A beta-glucosidase gene for increasing the content of tobacco squalene and its application and method

Abstract

The application discloses a beta-glucosidase gene for improving squalene content in tobacco, and application and method thereof, and belongs to the field of genetic engineering.The technical problem to be solved is that the squalene source is limited and the content in plants is low in the prior art.The technical solution points provide a beta-glucosidase gene (BGLU) separated from tobacco, the nucleotide sequence of which is shown in SEQ ID NO:1;the endogenous expression of the gene is enhanced by constructing an overexpression vector for the gene and transforming tobacco.The beneficial effects are that the squalene content in the transgenic tobacco plant is significantly improved compared with the wild type, and a new technical approach is provided for large-scale biosynthesis of squalene.The application is mainly used for cultivating new tobacco materials with high squalene content.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a β-glucosidase gene for increasing the squalene content in tobacco and its application and methods. Background Technology

[0002] Squalene is a linear triterpenoid compound with 30 carbon atoms and a key precursor in the biosynthesis of all sterols. Its unique unsaturated carbon chain structure endows it with excellent antioxidant, anti-inflammatory, and lipophilic biological activities, giving it high commercial value in the pharmaceutical, cosmetic, and health supplement industries. In the pharmaceutical field, squalene is a core ingredient in adjuvants for influenza and HPV vaccines, significantly enhancing the body's immune response to antigens. Simultaneously, its protective effect on hepatocytes and immunomodulatory function make it a core ingredient in high-end liver-protecting and immunity-boosting health supplements. In the cosmetics field, squalene, as a natural moisturizing factor, can quickly penetrate the skin's surface, repair the skin barrier, and maintain the skin's water-oil balance, making it an important additive in high-end skincare products.

[0003] Currently, the main sources of squalene are limited to deep-sea shark liver oil and a few plant oil extractions, which present significant technological bottlenecks and industry challenges. Shark liver oil extraction is the traditional mainstream method, but overfishing has led to a sharp decline in deep-sea shark populations, severely disrupting the marine ecological balance and facing ethical controversies; its sustainability can no longer meet market demand. Plant-derived squalene (such as olive oil, amaranth seed oil, and camellia seed oil) has environmental advantages, but its natural content is extremely low (usually only 0.01%-0.5%), and the extraction process requires multiple chromatography and purification steps, making the process complex, costly, and difficult to achieve large-scale production. Therefore, developing a sustainable, high-yield, and low-cost new biosynthetic pathway for squalene has become a key technological challenge that urgently needs to be overcome in this field.

[0004] Utilizing genetic engineering to modify plants to achieve the targeted synthesis and accumulation of high-value-added terpenoids is one of the important pathways to solve the aforementioned problems. Tobacco, as a model plant, has advantages such as a short growth cycle, large biomass, mature genetic transformation system, and low cultivation cost, making it an ideal host for constructing terpenoid bioreactors.

[0005] Relevant patent documents retrieved: (1) Country of publication: China, Publication number: CN105859364A, Publication date: August 20, 2016. This document discloses a tea seed cake fertilizer that optimizes the physiological environment for squalene biosynthesis by regulating the soil microenvironment for flue-cured tobacco growth, thereby increasing the squalene content in tobacco leaves.

[0006] (2) Country of origin: China, Publication number: CN106007860A, Publication date: October 12, 2016. This document discloses a liquid compound fertilizer that increases the squalene content in tobacco leaves by foliar spraying.

[0007] (3) Country of origin: China, Publication number: CN106045629A, Publication date: October 26, 2016. This document discloses a neem fertilizer, which can increase the content of squalene in tobacco leaves by applying the fertilizer to the roots during the growth process of tobacco.

[0008] Relevant non-patent literature retrieved: (1) Journal title: Tobacco Science and Technology, article title: Effects of overexpression of Panax notoginseng PnSS gene on the content of terpenoids and growth and development of tobacco, Volume No. 11, 2022. This article discloses that the squalene synthase gene (PnSS) of Panax notoginseng is transferred into tobacco, and overexpression of this gene can increase the content of terpenoids such as squalene in tobacco leaves.

[0009] (2) Journal title: Agronomy, article title: Expression of WsSQS and WsSQS2 in Tobacco Divergently Regulates Terpenoid Metabolism and Enhances SqualeneAccumulation, Volume 16, Issue 1, 2026. This article discloses the heterologous expression of the squalene synthase genes WsSQS and WsSQS2 from Withania somnifera in tobacco, which increased the squalene content to 2.05 times and 1.68 times that of the control, respectively.

[0010] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: First, the technical solutions disclosed in patent documents CN105859364A, CN106007860A, and CN106045629A belong to agronomic improvement measures, which promote squalene accumulation by adjusting the soil microenvironment or improving the physiological environment of tobacco growth. Their effects are greatly affected by factors such as soil conditions and climate, making them difficult to stabilize and control, and they fail to improve the squalene synthesis capacity of tobacco from a genetic perspective.

[0011] Second, the technical solutions disclosed in non-patent literature all focus on the squalene synthase gene (SQS) in the squalene synthesis pathway, that is, increasing squalene content by overexpressing heterologous SQS genes. There are no reports in the prior art of using tobacco endogenous non-SQS genes to increase squalene content.

[0012] In summary, there is a lack of existing technologies that utilize endogenous non-SQS genes in tobacco to stably increase squalene content through genetic engineering. Summary of the Invention

[0013] The purpose of this invention is to provide: The invention relates to the application of the tobacco endogenous β-glucosidase gene NtBGLU in increasing tobacco squalene content, and a method for increasing tobacco squalene content, in order to solve technical problems such as low tobacco squalene content, existing improvement schemes being limited to agronomic measures or heterologous SQS gene overexpression, and the lack of technical solutions for genetic improvement using tobacco endogenous non-SQS genes.

[0014] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0015] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0016] Unless otherwise stated, conventional methods within the scope of the art shall be used.

[0017] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0018] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0019] As used in this article, "squalene" refers to a linear triterpenoid compound with 30 carbon atoms and the chemical formula C3. 30 H 50 It is a key precursor in the biosynthesis of sterol compounds in cells and has biological activities such as antioxidant, anti-inflammatory and lipophilic properties.

[0020] The term “β-glucosidase” or “BGLU” used in this article refers to a class of hydrolases belonging to the glycosidic hydrolase family 1 (GH1), which can catalyze the hydrolysis of β-glycosidic bonds, release non-glycosidic ligands, and participate in the synthesis and regulation of plant secondary metabolites.

[0021] The term “NtBGLU” as used in this article refers to the β-glucosidase gene isolated and cloned from tobacco (Nicotiana tabacum), whose cDNA sequence is shown in SEQ ID NO: 1.

[0022] The term "overexpression" as used in this article refers to the process of using genetic engineering techniques to make the expression level of a target gene in the host cell significantly higher than its endogenous expression level.

[0023] The term "vector" as used in this article refers to a tool that can introduce a target gene into a host cell and enable its expression, including but not limited to plasmid vectors and viral vectors.

[0024] The term "pCAMBIA1300" as used in this article refers to a binary vector for plant genetic transformation containing a kanamycin resistance selection marker.

[0025] The technical solution of the present invention: In a first aspect, the present invention provides an isolated nucleic acid molecule.

[0026] The nucleic acid molecule encodes β-glucosidase, and the amino acid sequence of the β-glucosidase is shown in SEQ ID NO:2.

[0027] The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:1.

[0028] Based on a further solution to the technical problem of the present invention, a preferred embodiment of the technical solution provided in the first aspect of the present invention includes: The first preferred embodiment is an isolated nucleic acid molecule whose encoded β-glucosidase amino acid sequence is shown in SEQ ID NO:2. This technical solution solves the technical problem of "providing a nucleic acid molecule encoding β-glucosidase" and limits the scope of protection by specifying the nucleic acid molecule by its amino acid sequence.

[0029] The second preferred embodiment is an isolated nucleic acid molecule whose nucleotide sequence is shown in SEQ ID NO:1. This technical solution, while addressing the technical problem of "providing a nucleic acid molecule encoding β-glucosidase," further provides a specific nucleotide sequence, facilitating gene manipulation and vector construction.

[0030] Secondly, the present invention provides a recombinant vector.

[0031] The recombinant vector contains the nucleic acid molecules described in the first aspect.

[0032] The recombinant vector is a plant binary vector.

[0033] The plant binary vector is selected from at least one of the following: pCAMBIA series vectors, pGreen series vectors, pBI series vectors, pPZP series vectors, pNC series vectors, BIBAC vectors, or TAC vectors.

[0034] The plant binary vector is preferably a pCAMBIA series vector.

[0035] The pCAMBIA series carriers are further preferably pCAMBIA1300, pCAMBIA1301, pCAMBIA2300 or pCAMBIAsuper1300.

[0036] Among them, the pCAMBIA series vector is more preferably pCAMBIA1300.

[0037] Based on a further solution to the technical problem of the present invention, in the technical solution provided in the second aspect of the present invention, a preferred solution includes: The first preferred embodiment is a recombinant vector containing the nucleic acid molecules described in the first aspect. This technical solution, while solving the technical problem of "introducing nucleic acid molecules into plant cells," provides a basic scope of protection.

[0038] The second preferred option is a recombinant vector, specifically a plant binary vector. This solution, while addressing the technical challenge of "introducing nucleic acid molecules into plant cells," further improves the efficiency and stability of genetic transformation.

[0039] The third preferred embodiment is a recombinant vector, wherein the plant binary vector is selected from at least one of the pCAMBIA series vectors, pGreen series vectors, pBI series vectors, pPZP series vectors, pNC series vectors, BIBAC vectors, or TAC vectors. This technical solution, while solving the technical problem of "introducing nucleic acid molecules into plant cells," further provides a variety of optional vector types, expanding the scope of the technical solution's feasibility.

[0040] The fourth preferred option is a recombinant vector, wherein the plant binary vector is a pCAMBIA series vector. This technical solution, based on solving the technical problem of "introducing nucleic acid molecules into plant cells," further optimizes the pCAMBIA series vector.

[0041] The fifth preferred option is a recombinant vector, wherein the pCAMBIA series vector is pCAMBIA1300, pCAMBIA1301, pCAMBIA2300, or pCAMBIAsuper1300. This technical solution, while addressing the technical problem of "introducing nucleic acid molecules into plant cells," further provides a specific selection of pCAMBIA series vectors.

[0042] The sixth preferred embodiment: a recombinant vector, wherein the pCAMBIA series vector is pCAMBIA1300. This technical solution, while solving the technical problem of "introducing nucleic acid molecules into plant cells," further provides a specific vector corresponding to the embodiments, facilitating direct implementation by those skilled in the art.

[0043] Thirdly, the present invention provides a host cell.

[0044] The host cell contains the recombinant vector described in the second aspect.

[0045] The host cell is either Agrobacterium or Escherichia coli.

[0046] The Agrobacterium is selected from at least one of GV3101, LBA4404, EHA105 or EHA103.

[0047] The preferred strain of Agrobacterium is GV3101.

[0048] Based on a further solution to the technical problem of the present invention, a preferred embodiment of the technical solution provided in the third aspect of the present invention includes: The first preferred embodiment is a host cell containing the recombinant vector described in the second aspect. This technical solution provides a basic scope of protection while solving the technical problem of "amplifying and preserving the recombinant vector".

[0049] The second preferred option is a host cell, specifically Agrobacterium. This approach solves the technical problems of "amplifying and preserving the recombinant vector" and further facilitates subsequent transformation of tobacco plants.

[0050] The third preferred embodiment: a host cell, wherein the Agrobacterium is selected from at least one of GV3101, LBA4404, EHA105, or EHA103. This technical solution, while addressing the technical problem of "amplifying and preserving the recombinant vector," further provides a variety of selectable Agrobacterium strains.

[0051] The fourth preferred embodiment: a host cell, wherein the Agrobacterium is GV3101. This technical solution, while addressing the technical problem of "amplifying and preserving the recombinant vector," further provides a specific Agrobacterium strain corresponding to the examples, ensuring transformation efficiency.

[0052] Fourthly, the present invention provides the application of the nucleic acid molecule described in the first aspect, the recombinant vector described in the second aspect, or the host cell described in the third aspect in increasing the squalene content of tobacco.

[0053] The application involves constructing a recombinant vector to transform the nucleic acid molecules into tobacco plants, thereby enabling the nucleic acid molecules to be expressed in the tobacco plants.

[0054] Preferably, the application involves overexpressing the nucleic acid molecule in tobacco plants.

[0055] The recombinant vector used in the application is preferably a plant binary vector.

[0056] The recombinant vector used in the application is further preferably pCAMBIA1300.

[0057] The preferred transformation method used in the application is Agrobacterium-mediated genetic transformation.

[0058] The preferred Agrobacterium used in the application is GV3101.

[0059] Based on a further solution to the technical problem of the present invention, in the technical solution provided in the fourth aspect of the present invention, a preferred embodiment includes: The first preferred option is the application of the nucleic acid molecules described in the first aspect in increasing the squalene content of tobacco. This technical solution, having solved the technical problem of "increasing the squalene content of tobacco," directly uses nucleic acid molecules as the main application subject, thus offering the broadest scope of protection.

[0060] The second preferred option is the application of the recombinant carrier described in the second aspect in increasing the squalene content of tobacco. This technical solution, having solved the technical problem of "increasing the squalene content of tobacco," uses the recombinant carrier as the main application component, facilitating industrial application.

[0061] The third preferred option is the application of the host cells described in the third aspect in increasing the squalene content of tobacco. This technical solution, having solved the technical problem of "increasing the squalene content of tobacco," uses host cells as the primary application method, facilitating large-scale production and transformation.

[0062] The fourth preferred embodiment: the application of the nucleic acid molecule described in the first aspect in increasing the squalene content of tobacco, wherein the nucleic acid molecule is transformed into tobacco plants via Agrobacterium-mediated genetic transformation, and the nucleic acid molecule is overexpressed in the tobacco plants. This technical solution, while solving the technical problem of "increasing the squalene content of tobacco," further specifies the concrete implementation method and transformation method, corresponding to the examples.

[0063] Fifthly, the present invention provides a method for increasing the squalene content in tobacco.

[0064] The method includes the following steps: constructing a nucleic acid molecule encoding β-glucosidase into a recombinant vector, transforming it into tobacco plants, and overexpressing the nucleic acid molecule in the tobacco plants; the amino acid sequence of the β-glucosidase is shown in SEQ ID NO:2.

[0065] The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:1.

[0066] The tobacco is selected from K326, NC89, Yunyan 87 or Cuibi No. 1.

[0067] The tobacco is preferably K326.

[0068] Based on a further solution to the technical problem of the present invention, a preferred embodiment of the technical solution provided in the fifth aspect of the present invention includes: The first preferred embodiment is a method for increasing the squalene content in tobacco, comprising the following steps: constructing a nucleic acid molecule encoding β-glucosidase into a recombinant vector, transforming tobacco plants, and overexpressing the nucleic acid molecule in the tobacco plants; the amino acid sequence of the β-glucosidase is shown in SEQ ID NO:2. This technical solution solves the technical problem of "increasing the squalene content in tobacco" and, by limiting the nucleic acid molecule to its amino acid sequence, provides the broadest protection.

[0069] The second preferred embodiment: the method for increasing the squalene content of tobacco, wherein the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:1. This technical solution, while solving the technical problem of "increasing the squalene content of tobacco," further provides a specific nucleotide sequence, facilitating implementation by those skilled in the art.

[0070] The third preferred embodiment: the method for increasing the squalene content of tobacco, wherein the tobacco is selected from K326, NC89, Yunyan 87, or Cuibi No. 1. This technical solution, while solving the technical problem of "increasing the squalene content of tobacco," further provides tobacco varieties suitable for this method, ensuring the feasibility of the technical solution.

[0071] The fourth preferred embodiment: the method for increasing the squalene content in tobacco, wherein the recombinant vector is pCAMBIA1300, the transformation method is Agrobacterium-mediated genetic transformation, wherein the Agrobacterium is GV3101, and the tobacco is K326. This technical solution, while solving the technical problem of "increasing the squalene content in tobacco," further provides a specific implementation method that completely corresponds to the examples, ensuring the reproducibility of the technical solution's effects.

[0072] The present invention has at least the following beneficial effects: 1. Compared with existing agronomic improvement measures, this invention solves the problem of low squalene content in tobacco from a genetic perspective.

[0073] The agronomic improvement measures disclosed in the background art (CN105859364A, CN106007860A, CN106045629A) promote squalene accumulation by adjusting the soil microenvironment or the physiological environment of tobacco growth through fertilization. However, their effects are greatly influenced by environmental factors such as soil and climate, making them difficult to stabilize and control. This invention, by integrating the NtBGLU gene into the tobacco genome and overexpressing it, genetically enhances the squalene synthesis capacity of tobacco, unaffected by fluctuations in environmental factors.

[0074] 2. Compared with the existing technology of overexpressing heterosqualene synthase gene (SQS), the present invention adopts a completely new technical approach and achieves a higher squalene enhancement. Attached Figure Description

[0075] Figure 1 The relative expression levels of the NtBGLU gene are shown in the empty vector control plant (K326) and the NtBGLU gene overexpression plant (NtBGLU-OE). **, p<0.01.

[0076] Figure 2 This study compares the squalene content in leaves of empty vector control plants (K326) and NtBGLU gene overexpression plants (NtBGLU-OE). ***, p<0.001; ****, p<0.0001; *****, p<0.00001. Detailed Implementation

[0077] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0078] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0079] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0080] Main instruments, reagents and consumables used in this invention The instruments are shown in Table 1: Table 1 Instruments

[0081] Reagents: R014 Trizol Total RNA Extraction Kit (R014-50); Reverse Transcription Kit (Transcriptor First Strand cDNA Synthesis Kit, REF04379012001); DNA Polymerase (PrimeSTAR) ® GXL DNA Polymerase (R050A); Conventional agarose gel DNA recovery kit (Tiangen DP209); Agarose (Beyotime Biotechnology, ST004L); TBE electrophoresis buffer (Yisheng Biotechnology, 60144ES76); Plasmid mini-prep kit (Tiangen, DP103); Restriction endonuclease QuickCut ™ BamHI (Takara1605), QuickCut ™ SacI (Takara1627), QuickCut ™ BsrGI (Solebold Q2470), QuickCut ™ XhoI (Takara 1635).

[0082] Consumables: sterile centrifuge tubes (1.5mL, 50mL); sterile PCR tubes (0.2mL); sterile pipette tips (10μL, 100μL, 1000μL). All consumables are autoclaved to prevent contamination.

[0083] Biological materials: tobacco (K326) seeds (sterilely preserved); Agrobacterium strain GV3101 (Shanghai Weidi Biotechnology Co., Ltd., AC1001L); overexpression vector plasmid pCAMBIA1300 (Newp Biotechnology, V008765).

[0084] Example 1: Obtaining the tobacco β-glucosidase gene (BGLU) Based on the conserved sequence information of the tobacco BGLU gene reported in the public database nicomcs, sequence alignment was performed to identify conserved regions. Specific primers were designed (primer sequences were predicted by software to ensure specificity and avoid non-specific amplification). The full-length cDNA sequence of the NtBGLU gene was cloned from tobacco leaves using RT-PCR technology. PCR amplification conditions were optimized to improve the cloning efficiency of the target gene.

[0085] This invention employs PCR cloning technology. Using total RNA from tobacco leaves as a template, reverse transcription was performed according to the reverse transcription kit instructions to obtain high-quality cDNA (verified by agarose gel electrophoresis to ensure no degradation). PCR amplification was then performed using specific primers. The optimized amplification conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 45 cycles; and a final extension at 72℃ for 10 min to obtain the full-length cDNA sequence of the NtBGLU gene (sequence shown in SEQ ID NO: 1, corresponding amino acid sequence shown in SEQ ID NO: 2). The amplified gene fragment was separated by 1% agarose gel electrophoresis, recovered from the gel, ligated into the pMD19-T vector, transformed into *E. coli* DH5α competent cells, and positive clones were selected for sequencing verification, confirming the sequence was correct and free of base deletions and mismatches.

[0086] SEQ ID NO: 1: ATGACTTTTACCTGCTGGTTTTGCCAATCCCTTGATATTTGGAGACTATCCTGACGTAATGAAGAGGAATGCTGGCTCTAGATTGCCCAAATTCACAAGACAAGAATCTGCGCAAGTTAAAGGCGCTAT AGACTTCATAGCCCTGAACCATTATATGACAGTACATGTCACAGATAGCTCCAGCAGCTTGGAAACTGATATTAGAGACTTCAGTGCTGATGCAGCATTTCAATTTATTTGTAAGATCAATGACTAA.

[0087] SEQ ID NO: 2: MTFTCWFANPLIFGDYPDVMKRNAGSRLPKFTRQESAQVKGAIDFIALNHYMTVHTDSSSSLETDIRDFSADAAFQFICKIND.

[0088] Example 2 Construction of BGLU gene overexpression vector 1. Amplification and acquisition of the target sequence Based on the cloned gene sequence (SEQ ID NO: 1), specific primers were designed, with restriction endonucleases BamHI and SacI restriction sites introduced at both ends of the primers, respectively (the restriction sites were selected with reference to the multiple cloning site of the pCAMBIA1300 vector to avoid affecting the function of the vector promoter and terminator). Protective bases were added to the 5' end of the primers to verify the correct pMD19-T- sequence. NtBGLU Using the recombinant plasmid as a template, PCR amplification was performed under the same conditions as in Example 1 to obtain the full-length target fragment of the BGLU gene (SEQ ID NO: 1), which was then recovered by gel extraction for later use.

[0089] 2. pCAMBIA1300- NtBGLU Carrier construction The amplified target fragment and the overexpression vector pCAMBIA1300 (kanamycin resistant) were double-digested with restriction endonucleases BamHI (located at the 5' end) and SacI (located at the 3' end), respectively. The digestion system consisted of 3 μL DNA, 5 μL 10×QuickCut Buffer, 1 μL QuickCut BamHI, 1 μL QuickCut SacI, and 40 μL sterile water. The digestion products were separated by 1% agarose gel electrophoresis. The target fragment and linearized vector were recovered using a gel extraction kit. The concentrations were then measured using a nucleic acid quantification instrument to ensure a molar ratio of target fragment to vector of 3:1.

[0090] Using T4 DNA ligase, the recovered target fragment was ligated between the BamHI and SacI restriction sites of the pCAMBIA1300 vector. The ligation conditions were: 2 μL 10× ligation buffer, 2 μL pCAMBIA1300 vector, 1 μL target DNA fragment, 1 μL T4 DNA ligase, and 15 μL sterile water, incubated at 16°C for 5 hours. To ensure efficient ligation, the recombinant plasmid was constructed and named pCAMBIA1300- NtBGLU .

[0091] 3. Identification of recombinant plasmids The ligation product was transformed into *E. coli* competent cells DH5α using the heat shock transformation method. The cells were plated on LB medium containing kanamycin (50 mg / L) and incubated overnight at 37°C. Single colonies were selected and inoculated into LB liquid medium (containing 50 mg / L kanamycin), and cultured at 37°C with shaking at 200 rpm for 8 h. PCR verification was performed using the full-length primers for the BGLU gene (F-terminus: ATGACTTTTACCTGCTGGT (SEQ ID NO: 3), R-terminus: TTAGTCATTGATCTTACAAA (SEQ ID NO: 4)). Positive clones amplified the target band of the expected size.

[0092] Plasmids from positive clones were extracted and double-digested using BsrGI and XhoI restriction endonucleases located outside the insertion site on the pCAMBIA1300 vector. The digestion products were detected by 1% agarose gel electrophoresis, and the band size was consistent with expectations (linearized vector fragment + target gene fragment), indicating successful insertion of the target gene into the vector. Simultaneously, the positive clone plasmids were sent to a sequencing company for sequencing verification, confirming that the target gene sequence was free of mutations and the insertion direction was correct, indicating successful construction of pCAMBIA1300- NtBGLU Recombinant vector.

[0093] 4. Transformation of Agrobacterium with recombinant plasmids The sequencing verification of pCAMBIA1300- NtBGLU The recombinant plasmid and the pCAMBIA1300 empty vector plasmid were transformed into Agrobacterium GV3101 by the freeze-thaw method and then quickly added to LB liquid medium. The medium was cultured at 28°C and 180 r / min for 1 h with shaking. The medium was then plated on LB plates containing the corresponding antibiotics (50 mg / L kanamycin and 50 mg / L rifampin) and cultured at 28°C for 48 h. Positive Agrobacterium clones were screened, and after PCR verification, they were stored for later use.

[0094] Example 3 Agrobacterium-mediated genetic transformation of tobacco (1) K326 tobacco seeds were sown in seedling pots with a substrate of peat moss: perlite = 3:1 (sterilized). The seedlings were managed at 22℃ under 16 h light / 8 h darkness conditions with a light intensity of 2000 lux. The seedlings were watered and fertilized regularly until they had 4-6 true leaves. The healthy seedlings without pests or diseases were selected for conversion.

[0095] (2) The contents of pCAMBIA1300 (empty vector control) and pCAMBIA1300- NtBGLUSingle colonies of Agrobacterium were inoculated into LB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampin) and cultured overnight at 28°C with shaking at 180 r / min until the OD600 value of the bacterial culture was 0.8-1.0.

[0096] (3) Take 1 mL of bacterial culture and inoculate it into 50 mL of new LB liquid medium (containing the same antibiotic), and continue to shake and culture until the OD600 value is about 0.6-0.8 (which can improve the infection efficiency and reduce the contamination of other bacteria).

[0097] (4) Collect Agrobacterium by centrifugation at 5000 r / min and 4℃ for 10 min. Resuspend the cells in infection buffer (containing 10 mM MES, 10 mM MgCl2, 200-250 μM acetylsuccinone, pH=5.6), adjust the OD600 value to about 0.8, and place in the dark at room temperature for 120 minutes. Acetylsuccinone can promote the transfer of T-DNA on the Ti plasmid of Agrobacterium and improve the transformation efficiency.

[0098] (5) Select tobacco seedlings with uniform growth, cut leaves free from diseases and pests, rinse them three times with sterile water, remove the main vein in a sterile ultra-clean workbench, cut them into leaf discs of 0.5×0.5cm, and place them in the above Agrobacterium resuspension for 10-15 minutes, gently shaking them during the process to ensure that the leaf discs are fully in contact with the bacterial solution and improve the infection effect.

[0099] (6) Remove the infected leaf disc, blot the surface bacterial solution with sterile filter paper, inoculate it onto MS medium (containing 0.5 mg / L 6-BA and 0.1 mg / L NAA), and co-culture in the dark at 25°C for 2-3 days to avoid excessive growth of Agrobacterium causing leaf disc rot.

[0100] (7) After co-culture, the leaf discs were transferred to the screening medium (MS medium + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 50 mg / L kanamycin + 250 mg / L cephalosporin) to screen for resistant buds. Cephalosporin was used to inhibit the growth of Agrobacterium. The medium was changed every 2 weeks until resistant buds grew (about 4-6 weeks). During this period, miscellaneous bacteria and non-resistant tissues were removed in time.

[0101] (8) Cut off the resistant shoots and transfer them to rooting medium (1 / 2 MS medium + 50 mg / L kanamycin + 250 mg / L cephalosporin). Culture them at 22°C under 16 h light / 8 h dark conditions until a complete root system grows (about 2-3 weeks). Obtain transgenic tobacco plants and transplant them into seedling pots for acclimatization and culture for later use.

[0102] Example 4: Molecular identification of overexpressing plants (1) RT-qPCR analysis of BGLU gene expression level: The second true leaf (same location, same growth stage) of transgenic plants and empty vector control plants was collected. Total RNA was extracted and reverse transcribed into cDNA (method as in Example 1). The cDNA concentration was detected using a nucleic acid quantification instrument to ensure that the concentration of each sample was consistent. Using the tobacco 26S gene as an internal control, a targeted... NtBGLU Gene-specific primers were used for real-time quantitative PCR (RT-qPCR) analysis. The primer sequences were: F-terminus: CTGGTTTGCCAATCCCTT (SEQ ID NO: 5), R-terminus: AGCCAGCATTCCTCTTCATT (SEQ ID NO: 6). The total volume of the qPCR reaction system was 20 μL, and the specific composition was as follows: 10 μL of Roche FastStart Essential DNA Green Master premix, which contains FastStart Taq DNA polymerase, dNTPs, MgCl2, an optimized buffer system, and SYBR Green I dye; and 8 μL of sterile nuclease-free water. NtBGLU 0.5 μL each of the forward and reverse primers (10 μM) and 1 μL of cDNA template were used. All reactions were performed on a Roche LightCycler instrument. The qPCR amplification conditions were: 95℃ for 10 min; 95℃ denaturation for 10 s, 60℃ annealing for 20 s, 72℃ extension for 10 s, for a total of 45 cycles; 72℃ extension for 60 s.

[0103] The results showed that, compared with the control group, pCAMBIA1300- NtBGLU Overexpression in plants NtBGLU The relative expression level of the gene was significantly increased ( Figure 1 The highest expression level was more than 150 times that of the control, indicating that the overexpression construct effectively enhanced the transcriptional level of the tobacco BGLU gene, which met the expected design goal.

[0104] Example 5: Determination of squalene content in overexpression plants (1) Preprocessing Leaves from plants showing significant overexpression as identified by RT-qPCR in Example 4 and control plants were freeze-dried and ground into powder. 200 mg of powder was accurately weighed, and 3 mL of hexane was added as the extraction solvent. 10 μL of internal standard deuterated tridecanoic acid (1 mg / mL) was added, and the mixture was extracted by sonication at room temperature for 60 min. The extract was centrifuged at 13,000 rpm for 10 min. 500 μL of the supernatant was transferred to a new 1.5 mL sample vial, centrifuged, concentrated, and dried. 200 μL of derivatization reagent (BSTFA / DMF, 1 / 1, v / v) was added, and the mixture was reacted at 60 °C for 60 min. The sample was then injected for analysis.

[0105] (2) GC-MS analysis Analysis was performed on a Thermo TRACE 1300 / TSQ8000, with the gas phase section using a DB-5MS capillary column (30 m × 0.25 mm, id × 0.25 μm df). The temperature program was as follows: 80 °C (0 min), 20 °C / min; 215 °C (0 min), 0.5 °C / min; 220 °C (0 min), 15 °C / min; 310 °C (15 min). Multiple reaction monitoring (MRM) mode was used to precisely determine and quantify the precursor ion (m / z: 149) and daughter ion pair (m / z: 81 / 93) of squalene. The absolute content of squalene in the samples was calculated using the standard curve method. Squalene standards were purchased from Sigma-Aldrich (catalog number: S3626). Eight standard solutions with concentrations ranging from 0.244 ppm to 31.250 ppm were obtained through serial dilution. The peak areas of squalene in the eight standard solutions were obtained using the same GC-MS analytical method described above. A standard curve was plotted with the peak area after internal standard correction as the x-axis and the squalene concentration as the y-axis, yielding the linear regression equation y = 25.002x + 0.879 (R²). 2 =0.9946).

[0106] Table 2 Comparison of squalene content in overexpressed and control tobacco leaves.

[0107] Table 2 and Figure 2 Data shows that gene overexpression technology can enhance... NtBGLU Gene expression successfully increased the squalene content in tobacco leaves to 2.6 to 4.7 times that of the empty vector control, proving that the technical solution of the present invention can greatly promote the biosynthesis and accumulation of squalene in tobacco and achieve the expected technical effect.

[0108] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An isolated nucleic acid molecule, characterized in that, The enzyme encodes β-glucosidase, the amino acid sequence of which is shown in SEQ ID NO:

2.

2. The nucleic acid molecule according to claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

1.

3. A recombinant vector, characterized in that, Contains the nucleic acid molecule as described in claim 1 or 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector is a plant binary vector.

5. The recombinant vector according to claim 4, characterized in that, The plant binary vector is selected from at least one of the following: pCAMBIA series vectors, pGreen series vectors, pBI series vectors, pPZP series vectors, pNC series vectors, BIBAC vectors, or TAC vectors.

6. A host cell, characterized in that, Contains the recombinant vector according to any one of claims 3-5.

7. The host cell according to claim 6, characterized in that, The host cell is Agrobacterium or Escherichia coli.

8. The host cell according to claim 7, characterized in that, The Agrobacterium is selected from at least one of GV3101, LBA4404, EHA105 or EHA103.

9. The use of the nucleic acid molecule of claim 1 or 2, the recombinant vector of any one of claims 3-5, or the host cell of any one of claims 6-8 in increasing the squalene content of tobacco.

10. A method for increasing the squalene content in tobacco, characterized in that, Includes the following steps: The nucleic acid molecule encoding β-glucosidase was constructed into a recombinant vector and transformed into tobacco plants to overexpress the nucleic acid molecule in the tobacco plants; the amino acid sequence of the β-glucosidase is shown in SEQ ID NO:

2.

11. The method according to claim 10, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

1.

12. The method according to claim 10 or 11, characterized in that, The tobacco is selected from K326, NC89, Yunyan 87 or Cuibi No. 1.

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