassettes, significantly improving vector construction efficiency while ensuring the accuracy of the assembled sequence.

[0032] V. Enzyme activity regulation mechanism of HMGR phosphorylation site mutation

[0033] HMGR, the rate-limiting enzyme in squalene synthesis, has its activity negatively regulated by phosphorylation at sites S47 and T208. MAPK kinases catalyze phosphorylation at these sites, leading to conformational changes and inhibiting activity, while PP1 phosphatase can restore activity through dephosphorylation. In this invention, mutations in S47A and T208A, by replacing the hydroxyl amino acids at the phosphorylation sites, block MAPK-mediated phosphorylation, maintaining HMGR in a dephosphorylated state. This site-directed mutation does not affect the function of the enzyme's catalytic core and cofactor binding domains, but it relieves phosphorylation inhibition. Combined with the synergistic effect of PP1 dephosphorylation, it significantly increases the metabolic flux of the squalene synthesis pathway.

[0034] Compared with the prior art, the present invention has the following technical advantages:

[0035] This invention, by constructing a multi-gene co-expression vector system and applying it to the regulation of squalene synthesis in Camellia oleifera, overcomes the limitations of existing plant metabolic engineering technologies. It demonstrates significant advantages in regulatory mechanisms, technical methods, and application value, as detailed below:

[0036] I. Achieving synergistic regulation of the "signal-metabolism" pathway and overcoming the bottleneck of single-gene regulation.

[0037] This invention innovatively integrates MAPK kinase, PP1 phosphatase, and the rate-limiting enzyme HMGR into a single vector system. Through the synergistic expression of these three independent expression cassettes, a dynamic regulatory network of "phosphorylation activation - dephosphorylation deactivation - enzyme activity stabilization" is constructed. Specifically, MAPK and PP1 precisely regulate the activity state of HMGR through reversible phosphorylation modification, while mutant HMGR further amplifies metabolic flux by deactivating phosphorylation inhibition. This multi-target synergistic mechanism perfectly mimics the natural regulatory logic in plants, achieving seamless integration of signaling and metabolic pathways, fundamentally solving the challenge of metabolic network synergy that cannot be overcome by single-gene regulation.

[0038] II. Modular carrier design improves construction efficiency and versatility, overcoming the limitations of traditional carriers.

[0039] This invention employs a modular assembly strategy, designing promoters, gene coding regions, terminators, and isolation elements as independent functional modules, and achieving rapid assembly using efficient assembly technologies such as GoldenGate / Gibson. The introduction of the Insulator isolation element effectively blocks transcriptional interference between expression cassettes, ensuring that each gene is expressed at the expected level. The selection of the pCAMBIA1300 vector backbone endows the system with compatibility for both transient and stable transformations, making it adaptable to various plant systems such as tobacco and camellia. This modular design not only significantly improves vector construction efficiency but also enables flexible, plug-and-play expansion, providing a universal platform for subsequent research on the regulation of other triterpenoids and sterols, solving the problems of cumbersome and poorly versatile traditional vector construction.

[0040] III. Precisely regulate the activity state of HMGR enzyme to avoid non-specific interference from exogenous inducers.

[0041] This invention achieves precise regulation of HMGR enzyme activity through two mechanisms: first, by synergistic expression of MAPK and PP1, endogenous factors dynamically regulate the phosphorylation level of HMGR, avoiding non-specific interference from exogenous inducers; second, by site-directed mutagenesis of HMGR at S47A / T208A, the inhibition of enzyme activity by phosphorylation is directly relieved, ensuring its sustained catalytic activity. This dual strategy of endogenous regulation and site-directed mutagenesis enables precise targeted intervention in the rate-limiting step of squalene synthesis, effectively solving the problems of poor specificity and significant side effects associated with exogenous inducers.

[0042] IV. Adaptable to multiple plant systems and transformation methods, expanding application scenarios and industrialization potential.

[0043] This invention achieves compatibility with multiple plant systems by optimizing the vector framework and transformation strategy: on the one hand, Agrobacterium GV3101 is selected as the mediator, whose broad-spectrum infection capability can efficiently adapt to the transformation needs of tobacco and Camellia oleifera; on the other hand, it simultaneously supports transient and stable transformation, which can meet the needs of rapid functional verification and provide stable genetic cell lines or plants for industrial production. This multi-system adaptability significantly reduces the species limitations for technology promotion and provides a reusable technical framework for the large-scale production of Camellia oleifera squalene and the metabolic engineering of other plant natural products. Attached Figure Description

[0044] Figure 1 The image shows the effect of squalene accumulation and enzyme activity enhancement. Detailed Implementation

[0045] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0046] A method for constructing a multi-gene co-expression vector includes the following steps:

[0047] (1) MAPK, PP1, and HMGR gene fragments were extracted from Camellia oleifera tissue using RT-PCR technology;

[0048] (2) The extracted MAPK, PP1, and HMGR gene fragments are cloned into their respective expression cassettes, which include a promoter, a gene coding sequence, and a terminator.

[0049] (3) Insert an Insulator isolation element between the three expression boxes;

[0050] (4) The expression cassette with inserted isolation elements is integrated into the pCAMBIA1300 vector backbone using modular assembly technology to obtain a multi-gene co-expression vector.

[0051] To make the present invention more fully disclosed, more specific embodiments are described below.

[0052] Example 1: Construction of a multi-gene co-expression vector

[0053] 1. Gene origin and amplification

[0054] Total RNA was extracted from Camellia oleifera tissue, and gene fragments of MAPK3 (from Camellia oleifera), protein phosphatase 1C subtype (PP1), and HMGR (TRINITY_DN57861_c3_g1) were amplified by RT-PCR. The amplification primers contained specific linkage sites that were compatible with modular assembly.

[0055] 2. Expression Box Construction

[0056] (1) The MAPK3 gene was cloned into the "35S promoter-MAPK gene-NOS terminator" expression cassette;

[0057] (2) The PP1 gene was cloned into the "Ubi promoter-PP1 gene-NOS terminator" expression cassette;

[0058] (3) The HMGR gene (containing the S47A / T208A mutation) was cloned into the "double CaMV35S promoter-HMGR gene-NOS terminator" expression cassette;

[0059] (4) Insert an Insulator isolation element between the three expression boxes to avoid transcriptional interference.

[0060] 3. Carrier assembly

[0061] Using GoldenGate assembly technology, three expression cassettes with inserted isolation elements were tandemly integrated into the pCAMBIA1300 vector backbone via the BsaI restriction site to obtain a multi-gene co-expression vector.

[0062] Example 2: Enhanced Verification of Squalene Synthesis

[0063] 1. The multi-gene co-expression vector constructed in Example 1 was introduced into Agrobacterium GV3101, and positive clones were screened.

[0064] 2. Agrobacterium-mediated transient transformation was used to inject tobacco leaves with agro-infiltration.

[0065] 3. Samples were taken 72 hours after treatment, and the squalene content and HMGR enzyme activity were determined by GC-MS. The results are shown in the attached figure. Figure 1 The graph shows the effect of squalene accumulation and enzyme activity enhancement.

[0066] Example 3: Validation of mutant HMGR

[0067] 1. Construct multi-gene co-expression vectors containing wild-type HMGR and mutant HMGR (S47A / T208A), respectively. Other components are the same as in Example 1.

[0068] 2. Two vectors were introduced into tobacco leaves for transient expression, and the phosphorylation status of HMGR and its effect on downstream synthetic pathways were detected.

[0069] 3. Mutant HMGR can relieve phosphorylation inhibition, and its synergistic effect with MAPK and PP1 is more significant, demonstrating the necessity of phosphorylation site mutation.

[0070] Example 4: System applied to Camellia oleifera tissue

[0071] 1. Using Agrobacterium-mediated stable transformation, the vector constructed in Example 1 was introduced into Camellia oleifera callus tissue.

[0072] 2. After 2 weeks of culture, total RNA was extracted and the expression levels of MAPK3, PP1, and HMGR were detected. The three genes were stably and synergistically expressed in Camellia oleifera cells.

[0073] 3. The activation status of squalene synthesis-related pathways in Camellia oleifera callus was detected to verify the applicability of the carrier system to Camellia oleifera.

[0074] The above content should not be construed as limiting the specific implementation of this invention to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of this invention, and all such deductions or substitutions should be considered as falling within the patent protection scope defined by the submitted claims.

Claims

1. A method for constructing a multi-gene co-expression vector, characterized in that, Includes the following steps: (1) MAPK, PP1, and HMGR gene fragments were extracted from Camellia oleifera tissue using RT-PCR technology; (2) The extracted MAPK, PP1, and HMGR gene fragments are cloned into their respective expression cassettes, which include a promoter, a gene coding sequence, and a terminator. (3) Insert an Insulator isolation element between the three expression boxes; (4) The expression cassette with inserted isolation elements is integrated into the pCAMBIA1300 vector backbone using modular assembly technology to obtain a multi-gene co-expression vector.

2. The method for constructing a multi-gene co-expression vector according to claim 1, characterized in that, In step (1), MAPK refers to MAPK3 from Camellia oleifera, PP1 refers to the protein phosphatase 1C subtype from Camellia oleifera, and HMGR refers to the Camellia oleifera HMGR encoding gene with the number TRINITY_DN57861_c3_g1.

3. The method for constructing a multi-gene co-expression vector according to claim 1, characterized in that, The MAPK expression cassette described in step (2) is a 35S promoter-MAPK gene-NOS terminator, the PP1 expression cassette is a Ubi promoter-PP1 gene-NOS terminator, and the HMGR expression cassette is a dual CaMV 35S promoter-HMGR gene-NOS terminator. The construction of the MAPK expression cassette, PP1 expression cassette, and HMGR expression cassette includes inserting one or two of the tandem or parallel structures into a single vector backbone.

4. The method for constructing a multi-gene co-expression vector according to claim 3, characterized in that, The HMGR gene carries at least one phosphorylation site mutation, and the phosphorylation site mutation of the HMGR gene includes one or both of S47A and T208A.

5. The method for constructing a multi-gene co-expression vector according to claim 1, characterized in that, In step (4), modular assembly is performed using the BsaI restriction site for splicing.

6. The method for constructing a multi-gene co-expression vector according to claim 1, characterized in that, The modular assembly in step (4) includes one or both of the following assembly technologies: Golden Gate and Gibson.

7. The application of a method for constructing a multi-gene co-expression vector according to any one of claims 1-6 in improving the synthesis of squalene in Camellia oleifera, characterized in that, Introduce multi-gene co-expression vectors into plant cells.

8. The application of the method for constructing a multi-gene co-expression vector according to claim 7 in improving the synthesis of squalene in camellia oleifera, characterized in that, The plants mentioned include one or both of tobacco and camellia oleifera.

9. The application of the method for constructing a multi-gene co-expression vector according to claim 7 in improving the synthesis of squalene in camellia oleifera, characterized in that, The multi-gene co-expression vector is introduced into plant cells using one or both of Agrobacterium-mediated transient and stable transformation.

10. The application of the method for constructing a multi-gene co-expression vector according to claim 9 in improving the synthesis of squalene in camellia oleifera, characterized in that, The Agrobacterium used is GV3101.

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