Gene combination for heterologous production of salidroside in tobacco and application of gene combination

Abstract

The invention relates to the technical field of gene engineering, in particular to a gene combination for heterologously producing salidroside in tobacco and application of the gene combination. The invention relates to a gene combination for heterologous production of salidroside in tobacco. The gene combination comprises an Rr4HPAAS gene, an Rr4HPAR1 gene and an RrUGT33 gene, the nucleotide sequences of the Rr4HPAAS gene, the Rr4HPAR1 gene and the RrUGT33 gene are as shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. According to the invention, synthesis of the salidroside compound in transgenic tobacco is realized, the salidroside compound can be used as a natural product source of salidroside, the salidroside compound can be produced in heterologous plants, and the application rate of the salidroside compound is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a gene combination for heterologously producing salidroside in tobacco and an application thereof. Background Art

[0002] Rhodiola rosea is a precious edible herb with a long history and is known as Tibetan ginseng. Rhodiola plants thrive in cold regions and high altitudes, such as the slopes of the Hengduan Mountains and the Himalayas. Salidroside [2-(4-hydroxyphenyl)ethyl-β-D-pyranoglucopyranoside] is the primary bioactive component of Rhodiola plants. It exhibits a variety of biological properties, including anti-aging, hypoxia tolerance, anti-inflammatory activity, cardiovascular disease prevention, anti-cancer, and neuroprotective and brain cell protection. Salidroside has attracted widespread attention as a potential nutritional supplement.

[0003] There are three different methods for producing salidroside: direct extraction from plants or cell tissue cultures, chemical synthesis, and microbial synthesis. Direct extraction is limited by the scarcity of natural resources and the low content of plant tissues, while chemical synthesis has the disadvantages of using toxic chemicals and complex procedures.

[0004] Over the past 30 years, numerous transgenic plants with genetically modified single traits have been used to promote commercial cultivation in several countries and regions. With the development of metabolic engineering and synthetic biology, multigene transformation has replaced single-gene transformation to modulate biosynthesis and improve multiple biological traits, a new trend in genetic breeding. To date, genetically engineered plants have achieved numerous breakthroughs in the biofortification of micronutrients, phytonutrients, and bioactive components, such as β-carotene-enriched golden rice, potatoes, bananas, and rapeseed. Tomato fruits biofortified with complex anthocyanins, L-dopa, folic acid, flavonols, and betaine were developed through multigene transformation involving targeted metabolite biosynthesis pathways. Therefore, it is of great significance to transform different salidroside synthase genes into candidate plants to cultivate tobacco plants. These transgenic plants can also serve as materials for salidroside production.

[0005] After a multi-gene transformed plant is cultured into a complete plant under appropriate conditions, molecular tests such as PCR and qRT-PCR are performed on the regenerated plants to screen out transgenic plants expressing biologically active exogenous proteins. The fruits of some transgenic plants, such as tomatoes, cucumbers, lettuce, bananas, and strawberries, can be eaten raw or cooked, and can also be used as plant biofactories for natural compounds, which are simple to operate and low-cost. In summary, plant chassis are more suitable for the heterologous synthesis of complex natural products. Usually, one or more key enzyme genes involved in metabolic biosynthetic pathways are introduced into candidate plant chassis through anabolic engineering to synthesize natural products. This method not only helps to develop more functional and complex fortified varieties, but also supplements the nutrients needed by the human body on a daily basis and improves malnutrition problems. The application of plant chassis has a protective effect on some rare plant resources and is an important strategy for the future development of plant cell factories. Summary of the Invention

[0006] The present invention aims to provide a gene combination for heterologous production of salidroside in tobacco and its application. The present invention also provides a method for producing salidroside compounds using transgenic plants. Using a multi-gene assembly strategy based on In-Fusion and P2A polypeptide integration, three salidroside synthase genes were successfully transformed into tobacco to produce salidroside compounds. This method has significant application value, as heterologous production of salidroside in transgenic tobacco addresses issues such as low natural content and cultivation restrictions of salidroside compounds.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a gene combination for heterologously producing salidroside in tobacco, comprising an Rr4HPAAS gene, an Rr4HPAR1 gene and an RrUGT33 gene; the nucleotide sequences of the Rr4HPAAS gene, the Rr4HPAR1 gene and the RrUGT33 gene are shown in SEQ ID NOs: 1, 3 and 5, and the amino acid sequences are shown in SEQ ID NOs: 2, 4 and 6.

[0009] Wherein, the Rr4HPAAS gene sequence is:

[0010] atgggcagcttgccttctcctaatgatccatcaaacaccttcaaccccatggacctcaccgagttatccaccgagtcgaaactcgtcgtagatttcataactcagtactaccaaaccctagagacccgacccgtccagccacgggtcaagccaggtttcttaacgggccagcttccagataaagcaccctttcatggtgaatcaatggaagtaatattgtctgatgtaaatgagaagattgtccctggcctcactcattggcaaagccctaatttccatgcatactttccagccagttccagcaacgcagggctgttgggagagttactatgctccggactcagtgtcattgggttcacatggagctcctcccctgccgcgacggagcttgagaatgtcgtggttgactggatggccaagatgcttaaccttccatcctctttctgcttctccggcggaggcggtggcgttctgcaagcaaacacttgcgaggctgtgttgtgcactttagccgctgcgagggacaaggctcttaaccgggtgggagatgatcagatcaataaactggtcctctactgctccgaccaaacacatttcacaatccacaagggcgcaaagttgataggaatccgatcaaagaacataaaatcaatcactactaagaaagagaacgagtttaaactctgtcctaacgacctacgcgacgcgataaggagtgatctggaagcaggactagttccgttttacgtatgcggaacgattggaacgaccgcgttaggagttgtggatccgattaaagagctgggtaaggtggcaagagagtttgatttgtggttacatgttgatggagcttatggtggcagtgcatgcatatgccctgagtttcagcattaccttgatggagttgaccttgttgactcgatcagcatgaatgcacataaatggcttttatccaatctagattgctgcttcctgtggcttcaatctcctaacgccctaatcgaatccctggccgcagaagctaactttctgaaaggtggtagtgagatggtggattacaaggactggcagatatcgttgagtcgtcgatttagagcgatcaagatgtggatggtgataaggcgatacggtgtgagtaatctcattgagcatattcgatccgacgtgagcatggcggtgagattcgaagagatggtggcggcggacgaccggtttgaaatcgtgtttcctagaaagtttgcgcttgtttgcttcaagcttagtagcgagaagacaccaccgggccgcgactcggagttaactcgtgagctgatggagagagtcaactcgagtgggaaggcttacttgagtggagttcaaatgggtcggatcttcttcatcaggtgtgtgatcgggtcgagtttgactgaggagagacacgtcgataatctgtggaggctcattcaagaaacagctcaaagcatcgtgtct

[0011] The amino acid sequence is:

[0012] MGSLPSPNDPSNTFNPMDLTELSTESKLVVDFITQYYQTLETPVQPRVKPGFLTGQLPDKAPFHGESMEVILSDVNEKIVPGLTHWQSPNFHAYFPASSSNAGLLGELLCSGLSVIGFTWSSSPAATELENVVVDWMAKMLNLPSSFCFSGGGGVLQANTCEAVLCTLAAARDKALNRVGDDQINKLVLYCSDQTHFTIHKGAKLIGIRSKNIKSITTKKENEFKLCPNDLRDAIRSDLEAGL VPFYVCGTIGTTALGVVDPIKELGKVAREFDLWLHVDGAYGGSACICPEFQHYLDGVDLVDSISMNAHKWLLSNLDCCFLWLQSPNALIESLAAEANFLKGGSEMVDYKDWQISLSRRFRAIKMWMVIRRYGVSNLIEHIRSDVSMAVRFEEMVAADDRFEIVFPRKFALVCFKLSSEKTPPGRDSELTRELMERVNSSGKAYLSGVQMGRIFFIRCVIGSSLTEERHVDNLWRLIQSIVS

[0013] The Rr4HPAR1 gene sequence is:

[0014] Atgagtttaagcggagcggggaaggtggtttgcgttaccggcgcgtctggctacatagcgtcctggctcgtcaagcttcttctccagcgcggttataccgtcaaggcctccgttcgcgatcctaatgatccgaaaaagactcagcacttgacggcacttgatggagctaaggagaggctgcagttgtacaaagccaatttgcttgaacaaggctcgtttgatcccatagttgaaggatgtgaaggtgttttccacaccgcgtctcccttttatcatgcagtggatgatccgcaggccgagttaattgaccctgctgtcaagggaacactcaatgttctttcttcatgtgctaaagttgcgtctcttaaaagagtagtcctgacttcttcgattgctgctgttgcatataatgggaaaccccgtactccggaggttgtagttgacgagacttggttttctaacccagatgtttgtaaggagatgaagctttggtatgtcatatccaagacactcgctgaagaagcagcatggaagtttgtgaaagagaaaggaatagacatggttaccataaatccggccatggtgattggtccccttctgcaaccaacactcaataccagtgctgctgctattctgaacttgatcaatggatcggagacatacccaaatgcttcttttggatgggtcaatgtgaaagatgttgcagaagcacacgttcttgcatttgaggttcc ttcagctaatggtagatactgcttggtggaaagagttgcccacagttctgaagtggtgaacatgctccatgagctctaccctgatatcaaacttcccgccaagtgtgcagatgacaaaccatttgtgccaatttatcaagtttcaaaagaaaaggcacatactttaggggtaaaattcattcctttagaggtaagcctcaaggaaacagttgaaagcttgaaggaaaagggtttcgccaaactc

[0015] The amino acid sequence is:

[0016] MSLSGAGKVVCVTGASGYIASWLVKLLLQRGYTVKASVRDPNDPKKTQHLTALDGAKERLQLYKANLLEQGSFDPIVEGCEGVFHTASPFYHAVDDPQAELIDPAVKGTLNVLSSCAKVASLKRVVLTSSIAAVAYNGKPRTPEVVVDETWFSNPDVCKEMKL WYVISKTLAEEAAWKFVKEKGIDMVTINPAMVIGPLLQPTLNTSAAAILNLINGSETYPNASFGWVNVKDVAEAHVLAFEVPSANGRYCLVERVAHSSEVVNMLHELYPDIKLPAKCADDKPFVPIYQVSKEKAHTLGVKFIPLEVSLKETVESLKEKGFAKL

[0017] The RrUGT33 gene sequence is:

[0018]

[0019] The amino acid sequence is:

[0020] MSLIEKPLTAIETREKPHAVCIPYPAQGHINPMMQLAKLLHHSGFHITFVHTEYNYDRLVKSQGSACVAGLPDFRFEAIPDGLPSTNGDVTQDIPLLSSSTSKTCLKPFKELLKRLQDKCKELP DDVPPLSCIVSDAAMSFTIDASEEFGVPIALLWTASACGFLGYTHYPYLIDRGVIPLKDESQLTNGYLDMSIDGIPCMEGIRLRDLPSFLRTTDLDDMMFSYILHEIKQVSRGSAIILNTFEALD HDVLDSLSKIYQNVILPVGPLHVSLNKIPKHYPLQSLSSNLWKDDTDCIPWLSSKASKSVIYVNFGSITTVSPKQIVEFAWGLANSKHPFLWIIRPDLVAGEASIIPQDFMDETKGRGLLAGWC DQELVLNHPSIGGFLTHCGWNSIIESISAGVPTVCWPFFAEQQTNCWFACKKWCIGMEMHTDVKRDEVDKLLRELMEGDKGEELKRKATNWKRLAEEAVSSTGLSTLNFRTLVNQVLLSKTKHIR

[0021] The present invention also provides the use of the gene combination in heterologous production of salidroside in tobacco.

[0022] The present invention also provides an expression vector comprising an initial vector, a connecting peptide and the gene combination.

[0023] Preferably, the initial vector is pCAMBIA1304, and the purpose of the recombinant vector is achieved by replacing the sequence between the Nco I site and the Spe I site of the vector.

[0024] Preferably, the connecting peptide is a P2A polypeptide and / or a T2A polypeptide.

[0025] The present invention also provides a host transformed or transfected with the expression vector; the host is a microorganism.

[0026] The present invention also provides the use of the expression vector or the host in heterologous production of salidroside in tobacco.

[0027] The present invention also provides a method for heterologous production of salidroside, which uses the host to transfect into a target plant.

[0028] Preferably, the target plant is tobacco, rice, wheat, corn, cucumber, tomato, lettuce, watermelon, melon, pumpkin, apple, banana, pear or bitter melon; and the tobacco variety is k326.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] Theoretically, in order to successfully produce salidroside in a heterologous host, it is particularly important to use an appropriate multi-gene assembly strategy. In previous practices, re-transformation and co-transformation methods are often used to inject multiple genes into heterologous plant matrices so that they can be smoothly introduced into complete metabolic pathways. However, these methods not only consume a lot of time and energy, but are also not applicable to the simultaneous transformation of multiple genes. Therefore, fusion PCR technology stands out with its unique advantages. It can integrate multiple gene expression cassettes into the host chromosome genome through a single T-DNA insertion operation. To this end, the present invention successfully constructed a multi-gene expression vector containing three salidroside synthase genes - Rr4HPAAS, Rr4HPAR1, and RrUGT33 genes using fusion PCR technology and a multi-gene assembly strategy combined with a linker peptide. It was then transferred into tobacco K326 plants using Agrobacterium transformation, achieving the synthesis of salidroside compounds in transgenic tobacco. It can be used as a source of salidroside natural products, can produce salidroside compounds in heterologous plants, and improve the application rate of salidroside compounds.

[0031] The introduction of the 2A polypeptide element in this invention is crucial for reducing the presence of repetitive sequences in multigenes and improving vector stability. The P2A polypeptide exhibits optimal cleavage efficiency, reducing the constraints of repetitive sequences on multigene vectors and enabling co-expression of multiple genes. This approach is broadly adaptable to various plant species. This invention utilizes tobacco K326 as a model crop, which offers a short growth cycle, a clear genetic background, and simple transgenic manipulation, making it a promising biofactory.

[0032] The present invention develops a strategy for constructing multiple expression vectors for key enzyme genes in salidroside synthesis and transforms them into plants, enabling heterologous synthesis of salidroside. Stable transgenic crops prepared by the present method can be directly planted to produce large quantities of salidroside compounds, reducing investment in technology, equipment, and planting conditions, thereby lowering production costs. The present invention also addresses the regional limitations of Rhodiola rosea plant cultivation, improves the utilization rate of salidroside, and facilitates the industrialized production of salidroside compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0034] Figure 1 Figure a shows the strategy for constructing a multi-gene vector; b to d show the PCR identification images of Rr4HPAAS (1470 bp), (c) Rr4HPAR1 (978 bp), and (d) RrUGT33 (1494 bp) on the multi-gene vector pCAMBIA1304-Rr4HPAAS-Rr4HPAR1-RrUGT33, with a maker of 2000 bp.

[0035] Figure 2 Comparison of the growth status of tobacco k326 transgenic plants and wild-type plants.

[0036] Figure 3 Molecular identification of transgenic tobacco lines. PCR detection of transgenic tobacco plants. From left to right, the electrophoretic bands are: marker, Rr4HPAAS, Rr4hHPAR1, and RrUGT33. The first well after the marker is Rr4HPAAS, the second well is Rr4hHPAR1, and the third well is RrUGT33. Markers (from top to bottom): 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.

[0037] Figure 4 Subcellular localization of Rr4HPAAS, Rr4HPAR1, and RrUGT33; GFP: green fluorescent protein fluorescence; Autofluorescence: chloroplast autofluorescence; BrightField: bright field; Merge: overlay of chloroplast autofluorescence, green fluorescence, and bright field. Scale bar: 50 μm.

[0038] Figure 5 Figure 2. Analysis of salidroside accumulation in transgenic tobacco leaves. a. Standard curve for salidroside. b. Analysis of salidroside accumulation in transgenic tobacco lines. Data are presented as mean ± SD, with three replicates for each sample. DETAILED DESCRIPTION

[0039] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1 Construction of recombinant vector

[0041] like Figure 1 As shown, the present invention utilizes the method of combining InFusion and 2A polypeptide to construct a large vector pCAMBIA1304-Rr4HPAAS-Rr4HPAR1-RrUGT33 containing Rr4HPAAS, Rr4HPAR1 and RrUGT33 genes.

[0042] The preparation method of the vector comprises the following steps: using Rhodiola rosea Rr4HPAAS (MF_674522.1), Rr4HPAR1 (MF_674524.1) and RrUDP33 (MF674558.1) as seed sequences, performing local Blastp sequence alignment, and obtaining candidate genes Rt4HPAAS, RrUDP33 and R4HPAR1. PCR amplification primers (Rr4HPAAS-F: 5'-atgggcagcttgccttctc-3'; Rt4HPAAS-R: 5'-agctcaaagcatcgtgtct-3'; Rr4HPAR1-F: 5'-atgagtttaagcggag-3'; Rt4HPAR1-R: 5'-aaaagggtttcgccaaactc-3'; RrUGT33-F: 5'-atgagcttaattgaaa-3'; RrUGT33-F: 5'-acggatatgttttgtt-3') were designed as shown in SEQ ID NOs: 13-18. Gene amplification was performed using Rhodiola rosea cDNA as a template (Tables 1 and 2). PCR products were excised and recovered by 1% agarose gel electrophoresis. The recovered product was ligated with the 1304 vector (pCAMBIA1304, Cytofil) and transformed into DH5α Escherichia coli. Single clones with correct sequencing results were picked, and the plasmids were extracted and stored at -20°C for future use.

[0043] Table 1 Amplification procedure

[0044]

[0045] Table 2 Amplification system

[0046]

[0047] Using the 1304-Rr4HPAAS plasmid as a template, primers 1304-AAS-F and SP2A-R were used to amplify the 4HPAAS-P2A fragment (PCR product 1). Using the 1304-Rr4HPAR1 plasmid as a template, primers P2AR1-F and T2AR1-R were used to amplify the P2A-4HPAR1-1304 fragment (PCR product 2). Using the 1304-RrUGT33 plasmid as a template, primers T2AUGT33-F and 1304-UGT33-R were used to amplify the T2A-UGT33-1304 fragment (PCR product 3). Products 1, 2, and 3 were ligated using multi-fragment ligase, and the corresponding 1304-4HPAAS+4HPAR1+UGT33 plasmid was identified by polymerase chain reaction. This multigene expression vector is relatively large (approximately 16.5 kb in length from left to right border) and can be used to synthesize salidroside. The pCAMBIA1304-Rr4HPAAS-Rr4HPAR1-RrUGT33 vector was introduced into Agrobacterium tumefaciens GV3101 and genetic transformation was performed.

[0048] Table 3 Primer design for construction of multi-gene expression vector

[0049]

[0050]

[0051] As shown in SEQ ID NOs: 7-12.

[0052] Results: The ORF sequences of salidroside synthesis-related transcription factor genes Rr4HPAAS, Rr4HPAR1 and RrUGT33 without the stop codon were inserted into the NcoⅠ and SpeⅠ restriction sites of the 1304 vector by seamless cloning to obtain the plant overexpression vector pC1304Rr4HPAAS-Rr4HPAR1-RrUGT33. The schematic diagram of the constructed vector is shown in the figure. Figure 1 a is shown; b, c, and d are PCR identification diagrams of the vector.

[0053] Example 2 Obtaining transgenic plants

[0054] 1. Preparation of Transgenic Tobacco

[0055] The recombinant vector pC1304Rr4HPAAS-Rr4HPAR1-RrUGT33 was transformed into Agrobacterium tumefaciens GV3101 for verification. The Agrobacterium containing the recombinant vector pC1304Rr4HPAAS-Rr4HPAR1-RrUGT33 was named Agrobacterium-2. Transgenic tobacco plants containing the recombinant vector pC1304Rr4HPAAS-Rr4HPAR1-RrUGT33 were generated by co-cultivation with Agrobacterium-2. Additionally, wild-type tobacco plants that were not transfected with Agrobacterium were cultured as controls.

[0056] Specific methods include:

[0057] (1) Pre-culture tobacco seeds were sterilized in 75% alcohol for 1 min, washed with sterile water for 1 min, and then sterilized with diluted 84 disinfectant for 5 min. They were then washed with sterile water 3 times, 1 min each time. The sterilized tobacco seeds were sown on MS culture medium and cultured at 28°C with a 16h / 8h light / dark cycle for 3 weeks. Sterile tobacco leaves were cut into 0.5 cm pieces with a scalpel. 2 Small pieces were inoculated on pre-culture medium.

[0058] (2) Agrobacterium infection and co-cultivation Agrobacterium was picked up in the infection solution to prepare an Agrobacterium resuspension with an OD600 of 0.4. Tobacco leaves pre-cultured for 2 days were inoculated with the Agrobacterium suspension and infected for 10 minutes. The infected tobacco leaves were inoculated on filter paper, dried, and then inoculated on the co-culture medium and cultured in the dark for 3 days.

[0059] (3) Induction: After 2 days of co-cultivation, the leaves were transferred to the induction medium to induce callus, and the callus tissue was grown for about 10 days.

[0060] (4) Select callus tissue that meets the standards and inoculate it on the corresponding resistance screening medium for 30 days at a temperature of 25±2°C.

[0061] (5) Differentiation and rooting: Inoculate the actively growing positive callus tissue from the second sieve onto the differentiation medium, 5 calli / dish, and culture at 25℃ with 16h / 8h light / dark for 30 days. During the differentiation process, if seedlings are formed from the calli, inoculate them onto the seedling medium and grow them for 10 days. When the seedlings have grown more than 10 roots, gently remove the seedlings with tweezers, rinse the remaining culture medium with tap water, and transplant them into a seedling pot filled with sterilized seedling medium. Since the transgenic seedlings are relatively weak, cover the seedlings with a protective cover for about 5 days. Remove the protective cover and continue to culture for another week. Then transplant them into a large pot and place them in the greenhouse for normal management.

[0062] PCR identification: Take the leaves of transgenic tobacco ( Figure 2), genomic DNA was extracted; using the extracted genomic DNA as a template, primers 35s-seq: ttcatttggagagaacacgggggac; 35S-AAS-550R: ggctaaagtgcacaacacagcc (product fragment 550 bp) and UGT33-F: caaaaaatggtgcattgggatgga; UGT33-GFP-R335: catcaccttcaccctctccactg (product fragment 335 bp) were designed as shown in SEQ ID NOs: 19 to 22.

[0063] KOD OneTM PCRmasterMix was used to specifically amplify the Rr4HPAAS (1470bp), Rr4hHPAR1 (978bp), and RrUGT33 (1494bp) genes. PCR detection results showed that the vector and the target gene had been connected in the leaves of tobacco plants. ( Figure 3 PCR detection of transgenic tobacco plants. From left to right, the electrophoretic bands are: marker, Rr4HPAAS, Rr4hHPAR1, and RrUGT33. The first well after the marker is Rr4HPAAS, the second well is Rr4hHPAR1, and the third well is RrUGT33. Markers (from top to bottom): 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.

[0064] Example 3 Subcellular Localization of Rr4HPAAS, Rr4HPAR1, and RrUGT33

[0065] The specific method includes: To determine the localization of Rr4HPAAS, Rr4HPAR1, and RrUGT33 proteins in cells, the vector plasmid pCAMBIA1304-GFP was double-digested with Nco I and Spe I, and the CDS sequences of Rr4HPAAS, Rr4HPAR1, and RrUGT33 without the stop codon were amplified. These sequences were then homologously recombined with the linearized vector to generate 35S::Rr4HPAAS::GFP, 35S::Rr4HPAR1::GFP, and 35S::RrUGT33::GFP fusion expression vectors. The correctly sequenced recombinant plasmids and pCAMBIA1304-GFP were transformed into Agrobacterium tumefaciens GV3101 without the vector. Resistance screening was performed in YEB solid medium containing 50 μg / mL rifampicin (Rif) and 50 μg / mL kanamycin (Kana), and positive PCR was performed to identify the cells. After preparing the Agrobacterium resuspension, it was injected into Nicotiana benthamiana leaves that had grown normally for 5 weeks, cultured in the dark for 1 day, and then cultured normally for 2 days. The injected material was taken out and the green fluorescence signal was observed under a confocal microscope.

[0066] Analysis of subcellular localization results: To determine the expression sites of Rr4HPAAS, Rr4HPAR1 and RrUGT33 proteins, Agrobacterium carrying 35S::Rr4HPAAS-GFP, 35S::Rr4HPAR1-GFP and 35S::RrUGT33-GFP and Agrobacterium carrying 35S::GFP (negative control) were used to infect Nicotiana benthamiana leaves to detect the subcellular localization of Rr4HPAAS, Rr4HPAR1 and RrUGT33 proteins. Figure 4 As can be seen from the figure, Rr4HPAAS and Rr4HPAR1, RrUGT33 fusion proteins are all localized in chloroplasts and co-localized with chloroplast autofluorescence, while the control GFP signal is expressed in both the cell membrane and the nucleus, indicating that Rr4HPAAS and Rr4HPAR1, RrUGT33 proteins are localized in chloroplasts.

[0067] Example 4 HPLC-MS / MS Analysis of Salidroside Compounds in Transgenic Tobacco Plants

[0068] Extraction of salidroside compounds

[0069] Weigh 50 mg of each wild-type tobacco leaf sample and the transgenic leaf sample prepared in Example 2, grind into a powder with liquid nitrogen, add 1 mL of 50% methanol solution based on the sample size, mix thoroughly, and ultrasonically extract for 1 hour. Replace ultrasonic losses with methanol solution. Centrifuge at 5000 rpm for 5 minutes, remove the supernatant, add 50% methanol solution for replacement, and ultrasonicate at 40 kHz for 1 hour. Combine the supernatants and filter through a membrane for later use.

[0070] HPLC-MS / MS detection

[0071] The specific parameters of the HPLC-MS / MS instrument, column model, retention time, and ion source used in this experiment are shown in Tables 4 and 5.

[0072] Table 4 HPLC-MS / MS parameters of salidroside compounds

[0073]

[0074] Table 5 LC-MS / MS parameters

[0075]

[0076]

[0077] HPLC-MS / MS results showed that salidroside was accumulated in tobacco leaves ( Figure 5b), with an average content of approximately 42.26 μg / g, and no salidroside compounds were found in wild-type tobacco plants ( Figure 5 b).

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gene combination for heterologous production of salidroside in tobacco, characterized in that: It includes Rr4HPAAS gene, Rr4HPAR1 gene and RrUGT33 gene; the nucleotide sequences of the Rr4HPAAS gene, Rr4HPAR1 gene and RrUGT33 gene are shown in SEQ ID NOs: 1, 3 and 5.

2. Use of the gene combination according to claim 1 in heterologous production of salidroside in tobacco.

3. An expression vector, characterized in that: It comprises an initial vector, a connecting peptide and the gene combination according to claim 1.

4. The expression vector according to claim 3, characterized in that The original vector was pCAMBIA1304.

5. The expression vector according to claim 3, characterized in that The connecting peptide is a P2A polypeptide and / or a T2A polypeptide.

6. A host, characterized in that The host is transformed or transfected with the expression vector according to any one of claims 3 to 5; the host is a microorganism.

7. Use of the expression vector according to any one of claims 3 to 5 or the host according to claim 6 in heterologous production of salidroside in tobacco.

8. A method for heterologous production of salidroside, characterized in that: The host according to claim 6 is used to transfect into a target plant.

9. The method according to claim 8, characterized in that The target plant is tobacco, rice, wheat, corn, cucumber, tomato, lettuce, watermelon, melon, pumpkin, apple, banana, pear or bitter gourd.

10. The method according to claim 9, characterized in that The variety of the tobacco is k326.

Images