Metabolic engineering modified tobacco BY2 cell and application of metabolic engineering modified tobacco BY2 cell in salidroside production

Abstract

The invention relates to the technical field of biosynthesis of salidroside, in particular to a metabolic engineering modified tobacco BY2 cell and application of the metabolic engineering modified tobacco BY2 cell in production of salidroside. The metabolic engineering modified tobacco BY2 cell is constructed according to the following method: S1, connecting nucleotide sequences of an Rr4HPAAS gene, an Rr4HPAR gene and an RrUGT33 gene in series, and inserting into an expression vector to obtain a recombinant plasmid pK7FGW2-HHU; and S2, transferring the recombinant plasmid pK7FGW2-HHU into a tobacco BY2 cell through an agrobacterium tumefaciens mediated method, so as to obtain the metabolic engineering modified tobacco BY2 cell. The invention provides a metabolic engineering modified tobacco BY2 cell and application of the metabolic engineering modified tobacco BY2 cell in salidroside production, and aims to reconstruct a salidroside metabolic pathway, realize salidroside heterologous production and reduce the production cost of salidroside.

Description

Technical Field

[0001] The present application relates to the technical field of biosynthesis of salidroside, and in particular to a metabolically engineered tobacco BY2 cell and its application in the production of salidroside. Background Art

[0002] Rhodiola rosea L. is a perennial herb belonging to the genus Rhodiola of the Rhodiola family. It is an important Chinese herbal medicine with anti-fatigue, antioxidant, and cardiovascular protection effects. In recent years, Rhodiola rosea has gradually become a hot spot in health care product research, resulting in a sharp increase in market demand. Currently, Rhodiola rosea has been included in China's "National Secondary Protected Plant List".

[0003] Salidroside is the iconic active ingredient of Rhodiola rosea, a phenylethanol glycoside compound with multiple pharmacological activities such as anti-hypoxia, anti-fatigue, neuroprotection, and anti-tumor, and is widely used in the fields of medicine, functional food, and cosmetics. At present, salidroside is mainly extracted from wild Rhodiola rosea, and the content of salidroside in Rhodiola rosea is only 0.8% to 1%. Resources are becoming increasingly scarce, so the research on artificial synthesis to replace plant extraction has practical demand value.

[0004] Artificial synthesis of salidroside can be achieved through chemical synthesis and biosynthesis. Chemical synthesis of salidroside often requires harsh reaction conditions and expensive catalysts, and is prone to generate byproducts that are difficult to remove. The biological safety of salidroside is difficult to ensure. In contrast, biosynthesis has the advantages of low cost, high efficiency and selectivity, and less pollution. Summary of the invention

[0005] The present application provides a metabolically engineered tobacco BY2 cell and its application in the production of salidroside, so as to reconstruct the salidroside metabolic pathway, realize the heterologous production of salidroside, and reduce the production cost of salidroside.

[0006] In a first aspect, a metabolically engineered tobacco BY2 cell is provided, wherein the metabolically engineered tobacco BY2 cell is constructed according to the following method:

[0007] S1. Connect the nucleotide sequences of Rr4HPAAS gene, Rr4HPAR gene and RrUGT33 gene in series and insert them into an expression vector to obtain a recombinant plasmid pK7FGW2-HHU; the amino acid sequence encoded by the Rr4HPAAS gene is shown in SEQ ID NO: 1, the amino acid sequence encoded by the Rr4HPAR gene is shown in SEQ ID NO: 2, and the amino acid sequence encoded by the RrUGT33 gene is shown in SEQ ID NO: 3;

[0008] S2. The recombinant plasmid pK7FGW2-HHU is transferred into tobacco BY2 cells by Agrobacterium-mediated method: the Agrobacterium strain carrying the recombinant plasmid pK7FGW2-HHU is used to infect tobacco BY2 cells, and then the infected tobacco BY2 cells are spread on a solid culture medium plate, placed in a constant temperature incubator and cultured in the dark for 3 to 4 days, and then transferred to a solid resistance culture medium plate for culture and screening for 20 to 30 days until transformation-positive cell clusters grow out, thereby obtaining the metabolically engineered tobacco BY2 cells.

[0009] Preferably, the nucleotide sequence of the Rr4HPAAS gene is as shown in SEQ ID NO:4.

[0010] Preferably, the nucleotide sequence of the Rr4HPAR gene is as shown in SEQ ID NO:5.

[0011] Preferably, the nucleotide sequence of the RrUGT33 gene is as shown in SEQ ID NO:6.

[0012] Preferably, the expression vector is pK7FGW2 vector.

[0013] Preferably, the construction process of the recombinant plasmid is as follows:

[0014] S101, inserting the gene sequence of the Rr4HPAAS gene into the restriction nuclease cutting sites Hind III and EcoR I of the pUC19 vector by seamless cloning to obtain a pUC19-Rr4HPAAS plasmid; performing PCR amplification on the pUC19-Rr4HPAAS plasmid by designing primers Rr4HPAAS-F (nucleotide sequence as shown in SEQ ID NO: 7) and Rr4HPAAS-R (nucleotide sequence as shown in SEQ ID NO: 8) to obtain a Rr4HPAAS nucleotide sequence fragment;

[0015] S102, inserting the Rr4HPAR gene sequence into the restriction nuclease cutting sites Hind III and EcoR I of the pUC19 vector by seamless cloning to obtain a pUC19-Rr4HPAR plasmid; performing PCR amplification on the pUC19-Rr4HPAR (nucleotide sequence as shown in SEQ ID NO: 10) plasmid by designing primers Rr4HPAR-F (nucleotide sequence as shown in SEQ ID NO: 9) and Rr4HPAR-R to obtain a Rr4HPAR nucleotide sequence fragment;

[0016] S103, inserting the RrUGT33 gene sequence into the restriction nuclease cutting sites Hind III and EcoR I of the pUC19 vector by seamless cloning to obtain a pUC19-RrUGT33 plasmid; performing PCR amplification on the pUC19-RrUGT33 plasmid by designing primers RrUGT33-F (nucleotide sequence as shown in SEQ ID NO: 11) and RrUGT33-R (nucleotide sequence as shown in SEQ ID NO: 12) to obtain a RrUGT33 nucleotide sequence fragment;

[0017] S104, inserting the Rr4HPAAS, Rr4HPAR, RrUGT33 nucleotide sequence fragments into the restriction nuclease cutting sites Hind III and EcoR I of the pUC19 vector by seamless cloning to obtain the pUC19-HHU plasmid; performing PCR amplification on the pUC19-HHU plasmid by designing primers HHU-F (nucleotide sequence as shown in SEQ ID NO: 13) and HHU-R (nucleotide sequence as shown in SEQ ID NO: 14) to obtain the HHU nucleotide sequence fragment;

[0018] S105. Construct the HHU nucleotide sequence fragment into the plant expression vector pK7FGW2 through the Gateway cloning method to obtain the recombinant plasmid pK7FGW2-HHU.

[0019] In a second aspect, a use of a metabolically engineered tobacco BY2 cell in producing salidroside is provided, wherein the metabolically engineered tobacco BY2 cell comprises any of the metabolically engineered tobacco BY2 cells described above.

[0020] Preferably, the application includes:

[0021] Inoculating the metabolically engineered tobacco BY2 cells into a liquid growth medium, culturing at 25° C. for 7 days, and harvesting seed cells;

[0022] The seed cells are transferred into a liquid production culture medium and cultured at 27° C. for 5 to 14 days to produce salidroside.

[0023] Preferably, the liquid production medium consists of the following components at final concentrations: 10 g / L sugar, 4.43 g / L MS basal medium, water as solvent, and pH 5.8.

[0024] The beneficial effects of the technical solution provided by this application include:

[0025] The present application provides a metabolically engineered tobacco BY2 cell and its application in the production of salidroside. The tobacco cell BY2 is used as a chassis for metabolic modification, so that it can effectively carry an HHU expression plasmid, thereby effectively catalyzing L-tyrosine to produce salidroside, so that the tobacco BY2 cell has the ability to heterologously produce salidroside as a biological factory. Therefore, heterologous production of salidroside can be achieved, and the production cost of salidroside can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 Schematic diagram of the construction mode of the tandem expression vector of Rr4HPAAS, Rr4HPAR and RrUGT33 (HHU) genes provided in this application;

[0028] Figure 2 The images of HHU cells under a microscope provided for this application;

[0029] Figure 3 Agarose gel electrophoresis diagram of HHU fragment provided in this application;

[0030] Figure 4 Liquid phase (A) and mass spectrometry (B) test results of HHU cell samples provided in this application;

[0031] Figure 5 Schematic diagram of the effect of different sucrose concentrations on salidroside production provided in this application;

[0032] Figure 6 This is a schematic diagram of the effect of different culture times on salidroside production under the condition of 1% sucrose addition concentration provided in this application. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0034] Refer to the attached Figure 1 -Attached Figure 6(It should be noted that in the present application, HHU cells are cells that overexpress three genes, wherein the three genes are Rr4HPAAS gene, Rr4HPAR gene and RrUGT33 gene, and tobacco BY2 cells are chassis cells):

[0035] Example 1

[0036] This embodiment provides a metabolically engineered tobacco BY2 cell, wherein the metabolically engineered tobacco BY2 cell is constructed according to the following method:

[0037] S1. Connect the nucleotide sequences of Rr4HPAAS gene, Rr4HPAR gene and RrUGT33 gene in series and insert them into an expression vector to obtain a recombinant plasmid pK7FGW2-HHU; the amino acid sequence encoded by the Rr4HPAAS gene is shown in SEQ ID NO: 1, the amino acid sequence encoded by the Rr4HPAR gene is shown in SEQ ID NO: 2, and the amino acid sequence encoded by the RrUGT33 gene is shown in SEQ ID NO: 3;

[0038] The construction of the recombinant plasmid comprises the following steps:

[0039] S101, inserting the gene sequence of the Rr4HPAAS gene into the restriction nuclease cutting sites Hind III and EcoR I of the pUC19 vector by seamless cloning to obtain a pUC19-Rr4HPAAS plasmid; performing PCR amplification on the pUC19-Rr4HPAAS plasmid by designing primers Rr4HPAAS-F and Rr4HPAAS-R to obtain a Rr4HPAAS nucleotide sequence fragment;

[0040] S102, inserting the Rr4HPAR gene sequence into the restriction nuclease cutting sites Hind III and EcoR I of the pUC19 vector by seamless cloning to obtain a pUC19-Rr4HPAR plasmid; performing PCR amplification on the pUC19-Rr4HPAR plasmid by designing primers Rr4HPAR-F and Rr4HPAR-R to obtain a Rr4HPAR nucleotide sequence fragment;

[0041] S103, inserting the RrUGT33 gene sequence into the restriction nuclease cutting sites Hind III and EcoR I of the pUC19 vector by seamless cloning to obtain a pUC19-RrUGT33 plasmid; performing PCR amplification on the pUC19-RrUGT33 plasmid by designing primers RrUGT33-F and RrUGT33-R to obtain a RrUGT33 nucleotide sequence fragment;

[0042] S104, inserting the Rr4HPAAS, Rr4HPAR, and RrUGT33 nucleotide sequence fragments into the restriction nuclease cutting sites Hind III and EcoR I of the pUC19 vector by seamless cloning to obtain a pUC19-HHU plasmid; performing PCR amplification on the pUC19-HHU plasmid by designing primers HHU-F and HHU-R to obtain an HHU nucleotide sequence fragment;

[0043] S105. Construct the HHU nucleotide sequence fragment into the plant expression vector pK7FGW2 through the Gateway cloning method to obtain the recombinant plasmid pK7FGW2-HHU.

[0044] Specifically, the nucleotide sequence of the Rr4HPAAS gene is shown in SEQ ID NO: 4, the nucleotide sequence of the Rr4HPAR is shown in SEQ ID NO: 5, and the nucleotide sequence of the RrUGT33 is shown in SEQ ID NO: 6.

[0045] S2. The recombinant plasmid pK7FGW2-HHU is transferred into tobacco BY2 cells by Agrobacterium-mediated method: the Agrobacterium strain carrying the recombinant plasmid pK7FGW2-HHU is used to infect tobacco BY2 cells, and then the infected tobacco BY2 cells are spread on a solid culture medium plate, placed in a constant temperature incubator and cultured in the dark for 3 days, and then transferred to a solid resistance culture medium plate for culture and screening for 30 days until transformation-positive cell clusters grow out, thereby obtaining the metabolically engineered tobacco BY2 cells.

[0046] Furthermore, the expression vector is a pK7FGW2 vector.

[0047] The relevant primers are shown in Table 1.

[0048] Table 1

[0049]

[0050] Example 2

[0051] This embodiment provides an application of metabolically engineered tobacco BY2 cells in the production of salidroside.

[0052] The application comprises the following steps:

[0053] Inoculating the metabolically engineered tobacco BY2 cells into a liquid growth medium, culturing at 25° C. for 7 days, and harvesting seed cells;

[0054] The seed cells were transferred into a liquid production culture medium and cultured at 27° C. for 14 days to produce salidroside.

[0055] The liquid production medium is composed of the following components at the final concentrations: 10 g / L sugar, 4.43 g / L MS basal medium, water as solvent, and pH 5.8.

[0056] Example 3 Production Test of Salidroside

[0057] The constructed recombinant plasmid pK7FGW2-HHU carries Rr4HPAAS, Rr4HPAR and RrUGT33, and a green fluorescent protein (GFP) is fused to the C-terminus of the last gene to express positive transformed cells under a microscope.

[0058] The positively transformed callus was selected, subcultured twice on a solid medium containing the antibiotic kanamycin, and then transferred to a liquid medium for culture to establish a suspension cell line. The GFP signal was observed under a microscope, and the full-length bands of three exogenous genes were amplified by PCR for agarose gel electrophoresis to verify whether the HHU overexpression cell line was stably transcribed. Among them, the culture temperature of the positively transformed callus on the solid medium was 25°C, and the constant temperature incubator was dark cultured, and the culture time was 30 days for one generation. After one generation of culture, the positively transformed callus was transferred to a new solid medium and cultured under the same conditions; the positively transformed callus was transferred to a liquid medium and placed in a constant temperature shaker, the culture temperature was 25°C, the speed was 150rpm, and the dark culture was 7 days for one generation, that is, the seed cells, and then 1mL of seed cells was transferred to a new liquid medium and cultured under the same conditions.

[0059] The solid culture medium is composed of the following components in final concentrations: 30 g / L sugar, 4.43 g / L MS basic culture medium, 50 mg / L kanamycin, 7 g / L agar, water as solvent, and pH 5.8; the liquid culture medium is composed of the following components in final concentrations: 30 g / L sugar, 4.43 g / L MS basic culture medium, 50 mg / L kanamycin, water as solvent, and pH 5.8.

[0060] See also Figure 2 As shown, Figure 2 This is a picture of HHU cells under a microscope. Figure 2 Clear green fluorescence can be observed in the samples, indicating that tobacco BY2 cells carry the HHU expression plasmid and express it stably and efficiently.

[0061] See also Figure 3 As shown, Figure 3 This is the agarose gel electrophoresis diagram of HHU cells, i.e., the amplification results of Rr4HPAAS, Rr4HPAR and RrUGT33 genes in the HHU cell transcription template, indicating that agarose gel can separate the target bands of Rr4HPAAS, Rr4HPAR and RrUGT33 from the cDNA template of HHU overexpressing cells, indicating that the transcription levels of these genes are high.

[0062] Test the production capacity of salidroside:

[0063] For evaluation of tobacco BY2 cells as a host for heterologous biosynthesis see Figure 4 shown. Figure 4 The results of liquid chromatography (A) and mass spectrometry (B) detection of HHU cell samples.

[0064] It can be seen that the target peak with a retention time of 11.8min can be detected in the HHU cell sample, and further confirmed by mass spectrometry at m / z299.1129. This shows that the exogenous pathway can correctly perform the synthesis function and effectively catalyze L-tyrosine to produce salidroside. Tobacco BY2 cells have the ability to produce salidroside as a biological factory.

[0065] Using sucrose as carbon source, salidroside was cultured in HHU cell production medium, see Figure 5 As shown, when the sucrose concentration is 1%, the yield of salidroside after 14 days of culture is the highest, which is 357.69 μg / g·FW. When the sucrose addition is 0.5%, the salidroside content accumulates rapidly after 3 days. However, as the culture time increases, it cannot maintain this rapid accumulation, and the salidroside content is 229.52 μg / g·FW, which is lower than the salidroside yield when the sucrose addition is 1%. When the sucrose addition is increased to 3%, 5% and 7%, the accumulation of salidroside is significantly reduced. Therefore, the metabolically engineered tobacco BY2 cells prepared in the present application can achieve rapid increase in a short period of time in the heterologous production of salidroside.

[0066] Specifically, the HHU cell production medium contains 4.43 g / L of MS basal medium, wherein the sucrose concentration is relative to the mass of the HHU cell production medium. Here, the density of the HHU cell production medium is determined to be 1 g / mL, that is, when the sucrose concentration is 1%, 10 g of sucrose is added to every 1000 mL of HHU cell production medium.

[0067] Therefore, the addition of sucrose to the HHU cell production medium is a key factor in the heterologous production of secondary metabolites in segmented culture. A lower addition amount can allow the carbon source to flow rapidly into the secondary metabolites, but its production is limited by the addition amount. A higher addition amount can provide cells with sufficient carbon source, but inhibits the secondary metabolic diversion. In addition, too high a sucrose addition amount may affect the osmotic pressure of the cells, thereby reducing the biosynthesis of secondary metabolites.

[0068] See also Figure 6As shown in the figure, the yield of salidroside increased with the extension of the culture time, reaching the highest content of 377.53 μg / g·FW on the 11th day, which has exceeded the content of its original host Rhodiola rosea. A rapid increase phase occurred from the 1st day to the 5th day, and the salidroside content accumulated from 21.48 μg / g·FW to 249.75 μg / g·FW, an increase of 11.6 times in 4 days. This is consistent with the advantage of plant suspension cells as a short production cycle of biological factories.

[0069] SEQ ID NO: 1

[0070] MGSLPPSPNDPSNTFNPMDLTELSTESKLVVDFITQYYQTLETRPVQPRVKPGFLTGQLPDKAPFHGESMEVILSDVNEKIVPGLTHWQSPNFHAYFPASSSNAGLLGELLCSGLSVIGFTWS SSPAATELENVVVDWMAKMLNLPSSFCFSGGGGGVLQANTCEAVLCTLAAARDKALNRVGDDQINKLVLYCSDQTHFTIHKGAKLIGIRSKNIKSITTKKENEFKLCPNDLRDAIRSDLEAGL VPFYVCGTIGTTALGVVDPIKELGKVAREFDLWLHVDGAYGGSACICPEFQHYLDGVDLVDSISMNAHKWLLSNLDCCFLWLQSPNALIESLAAENFLKGGSEMVDYKDWQISLSRRFRAIK MWMVIRRYGVSNLIEHIRSDVSMAVRFEEMVAADDRFEIVFPRKFALVCFKLSSEKTPPGRDSELTRELMERVNSSGKAYLSGVQMGRIFFIRCVIGSSLTEERHVDNLWRLIQETAQSIVS.

[0071] SEQ ID NO: 2

[0072] MSLSGAGKVVCVTGASGYIASWLVKLLLQRGYTVKASVRDPNDPKKTQHLTALDGAKERLQLYKANLLEQGSFDPIVEGCEGVFHTASPFYHAVDDPQAELIDPAVKGTLNVLSSCAKVASLKRVVLTSSIAAVAYNGKPRTPEVVVDETWFSNPDVCKEMKLWYVISKTLAEEAAWKFVKEKGIDMVTINPAMVIGPLLQPTLNTSAAAILNLINGSETYPNASFGWVNVKDVAEAHVLAFEVPSANGRYCLVERVAHSSEVVNMLHELYPDIKLPAKCADDKPFVPIYQVSKEKAHTLGVKFIPLEVSLKETVESLKEKGFAKL。

[0073] SEQ ID NO:3

[0074] MSLIEKPLTAIETREKPHAVCIPYPAQGHINPMMQLAKLLHHSGFHITFVHTEYNYDRLVKSQGSACVAGLPDFRFEAIPDGLPSTNGDVTQDIPLLSSSTSKTCLKPFKELLKRLQDKCKELPDDVPPLSCIVSDAAMSFTIDASEEFGVPIALLWTASACGFLGYTHYPYLIDRGVIPLKDESQLTNGYLDMSIDGIPCMEGIRLRDLPSFLRTTDLDDMMFSYILHEIKQVSRGSAIILNTFEALDHDVLDSLSKIYQ NVILPVGPLHVSLNKIPKHYPLQSLSSNLWKDDTDCIPWLSSKASKSVIYVNFGSITTVSPKQIVEFAWGLANSKHPFLWIIRPDLVAGEASIIPQDFMDETKGRGLLAGWCDQELVLNHPSIGGFLTHCGWNSIIESISAGVPTVCWPFFAEQQTNCWFACKKWCIGMEMHTDVKRDEVDKLLRELMEGDKGEELKRKATNWKRLAEEAVSSTGLSTLNFRTLVNQVLLSKTKHIR。

[0075] SEQ ID NO:4

[0076]

[0077] SEQ ID NO:5

[0078] ATGAGTTTAAGCGGAGCGGGGAAGGTGGTTTGCGTTACCGGCGCGTCTGGCTACATAGCGTCCTGGCTCGTCAAGCTTCTTCTCCAGCGCGGTTATACCGTCAAGGCCTCCGTTCGCGATCCTAATGATCCGAAAAAGACTCAGCACTTGACGGCACTTGATGGAGCTAAGGAGAGGCTGCAGTTGTACAAAGCCAATTTGCTTGAACAAGGCTCGTTTGATCCCATAGTTGAAGGATGTGAAGGTGTTTTCCACACCGCGTCTCCCTTTTATCATGCAGTGGATGATCCGCAGGCCGAGTTAATTGACCCTGCTGTCAAGGGAACACTCAATGTTCTTTCTTCATGTGCTAAAGTTGCGTCTCTTAAAAGAGTAGTCCTGACTTCTTCGATTGCTGCTGTTGCATATAATGGGAAACCCCGTACTCCGGAGGTTGTAGTTGACGAGACTTGGTTTTCTAACCCAGATGTTTGTAAGGAGATGAAGCTTTGGTATGTCATATCCAAGACACTCGCTGAAGAAGCAGCATGGAAGTTTGTGAAAGAGAAAGGAATAGACATGGTTACCATAAATCCGGCCATGGTGATTGGTCCCCTTCTGCAACCAACACTCAATACCAGTGCTGCTGCTATTCTGAACTTGATCAATGGATCGGAGACATACCCAAATGCTTCTTTTGGATGGGTCAATGTGAAAGATGTTGCAGAAGCACACGTTCTTGCATTTGAGGTTCCTTCAGCTAATGGTAGATACTGCTTGGTGGAAAGAGTTGCCCACAGTTCTGAAGTGGTGAACATGCTCCATGAGCTCTACCCTGATATCAAACTTCCCGCCAAGTGTG CAGATGACAAACCATTTGTGCCAATTTATCAAGTTTCAAAAGAAAAGGCACATACTTTAGGGGTAAAATTCATTCCTTTAGAGGTAAGCCTCAAGGAAACAGTTGAAAGCTTGAAGGAAAAGGGTTTCGCCAAACTCTGA。

[0079] SEQ ID NO:6

[0080]

[0081] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A metabolically engineered tobacco BY2 cell, characterized in that: The metabolically engineered tobacco BY2 cells were constructed as follows: S1. Connect the nucleotide sequences of Rr4HPAAS gene, Rr4HPAR gene and RrUGT33 gene in series and insert them into an expression vector to obtain a recombinant plasmid pK7FGW2-HHU; the amino acid sequence encoded by the Rr4HPAAS gene is shown in SEQ ID NO: 1, the amino acid sequence encoded by the Rr4HPAR gene is shown in SEQ ID NO: 2, and the amino acid sequence encoded by the RrUGT33 gene is shown in SEQ ID NO: 3; S2. The recombinant plasmid pK7FGW2-HHU is transferred into tobacco BY2 cells by Agrobacterium-mediated method: the Agrobacterium strain carrying the recombinant plasmid pK7FGW2-HHU is used to infect tobacco BY2 cells, and then the infected tobacco BY2 cells are spread on a solid culture medium plate, placed in a constant temperature incubator and cultured in the dark for 3 to 4 days, and then transferred to a solid resistance culture medium plate for culture and screening for 20 to 30 days until transformation-positive cell clusters grow out, thereby obtaining the metabolically engineered tobacco BY2 cells.

2. The metabolically engineered tobacco BY2 cell according to claim 1, characterized in that: The nucleotide sequence of the Rr4HPAAS gene is shown in SEQ ID NO:

4.

3. The metabolically engineered tobacco BY2 cell according to claim 1, characterized in that: The nucleotide sequence of the Rr4HPAR gene is shown in SEQ ID NO:

5.

4. The metabolically engineered tobacco BY2 cell according to claim 1, characterized in that: The nucleotide sequence of the RrUGT33 gene is shown in SEQ ID NO:

6.

5. The metabolically engineered tobacco BY2 cell according to claim 1, characterized in that: The expression vector is pK7FGW2 vector.

6. The metabolically engineered tobacco BY2 cell according to claim 1, characterized in that: The construction process of the recombinant plasmid is as follows: S101, inserting the gene sequence of the Rr4HPAAS gene into the restriction nuclease cutting sites Hind III and EcoR I of the pUC19 vector by seamless cloning to obtain a pUC19-Rr4HPAAS plasmid; performing PCR amplification on the pUC19-Rr4HPAAS plasmid by designing primers Rr4HPAAS-F (nucleotide sequence as shown in SEQ ID NO: 7) and Rr4HPAAS-R (nucleotide sequence as shown in SEQ ID NO: 8) to obtain a Rr4HPAAS nucleotide sequence fragment; S102, inserting the Rr4HPAR gene sequence into the restriction nuclease cutting sites Hind III and EcoR I of the pUC19 vector by seamless cloning to obtain a pUC19-Rr4HPAR plasmid; performing PCR amplification on the pUC19-Rr4HPAR plasmid by designing primers Rr4HPAR-F (nucleotide sequence as shown in SEQ ID NO: 9) and Rr4HPAR-R (nucleotide sequence as shown in SEQ ID NO: 10) to obtain a Rr4HPAR nucleotide sequence fragment; S103, inserting the RrUGT33 gene sequence into the restriction nuclease cutting sites Hind III and EcoR I of the pUC19 vector by seamless cloning to obtain a pUC19-RrUGT33 plasmid; performing PCR amplification on the pUC19-RrUGT33 plasmid by designing primers RrUGT33-F (nucleotide sequence as shown in SEQ ID NO: 11) and RrUGT33-R (nucleotide sequence as shown in SEQ ID NO: 12) to obtain a RrUGT33 nucleotide sequence fragment; S104, inserting the Rr4HPAAS nucleotide sequence fragment, the Rr4HPAR nucleotide sequence fragment, and the RrUGT33 nucleotide sequence fragment into the restriction nuclease cutting sites Hind III and EcoR I of the pUC19 vector by seamless cloning to obtain a pUC19-HHU plasmid; performing PCR amplification on the pUC19-HHU plasmid by designing primers HHU-F (nucleotide sequence as shown in SEQ ID NO: 13) and HHU-R (nucleotide sequence as shown in SEQ ID NO: 14) to obtain a HHU nucleotide sequence fragment; S105. Construct the HHU nucleotide sequence fragment into the plant expression vector pK7FGW2 by Gateway cloning method to obtain the recombinant plasmid pK7FGW2-HHU.

7. Use of the metabolically engineered tobacco BY2 cell as claimed in claim 1 in producing salidroside.

8. The use according to claim 7, characterized in that The applications include: Inoculating the metabolically engineered tobacco BY2 cells into a liquid growth medium, culturing at 25° C. for 7 days, and harvesting seed cells; The harvested seed cells are transferred into a liquid production culture medium and cultured at 27° C. for 5-14 days to produce salidroside.

9. The use according to claim 8, characterized in that The liquid growth medium is composed of the following components at final concentrations: 10 g / L sugar, 4.43 g / L MS basal medium, water as solvent, and pH 5.8.

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