Application of a plant nucleoside hydrolase protein and its encoding gene in regulating nicotinamide ribose content in plants

Abstract

This invention discloses the application of plant nucleoside hydrolase proteins and their encoding genes in regulating the nicotinamide ribose content of plants. The plant nucleoside hydrolase proteins are kale nucleoside hydrolases BolNSH1, BolNSH2, and BolNSH3, and rice nucleoside hydrolases OsNSH1, OsNSH2, and OsNSH3. The amino acid sequences of kale nucleoside hydrolases BolNSH1, BolNSH2, and BolNSH3 are shown in SEQ ID NO: 1–3, and the amino acid sequences of rice nucleoside hydrolases OsNSH1, OsNSH2, and OsNSH3 are shown in SEQ ID NO: 4–6. This invention is the first to discover that kale nucleoside hydrolases BolNSH1, BolNSH2, and BolNSH3 and rice nucleoside hydrolases OsNSH1, OsNSH2, and OsNSH3 can catalyze the hydrolysis of pyridine nucleoside nicotinamide ribose to obtain nicotinamide. This invention silences the expression of this gene in kale, increasing the nicotinamide ribose content by 1.5-2.5 times, further enhancing the value of the kale plant. The novel gene and method provided by this invention lay the foundation for creating kale germplasm with high nicotinamide ribose content and have broad application prospects.

Description

Technical Field

[0001] This invention relates to the application of a plant nucleoside hydrolase protein and its encoding gene in regulating the content of nicotinamide ribose in plants, and belongs to the field of genetic engineering. Background Technology

[0002] Kale (Brassica oleraceae L. var. acephala DC.) is a herbaceous plant belonging to the Brassicaceae family and the Brassica genus. It is both ornamental and edible, and is considered one of the healthiest vegetables in the world. Many of its natural products (such as polyphenols, vitamins, and carotenoids) have important medicinal value, including anti-cancer, anti-aging, stomach ulcer improvement, and liver protection. (et al., 2023). Therefore, increasing the content of important natural products in kale is of great significance for improving its quality.

[0003] Nicotinamide ribose (NR) is a naturally occurring vitamin B3 derivative found in milk and is an important coenzyme for redox reactions in the body—nicotinamide adenine dinucleotide (NAD). + Nicotinamide riboside is a precursor to NMR. In recent years, increasing research has shown that NR plays an important role in the treatment of cardiovascular diseases, neurodegenerative diseases, metabolic diseases, lifespan extension, and immune regulation. Nicotinamide riboside can activate NMRK2 and SIRTs to maintain NAD+. + Nicotinamide riboside can protect cardiac function and limit adverse remodeling in mice with heart failure by maintaining homeostasis (Mehmelet et al., 2020); NR supplementation can treat Alzheimer's and Parkinson's diseases induced by DNA damage and oxidative stress (Gong et al., 2013; Berven et al., 2023); Nicotinamide riboside can effectively improve a mouse model of muscle atrophy by increasing muscle strength, revitalizing senescent muscle stem cells, and reducing inflammation and fibrosis (Zhang et al., 2016); Nicotinamide riboside can also improve NAD+ homeostasis by increasing NAD+ homeostasis. + Levels of nicotinamide ribose (NR) can improve type 2 diabetes, metabolic syndrome, and non-alcoholic fatty liver disease caused by metabolic disorders (Trammell et al., 2016); NR also has potential therapeutic effects against SARS-CoV-2 and COVID-19 infections. Nicotinamide ribose is found in low amounts in kale; if its degradation pathway can be blocked to further increase its content and obtain it from food, the application of nicotinamide ribose will be promoted.

[0004] In plants, the degradation of nicotinamide riboside is related to NAD. +The salvage synthesis pathway is closely linked to this pathway. Research on this pathway in plants has primarily relied on isotope tracing. Ashihara et al. used isotope tracing to hypothesize that the degradation of nicotinamide ribose to nicotinamide in plants is catalyzed by nucleoside enzymes, but they did not clone the relevant gene (Ashihara et al., 2012). In plants, only Arabidopsis thaliana has reported five nucleoside enzymes: AtNSH1, AtNSH2, AtNSH3, AtNSH4, and AtNSH5 (Jung et al., 2011). AtNSH1, AtNSH2, and AtNSH3 can catalyze the hydrolysis of purine and pyrimidine nucleosides (including uridine, adenosine, inosine, and xanthosine), but whether they catalyze the degradation of nicotinamide ribose and their application in increasing the nicotinamide ribose content in plants have not been reported. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide an application of plant nucleoside hydrolase protein and its encoding gene in regulating the content of nicotinamide ribose in plants.

[0006] Technical Solution: This invention provides the application of plant nucleoside hydrolase proteins in regulating the nicotinamide ribose content of plants. The plant nucleoside hydrolase proteins are kale nucleoside hydrolases BolNSH1, BolNSH2, and BolNSH3, and rice nucleoside hydrolases OsNSH1, OsNSH2, and OsNSH3. The amino acid sequences of kale nucleoside hydrolases BolNSH1, BolNSH2, and BolNSH3 are shown in SEQ ID NO:1-3, and the amino acid sequences of rice nucleoside hydrolases OsNSH1, OsNSH2, and OsNSH3 are shown in SEQ ID NO:4-6.

[0007] This invention also provides the application of the genes encoding the above-mentioned plant nucleoside hydrolase proteins in regulating the nicotinamide ribose content of plants. The nucleotide sequences of the kale nucleoside hydrolases BolNSH1, BolNSH2 and BolNSH3 are shown in SEQ ID NO:7-9, and the nucleotide sequences of the rice nucleoside hydrolases OsNSH1, OsNSH2 and OsNSH3 are shown in SEQ ID NO:10-12.

[0008] Furthermore, the plants mentioned are kale and rice.

[0009] This invention also provides the application of silencing vectors or silent strains of the kale nucleoside hydrolases BolNSH1, BolNSH2 and BolNSH3 genes in increasing the nicotinamide ribose content in kale.

[0010] Furthermore, the silencing vector is obtained by constructing the nucleotide sequences of the BolNSH1, BolNSH2 and BolNSH3 genes into the vector PCVA.

[0011] Furthermore, the silenced strain is obtained by introducing a silencing vector of the BolNSH1, BolNSH2 and BolNSH3 genes into Agrobacterium.

[0012] Furthermore, the primer sequences used to construct the silencing vector for the BolNSH1 gene are shown in SEQ ID NO:13-14.

[0013] Furthermore, the primer sequences used to construct the silencing vector for the BolNSH2 gene are shown in SEQ ID NO:15-16.

[0014] Furthermore, the primer sequences used to construct the silencing vector for the BolNSH2 gene are shown in SEQ ID NO:15-16.

[0015] This invention also provides the application of silencing vectors or silent strains of rice nucleoside hydrolases OsNSH1, OsNSH2 and OsNSH3 genes in increasing the nicotinamide ribose content in rice.

[0016] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention is the first to discover that kale nucleoside hydrolases BolNSH1, BolNSH2, and BolNSH3, and rice nucleoside hydrolases OsNSH1, OsNSH2, and OsNSH3, can catalyze the hydrolysis of pyridine nucleoside nicotinamide ribose to obtain nicotinamide. This invention silences the expression of this gene in kale, increasing the nicotinamide ribose content by 1.5-2.5 times, further enhancing the value of kale plants. The new gene and method provided by this invention lay the foundation for creating kale germplasm with high nicotinamide ribose content and have broad application prospects. Attached Figure Description

[0017] Figure 1 Electrophoresis images of kale and rice nucleoside hydrolase proteins.

[0018] Figure 2 This is a standard curve for determining protein concentration using an enzyme-linked immunosorbent assay (ELISA) reader.

[0019] Figure 3 This is an HPLC chromatogram showing the in vitro catalysis of nicotinamide ribose by the kale nucleoside hydrolases BolNSH1, BolNSH2, and BolNSH3.

[0020] Figure 4This is an HPLC chromatogram showing the in vitro catalysis of nicotinamide ribose by rice nucleoside hydrolases OsNSH1, OsNSH2, and OsNSH3.

[0021] Figure 5 LC-MS results for the in vitro catalytic conversion of nicotinamide from ribonucleotides by kale and rice nucleoside hydrolase.

[0022] Figure 6 The results show the gene expression levels and NR content of BolNSH1, BolNSH2 and BolNSH3 in VIGS-induced gene silencing kale. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0024] Example 1: Cloning of plant nucleoside hydrolase gene and expression of target protein

[0025] 1. Cloning of plant nucleoside hydrolase genes

[0026] Approximately 2 grams of plant leaves ("Beta" kale or "Nipponbare" rice) were weighed and RNA was extracted using the OminiPlant RNAKit (DNase I) kit (Jiangsu Kangwei Century Biotechnology Co., Ltd.). cDNA was then synthesized via reverse transcription using Vazyme's HIScript IIQ RT SuperMix for qPCR (+gDNA wiper). PCR amplification was performed using the primer pairs listed in Table 1. The PCR system (50 μL) consisted of: 25 μL of 2×Phanta Max Buffer, 1 μL of Phanta Max Super-Fidelity DNA Polymerase, 2 μL of dNTP Mix (2.5 mM), 2 μL each of forward and reverse primers (10 μmol / L), 1 μL of cDNA template, and 17 μL of ddH2O. The PCR reaction program was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 53℃ annealing for 15 s; 72℃ extension for 1 min, for a total of 35 cycles; and a final extension at 72℃ for 5 min. The target gene fragments BolNSH1, BolNSH2, BolNSH3, OsNSH1, OsNSH2, and OsNSH3 were obtained respectively, and the PCR products were purified using the EasyPure PCR Purification Kit (Beijing TransGen Biotech Co., Ltd.).

[0027] Table 1 Primer sequences for target gene amplification

[0028]

[0029] The nucleotide sequence (SEQ ID NO:7) of the target gene BolNSH1 is as follows:

[0030] ATGGAGTTTTGCAACGGTGGGATCTCAAACGGCGACGTTTTGGGTCTCTCTTCTAAACGCGAGAAGCTCATTATCGATACAGACCCAGGAATCGATGATAGCATGGCGATAATGATGGCGTTTCAAACACCAGAGCTGGAGATAGTAGGACTCACTACCGTCTTTGGTAATGTTTCTACGCAAGATGCTACCCGCAACGCCTTACTCCTGTGTGAGGTTGCTGGCTTCCCTGATGTTCCTGTGGCAGAAGGAAGCTCCGAACCCTTAAAGGGTGGGATTCCACGTGTTGCTGATTTTGTGCACGGTAAAAACGGACTAGGAGAGGTCTCTGTTCCTCTTCCTAGTAGAAAGAAATGTGACAAAAGTGCAGCTGAGTTTCTAGTTGAGAAGGTCTCCGAGTATCCTGGTGAAGTCACCATCCTCGCCCTTGGACCTCTAACCAACCTTGCAATAGCCATCAAGCGAGATAGTTCATTTGCGAGTAAGGTGAAGAAAATTGTTGTACTCGGTGGAGCTTTCTTTGCTTTGGGAAATGTCAATCCTGCAGCTGAGGCCAATATATATGGTGACCCGGAAGCAGCTGATGTTGTGTTCACGTGTGGTGCTGATATAACTGTTGTTGGGATAAACATCACAACACAACTTAAACTAACAGATGATGACCTCTTAGCCCTGCGTGACTCCAAGGGCAAACACGCTAAGTTGTTAAGTGACATGTGCAAGTTTTATAGAGACTGGCACGTCAAGTCTGATGGTGTTTACGGAGTTTACCTCCATGACCCGGTCAGCTTTGTGGCTGTAGTACGGCCTGATCTATTCACGTATAAGAAAGGCGTTGTTAGGGTGGAGACTCAAGGAATATGTGTTGGCCACACTCTCATGGACCAAGGCTCAAGAGATGGAATGGGAGCAATCCATGGGTGGGATATTCACCAGTATCAGTGGCATGGACGGTAGATGTAGATGGAGTGTTGGAATATATCAAAGGCATGCTGATGAAGCCTTAA

[0031] The nucleotide sequence (SEQ ID NO:8) of the target gene BolNSH2 is as follows:

[0032]

[0033] The nucleotide sequence (SEQ ID NO:9) of the target gene BolNSH3 is as follows:

[0034] ATGGTGGGAGAGCAGAGCAAGAAGATTATCATCGATACTGATCCTGGAATCGATGATGCAATGGCGATATTCGTGGCGCTAAACTCACCAGAGGTTGATGTGATTGGACTCACTACTATCTACGGCAACGTTTACACCACTCTCGCCACTCGTAACGCCTTGCATTTGTTGGAGGTTGCGGGTAGGACAGACATTCCTGTAGCTGAAGGAACACATA AAACTATCATGAATGGCACAAAGCTTCGAATAGCTGACTTCGTTCACGGTAAAGATGGGCTTGGCAACCAAAACTTCCCTCCACCTAAAGGGAAGCCAATTGAAAAATCTGCACCTGAGTTCTTAGTTGAACAAGCTAAGCTTCACCCTGGTGAAATCACTGTTGTTGCTTTGGGACCTCTCACAAATATAGCACTGGCTATTCAGCTTGATCCTGAGTTTTCCAAAAATGTTGGACAAATTGTTCTTCTTGGTGGAGCATTTGCAGTAAATGGAAATGTGAATCCAGCATCAGAAGCTAATATTTTTGGTGATCCAGAAGCTGCGGATATTGTGTTCACATGTGGTGCTGATGTGATTGCTGTGGGAATCAACGTGACTCATCAAGTTATTATGACAGCTGATGATAGGGACAAATTGGCAGCATCGAACGGGAAATTAGCTCAATACCTCTGTAAAATCCTTGGTGTGTACTATTCTTATCATCTTGACGCTTATGAGATTGAAGGTGTTTATCTTCATGATCCTACAACGATACTTGCGGCTTTCCTTCCTTCTCTATTCACTTATACTGAAGGAGTTGTTAGAGTGCAAACTAACGGTATCACTAGAGGACTCACTTTACTGTACAACAATCGAAAAAGGTTTGAGGAAGTGACCGAGTGGACTGACAAACCATCGGTAAAAGTGGCAGTGACGGTTGATGCACCAGCGGTCTTGAAGCTCATAATGGATAGGCTTATGGAGTCTTGA

[0035] The nucleotide sequence (SEQ ID NO:10) of the target gene OsNSH1 is as follows:

[0036] ATGGGGTCGAACGAGCAGATCCACCGGGACAAGCTCATCATCGACACGGATCCCGGCATCGATGATAGCATGACAATCCTTATGGCGTTCAGAGCTCCAACTGTGGAGATCATAGGGCTCACAACCATATTTGGCAACACCACCACAAAGAATGCGACTCAGAATGCGCTCTTGCTGTGTGAGAGAGCAGGACATCCTGAGGTTCCAGTAGCAGAGGGCAGCGCAGAGCCTCTCAAGGGAGGAGAGCCACGTGTTGCTGACTTTGTTCATGGGTCTGATGGCCTGGGGAATTTGTTTCTTCCTGCACCCACTAGTAAGAAGGTTGATGAAAATGCTGCTGAGTTCATGGTTAATAAGGTCTCTCAATTTCCTGGAGAAGTTTCTATACTCGCATTGGGACCCCTGACAAACGTGGCGCTGGCCATCAAAAGGGACCCTTCCTTTGCAAGTAAGGTGAAGAAAATAGTTGTACTGGGTGGTGCTTTCTTTGCGGCTGGAAATGTCAGCCCAGCTGCTGAAGCAAATATATATGGAGATCCAGAAGCAGCTGACATAGTTTTTACCTCAGGAGCAGATGTTGATGTGGTTGGCATTAACATAACAACTCAAGTCTGTTTTACAGATGAGGATCTCCTGGAGCTAAGAAACTCAAAAGGGAAGCATGCACAGTTCTTATGTGACATGTGCCAGTTCTATAGAGACTGGCATGCCGAATCCGATGGCTTTCATGGAATTTTCCTCCATGATCCTGTGAGCTTCACTGCCC TAGTTCACCCTGAGTACTTCACGTTCAAGAAGGGTGTTGTGAGAGTAGAAACCCAGGGCATTTGCACTGGTCACACTTTAATGGACCAAGGATTGAAAAAGTGGAATTCAGAGAATCCATGGTCTGGCTACAAGCCAATCTCAGTTGCATGGACAGTTGATGTGCCAAATGTTCTCGCATTCGTGAAGGAACTCCTCATGGCTCCATGA

[0037] The nucleotide sequence (SEQ ID NO:11) of the target gene OsNSH2 is as follows:

[0038] ATGACGACGACGAAGAAGAAGCTCGTCATCGACACCGACCCGGGAATCGATGACGCCATGGCCATCTTCGTGGCGCTGAGGTCGCCGGAGGTGGAGCTGCTGGGCCTCACCACCATCTTCGGCAACGTCTACACCACCCTCGCCACCCGCAACGCCCTCCACCTGCTGGAGGCTGTTGGGAGGACTGACATCCCCGTGGCAGAGGGATCCCATGTAACAATCAAGAAAGCCACCAAGCTACGGATTGCGAGCTTTGTTCATGGCTCGGATGGTCTAGGAAATCAGAACTTTCCTCCGCCAACTGGGAAGCCCTTAGATCAATCGGCCGCCGCGTTTCTTGTCGAGCAGGCAAATCTGTACCCTGGACAAGTTACTGTGGTTGCCCTTGGTCCACTTACCAACCTAGCTTTGGCTATAGAACTTGATCCTTCTTTCCCGAAGAAGATTGGGCAGATTGTGATCCTTGGTGGTGCGTATTCAGTTAATGGAAATGTCAACCCTGCAGCTGAGGCAAATATATTTGGTGACCCTGATGCAGCTGATATAGTTTTCACCTCCGGTGCTGATATTTTGGCTGTGGGAATAAATATTACACACCAAGTAGTTCTTTCTGATGCTGACCGGGAAAAGCTTGAACAGTCTGATAGCAAATATGCCCGATACTTGAGCAAGATCCTGGGTCTTTATTATGATTACCACAAGGACGCCTACTTCATAAAAGGAGTGTATCTTCACGATCCAGCAACACTTATTGCTGCGGTAGATCCGTCACTGATGACTTACACGGAAGGTGTAGTGAGGGTGCAGACTGATGGCATCACAAAGGGTCTCACAGTTTTCGACACAACCAAGAAAAGGTATGGGGAGATAACTGCGTGGACCGGTAAGCCAACCGTGAAGGTTGCTGTGACCGTTGATGCTCCTGCCGTTGTGGAGATGATCATGCAGAGGCTTACGACGGATGATTAG

[0039] The nucleotide sequence (SEQ ID NO:12) of the target gene OsNSH3 is as follows:

[0040] ATGGATCTGCAGGAGGCGGCGATGGAAGCCAGGAACGGCCATCGGATCCCGCCGACGGAGGAGAAGGTCATCATCGACACGGATCCAGGGATCGATGACAGCGTGGCGATCATGATGGCGTTCGAAGCTCCCGGCGTGAAGGTCGTCGGACTCACCACCATCTTCGGCAACTGCACTACCTCCCACGCCACCCGCAATGCCCTCATCCTGTGCGACAGAGCAGGCCGTCCTGAGGTGCCGGTAGCAGAGGGCAGCGCTGAGCCTCTCAAGGGAGGGAAACCTCACGTTGCTGATTTTGTTCATGGATCGGATGGACTTGGTAACACGTCCTTTCCTGATCCTACCACCACCAACAAAGTTGAGCAGTCAGCTGCAGAGTTTCTGGTTGATAAGGTATCAGAATCTCCAGGAGAGATCTCTGTACTCGCCTTGGGCCCTCTCACTAATATCGCATTGGCCATGAAGAAAGACTCCTCCTTTGCAAGCAAGGTTAAGAGGATAGTTGTGCTGGGTGGAGCTTTCTTTGCAGCTGGGAATGCCACCCCTTCAGCTGAAGCAAATATCCATAGCGACCCAGAAGCAGCTGACATAGTTTTCACTTCTGGGGCAGATATCTACGTAGTTGGCCTTAACATAACAACCCAAGTCTACTTCACAGACAAGGATATGTTGGAGCTAAGGAACTCGAAAGGGAAGCACGCACAATTCCTTTGTGACATCTGCAAGTTCTACAGAGACTGGCATGTCCACTCCTACGGCGTCGATGGTCCGCACATGAAACATCTCTCCTTTCCTTCTTACAAGTTCAGGCTTTGGGAAATTAACGTTCCAGCTCACATTTGTGTGATTTCTTCAGCTCTTTTTCTCCATGATCCTGTGAGTTTCACCGCGCTAGTTCACCCCGAGTACTTCACGTTCAAGAAGGGCGTGGTGAGGGTTGAGACCCAGGGCATCTGCAAGGGGCATACTTCCATGGACATGGGACTGAAGAAGTGGAATTCAGACAACCCGTGGACTGGTTACTCTCCGATCTCGGTTGCTTGGACTGTTGATGTTCCTAAGGTTCTGGCATATGCCAAGGAGCTACTCTTCAACGCACAATGA

[0041] 2. Construction of recombinant vector

[0042] The pET29a(+) (laboratory-preserved) vector was double-digested with Nde I and Xho I. The purified digested product was ligated with the recovered target gene fragment and transformed into E. coli DH5α competent cells. The cells were plated on LB solid medium (containing 50 μg / mL kanamycin). Single clones were picked and colony PCR was performed using the pET29a-F / pET29a-R primer pair (Table 2). Positive clones were screened, and plasmids were extracted and sequenced. Those with correct sequencing were identified as recombinant vectors.

[0043] Table 2 Primer sequences for recombinant vector identification

[0044]

[0045] 3. Construction of recombinant strains

[0046] The recombinant vector was transformed into Escherichia coli BL21(DE3) to obtain a recombinant strain containing the target gene.

[0047] 4. Expression of target protein

[0048] (1) Randomly select recombinant Escherichia coli BL21(DE3) monoclonal strains and incubate them overnight at 37°C with shaking at 220 rpm in 3 mL LB medium containing kanamycin (Kan). Add 2 mL of activated bacterial solution (concentration 1×10⁻⁶) 6 ~10 7 (CFU / ml) was transferred at a 1:100 ratio to a 200mL LB medium bottle and incubated on a shaker at 37°C and 220rpm. After 2.5-3 hours, 60μL of 1M IPTG inducer was added to the 200mL medium, and incubation was carried out at 16°C and 180rpm for 15 hours. The remaining 1mL of bacterial culture was used for preservation.

[0049] (2) Put 200 mL of bacterial solution into a 50 mL round-bottom centrifuge tube, centrifuge at 4℃, 12000 rpm for 1 min, discard the supernatant and collect the bacterial cells.

[0050] (3) Resuspend the bacterial cells in 20 mL of 10 mM PBS (pH 7.4) buffer, centrifuge at 4°C and 12,000 rpm for 1 min, discard the supernatant, and wash the bacterial cells once.

[0051] (4) Resuspend the bacterial cells: Resuspend the bacterial cells in 15 mL of 10 mM PBS (pH 7.4) buffer and place on ice.

[0052] (5) The bacterial cells were broken using a Branson Sonifier SFX550 ultrasonic disruptor. The parameters were set as follows: PULSE, 30% power, ON 1s, OFF 2s, TOTAL 3min.

[0053] (6) Obtaining crude enzyme solution: The ultrasonically broken sample was centrifuged at 4℃, 12000rpm for 30min. The supernatant was transferred to a clean centrifuge tube. The supernatant is the crude enzyme solution.

[0054] 5. Purification of the target protein (all the following operations were performed at 4°C)

[0055] (1) Equilibrate the Co column (GE His GraviTrap TALON, Sinopharm Chemical Reagent Co., Ltd.): Discard the 20% ethanol in the Co column, add 10 mL Binding Buffer (50 mM Na3PO4, 300 mM NaCl, pH adjusted to 7.4 with phosphoric acid) and pass it through the column until the column is empty.

[0056] (2) Target protein binding to the column: After the Binding Buffer has been completely drained, add all the crude enzyme solution into the chromatography column and flush once to allow the protein to bind to the resin.

[0057] (3) Elution of impurities: Add 10 mL Wash Buffer (50 mM Na3PO4, 300 mM NaCl, 10 mM imidazole, pH 7.4) to the chromatography column to elute impurities.

[0058] (4) Elution of target protein: Add 3 mL of Elution Buffer (50 mM Na3PO4, 300 mM NaCl, 500 mM imidazole, pH 7.4) to the chromatography column and elute to obtain the target protein.

[0059] (5) Recovery and preservation of Co column: Pass the column through 10-20 mL Binding Buffer, seal the column with 20% ethanol, and store at 4°C.

[0060] (6) Collect the target protein using SurePAGE. TM Electrophoresis was performed using a precast gel (concentration 4-20%, GenScript Biotech Inc.) to obtain bands of corresponding sizes, such as... Figure 1 As shown, M represents the protein marker. Figure 1 A is the target protein band of BolNSH1, with a size of 36.0 kDa; Figure 1 B is the target protein band of BolNSH2, with a size of 36.5 kDa; Figure 1 C represents the target protein band of BolNSH3, with a size of 34.7 kDa; Figure 1 D represents the target protein band of OsNSH1, with a size of 36.1 kDa; Figure 1 E represents the target protein band of OsNSH2, with a size of 34.5 kDa; Figure 1F represents the target protein band of OsNSH3, with a size of 39.9 kDa.

[0061] 6. Target protein dialysis

[0062] The eluted target protein was placed into a regenerated cellulose dialysis bag (Shanghai Yuanye Biotechnology Co., Ltd.), and then placed in 300 mL of pre-chilled dialysis buffer (10 mM PBS (pH 7.4)) and stored at 4°C. The dialysis buffer was replaced after 2-4 hours, and dialysis was continued for at least 2 hours or overnight.

[0063] 7. Protein quantification (BCA method protein concentration assay kit, Sangon Biotech (Shanghai) Co., Ltd.)

[0064] (1) Establishing a standard curve

[0065] First, dilute 5 mg / mL BSA to 0.5 mg / mL BSA to prepare a series of standard protein BSA concentration gradients (30 μL), as shown in Table 3 below:

[0066] Table 3. Standard Protein BSA Concentration Gradient

[0067]

[0068] (2) Prepare protein samples

[0069] Based on the protein electrophoresis results, the target protein was prepared with 10mM PBS at different dilutions, such as: undiluted, diluted 2 times, diluted 5 times, and diluted 10 times.

[0070] (3) Preparation of BCA working solution

[0071] Based on the required total volume of BCA working solution, take a quantitative amount of solution A: solution B = 50:1, mix well, and prepare BCA working solution.

[0072] (4) Protein concentration determination by enzyme-linked immunosorbent assay (ELISA)

[0073] Add 20 μL of BSA standard protein and target protein of various concentrations sequentially to the microplate, followed by 200 μL of BCA working solution. Perform two replicates for each sample. Incubate at 37°C for 30 min. After cooling to room temperature, measure A using a microplate reader. 562 value.

[0074] Using standard group of holes A 562 A standard curve is plotted in Microsoft Excel, with the average value on the ordinate and the corresponding protein concentration on the abscissa. Figure 2 According to two identical sample dilutions A 562The average value is used to calculate the protein concentration of the diluted sample from the standard curve. A sample with an appropriate dilution is selected to calculate the final protein concentration, and then the original sample protein concentration is calculated from the dilution factor.

[0075] The amino acid sequence (SEQ ID NO:1) of the target protein BolNSH1 is as follows:

[0076] MEFCNGGISNGDVLGLSSKREKLIIDTDPGIDDSMAIMMAFQTPELEIVGLTTVFGNVSTQDATRNALLLCEVAGFPDVPVAEGSSEPLKGGIPRVADFVHGKNGLGEVSVLPSRKKCDKSAAEFLVEKVSEYPGEVTILALGPLTNLAIAIKRDSSFASKVKKIVV LGGAFFALGNVNPAAEANIYGDPEAADVVFTCGADITVVGINITTQLKLTDDDLLALRDSKGKHAKLLSDMCKFYRDWHVKSDGVYGVYLHDPVSFVAVVRPDLFTYKKGVVRVETQGICVGHTLMDQGLKRWNGSNPWVGYSPVSVAWTVDVDGVLEYIKGMLMKP.

[0077] The amino acid sequence (SEQ ID NO:2) of the target protein of BolNSH2 is as follows:

[0078] MDSTMENCNGGISNGDVSGLSSKREKLIIDTDPGIDDSMAIMMAFQTRELDILGLTTVFGNVQTQDATRNALLLCEIAGFPDLPVAEGSSEPLKGGIPRVADFVHGKNGLGDVSVPSPCRKKCDKSAAEFLVDKVSQYPGEVTVLALGPLTNLAIAIKRDSSFASKVKKI VILGGAFFSLGNVNPAAEANIYNDPEAADVVFTCGADITVVGINITTQLMLSDDLLSLRESKGKHAKLLSDMCKFYRDWHVKSDGVYGVYLHDPVSFVAVVRPDLFTYKKGVVRVETQGICVGHTLMDQGLKRWNGSNPWVGYSPVSVAWTVDVDGVLEYIKGMLMKP.

[0079] The amino acid sequence (SEQ ID NO:3) of the target protein BolNSH3 is as follows:

[0080] MVGEQSKKIIIDTDPGIDDAMAIFVALNSPEVDVIGLTTIYGNVYTTLATRNALHLLEVAGRTDIPVAEGTHKTIMNGTKLRIADFVHGKDGLGNQNFPPPKGKPIEKSAPEFLVEQAKLHPGEITVVALGPLTNIALAIQLDPEFSKNVGQIVLLGGAFAVNGNVNPASEANIFGDPEAADIVFTCGADVIAVGINVTHQVIMTADDRDKLAASNGKLAQYLCKILGVYYSYHLDAYEIEGVYLHDPTTILAAFLPSLFTYTEGVVRVQTNGITRGLTLLYNNRKRFEEVTEWTDKPSVKVAVTVDAPAVLKLIMDRLMES。

[0081] The amino acid sequence of the target protein OsNSH1 (SEQ ID NO:4) is:

[0082] MGSNEQIHRDKLIIDTDPGIDDSMTILMAFRAPTVEIIGLTTIFGNTTTKNATQNALLLCERAGHPEVPVAEGSAEPLKGGEPRVADFVHGSDGLGNLFLPAPTSKKVDENAAEFMVNKVSQFPGEVSILALGPLTNVALAIKRDPSFASKVKKIVVLGGAFFAAGNVSPAAEANIYGDPEAADIVFTSGADVDVVGINITTQVCFTDEDLLELRNSKGKHAQFLCDMCQFYRDWHAESDGFHGIFLHDPVSFTALVHPEYFTFKKGVVRVETQGICTGHTLMDQGLKKWNSENPWSGYKPISVAWTVDVPNVLAFVKELLMAP。

[0083] The amino acid sequence of the target protein OsNSH2 (SEQ ID NO:5) is:

[0084] MTTTKKKLVIDTDPGIDDAMAIFVALRSPEVELLGLTTIFGNVYTTLATRNALHLLEAVGRTDIPVAEGSHVTIKKATKLRIASFVHGSDGLGNQNFPPPTGKPLDQSAAAFLVEQANLYPGQVTVVALGPLTNLALAIELDPSFPKKIGQIVILGGAYSVNGNVNPAAEANIFGDPDAADIVFTSGADILAVGINITHQVVLSDADREKLEQSDSKYARYLSKILGLYYDYHKDAYFIKGVYLHDPATLIAAVDPSLMTYTEGVVRVQTDGITKGLTVFDTTKKRYGEITAWTGKPTVKVAVTVDAPAVVEMIMQRLTTDD。

[0085] The amino acid sequence of the target protein OsNSH3 (SEQ ID NO: 6) is as follows:<000020​​​​Among them, the amino acid sequence similarity between BolNSH1 and OsNSH1 was 74%, the amino acid sequence similarity between BolNSH1 and OsNSH3 was 64%, and the amino acid sequence similarity between BolNSH3 and OsNSH2 was 73%. The final protein sample concentrations were: BolNSH1: 1020.066 μg / mL; BolNSH2: 803.750 μg / mL; BolNSH3: 862.206 μg / mL; OsNSH1: 792.325 μg / mL; OsNSH2: 889.981 μg / mL; OsNSH3: 732.406 μg / mL.

[0088] Example 2: In vitro enzymatic catalytic reaction

[0089] 1. In vitro enzymatic catalytic reaction

[0090] Prepare a 300 μL reaction system containing 10 mM PBS buffer (pH 7.4), 10 mM MgCl2, 1 mM DTT, 100 μM nicotinamide ribose or nicotinic acid ribose, and 10 μM enzyme (one of the six nucleoside hydrolases prepared in Example 1). The reaction temperature is 30 °C, and the reaction time is 10 min. After the reaction, add an equal volume of chloroform, mix well, and centrifuge at 12,000 rpm for 10 min. Repeat this process three times to terminate the enzyme reaction and remove the protein.

[0091] 2. HPLC detection results of enzymatic catalytic reactions

[0092] The results of in vitro enzymatic catalysis were analyzed using a Waters HPLC system with a ZORBAX SB-Aq C18 column (4.6 mm × 250 mm, 5 μm, Agilent Technologies). Detection method: Mobile phase A was 0.1% formic acid-water, mobile phase B was methanol, flow rate was 0.5 mL / min, column temperature was 25℃, and injection volume was 50 μL. Gradient elution conditions: 2 min B:A volume ratio 1:99, 25 min B:A volume ratio 30:70, 30 min B:A volume ratio 100:0, 35 min B:A volume ratio 100:0, 36 min B:A volume ratio 1:99, and the program ended at 40 min. Detection wavelength was 254 nm. The HPLC detection results of nicotinamide ribose catalyzed by BolNSH1, BolNSH2, and BolNSH3 are shown below. Figure 3As shown, "Boiled" indicates the corresponding protein was boiled as a negative control; "BolNSH1 / 2 / 3" indicates the catalytic reaction; "NR Standard" indicates nicotinamide ribose standard; and "NamStandard" indicates nicotinamide standard. The results show that BolNSH1, BolNSH2, and BolNSH3 can all catalyze the hydrolysis of nicotinamide ribose. The HPLC detection results of OsNSH1, OsNSH2, and OsNSH3 catalyzing nicotinamide ribose are shown below. Figure 4 As shown, the results indicate that OsNSH1, OsNSH2, and OsNSH3 can catalyze the hydrolysis of nicotinamide ribose to generate nicotinamide.

[0093] 3. LC-MS analysis of the results of the enzyme-catalyzed reaction

[0094] The results of in vitro enzymatic catalytic reactions were analyzed by LC-MS using a Waters Xevo TQ mass spectrometer equipped with an ACQUITYUPLC H-Class system and an ACQUITYUPLC BEHHSS T3 (2.10 mm × 100 mm, 1.8 μm; Waters) analytical column. Analytical methods: Mobile phase A was 0.1% formic acid in water, mobile phase B was methanol, flow rate was 0.3 mL / min, column temperature was 25 °C, and injection volume was 1 μL. Gradient elution conditions: 0 min B:A volume ratio 1:99, 6 min B:A volume ratio 1:99, 6.01 min B:A volume ratio 60:40, 9 min B:A volume ratio 60:40, 9.1 min B:A volume ratio 1:99, 13 min B:A volume ratio 1:99, and the program ended at 13 min. Positive ions were detected by mass spectrometry in selected ion monitoring (SIR) mode. LC-MS results of nicotinamide to nicotinamide catalyzed by nucleoside hydrolase are as follows: Figure 5 As shown in the figure, A represents the nicotinamide standard; B, C, and D represent the analysis of the product peaks catalyzed by BolNSH1, BolNSH2, and BolNSH3 in kale, respectively, for the nicotinamide ribose; and E, F, and G represent the analysis of the product peaks catalyzed by OsNSH1, OsNSH2, and OsNSH3 in rice, respectively. The figure shows that the product peaks catalyzed by nucleoside hydrolases for nicotinamide ribose have the same molecular weight as the nicotinamide standard. These results indicate that plant nucleoside hydrolases can catalyze the conversion of nicotinamide ribose to nicotinamide.

[0095] Example 3: Virus-induced gene silencing increases nicotinamide riboside content in kale

[0096] 1. Construction of virus-induced gene silencing vectors

[0097] Referring to the method of constructing a virus-induced gene silencing vector in cabbage by Xiao et al. (Xiao, ZL et al. (2020). An efficient virus-induced genesilencing (VIGS) system for functional genomics in Brassicas using a cabbageleafcurl virus (CaLCuV)-based vector. Planta, 252(3)), the following primer pairs were used to amplify 500bp fragments of the BolNSH1, BolNSH2 and BolNSH3 genes (nucleotide sequences such as SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9) in kale and ligated them into the PCVA vector (provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences).

[0098] Table 4 Primer sequences for amplifying the BolNSH1, BolNSH2, and BolNSH3 gene silencing fragments.

[0099]

[0100] 2. Construction of genetically modified kale

[0101] The method for constructing virus-induced gene silencing transgenic cabbage in cabbage was slightly modified based on the method of Xiao et al. (Xiao, ZL et al. (2020). An efficient virus-induced genesilencing (VIGS) system for functional genomics in Brassicas using a cabbageleafcurl virus (CaLCuV)-based vector. Planta, 252(3)).

[0102] (1) The PCVA vector containing the target gene, the empty PCVA vector, and the empty PCVB vector (provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences) were transformed into Agrobacterium GV3101, respectively.

[0103] (2) Select positive Agrobacterium clones and inoculate them into 2 mL of LB liquid medium (containing 50 μg / mL kanamycin and 25 μg / mL rifamycin), and incubate at 28℃ for 10-12 h;

[0104] (3) Add 200 μL of the above culture medium to 5 ml of LB liquid medium (containing 50 μg / mL kanamycin and 25 μg / mL rifamycin), and incubate at 28℃ for 6-10 h until OD. 600 It is 1.0;

[0105] (4) Collect 2 mL of the above culture medium, centrifuge at 6000 g for 8 min at 4℃, and discard the supernatant;

[0106] (5) Use 2 mL of infection buffer (containing 5 mg / mL) to precipitate the bacterial cells. D Resuspend in glucose, 10 mM MES, 100 μM Macetosyringone, 2 mM Na3PO4·12H2O, centrifuge at 4 °C, 6000 g for 8 min and discard the supernatant.

[0107] (6) The bacterial pellet was re-soaked in 2 mL of infection buffer (containing 5 mg / mL) D The bacterial culture was resuspended in a solution of glucose, 10 mM MES, 100 μM Macetosyringone, and 2 mM Na3PO4·12H2O, and then incubated at room temperature in the dark for 3-4 hours.

[0108] (7) The PCVA vector bacterial solution containing the target gene and the PCVB empty vector bacterial solution were mixed at a ratio of 1:1 (V / V), and the PCVA empty vector bacterial solution and the PCVB empty vector bacterial solution were mixed at a ratio of 1:1 (V / V) as a control. Then, 0.5 mL of the mixture was injected into the leaves of kale at the cotyledon stage using a 1 mL syringe, and the plants were kept in the dark for 24 h.

[0109] (8) Analyze gene silencing status after 3 weeks.

[0110] 3. Analysis of BolNSH1, BolNSH2 and BolNSH3 gene expression in transgenic kale

[0111] The expression of the BolNSH3 gene in transgenic kale was detected using real-time quantitative PCR (qRT-PCR). Total RNA was extracted from transgenic kale leaves, and cDNA was synthesized by reverse transcription using Vazyme's HIScript IIQ RT SuperMix for qPCR (+gDNA wiper). Quantitative reagents used were Vazyme's ChamQ Blue Universal SYBR qPCR Master Mix. Using Bol-Actin as an internal control gene, the specific primer pairs for the BolNSH1, BolNSH2, and BolNSH3 genes based on qRT-PCR are shown in Table 4. The reaction system (15 μL) consisted of: 7.5 μL of 2×ChamQ Universal SYBR qPCR Master Mix; 0.3 μL each of forward and reverse primers (10 μmol / L); 1 μL of cDNA; and 5.9 μL of ddH2O. Specific conditions were as follows: 95℃ pre-denaturation for 6 min; 95℃ for 10 s, 60℃ for 20 s, 40 cycles; three replicates per sample. Exploit 2 -ΔΔCT The method (Livak et al., 2001) was used to analyze the relative transcriptional levels of genes. The results are as follows: Figure 6 As shown in Figure A, the expression levels of the BolNSH1, BolNSH2, and BolNSH3 genes were significantly reduced.

[0112] Table 5 qRT-PCR primer sequences

[0113]

[0114] 4. Detection of nicotinamide ribose content in genetically modified kale

[0115] Leaves from transgenic kale with reduced gene expression were collected, frozen in liquid nitrogen, and then ground into powder using a mortar and pestle. A certain amount of 50% methanol (W:V = 1:5) was added to the powder, and nicotinamide ribose was extracted by sonication for 30 min. The mixture was then centrifuged at 12000 rpm for 30 min, and the supernatant was collected. Finally, the content of nicotinamide ribose in the transgenic kale was quantitatively analyzed by LC-MS. Figure 6 As shown in B, the nicotinamide ribose content increased by 1.5-2.5 times, greatly enhancing the nutritional value of kale.

Claims

1. Kale nucleoside hydrolase BolNSH1、BolNSH2 or BolNSH3 The application of gene silencing vectors or silencing strains in increasing the nicotinamide ribose content in kale, characterized by: The kale nucleoside hydrolase BolNSH1、 BolNSH2 and BolNSH3 The nucleotide sequence of the gene is shown in SEQ ID NO:7~9, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:1~3.

2. The application according to claim 1, characterized in that, The silent strain is... BolNSH1、BolNSH2 or BolNSH3 The gene silencing vector was introduced into Agrobacterium.

3. The application according to claim 1, characterized in that, Used for building BolNSH1 The primer sequences used for the gene silencing vector are BolNSH1-vigas-F: GCGAGCTCAGATCTGGATCCTCTAGACGATGATAGCATGGCGATAA; BolNSH1-vigas-R: CGGCTAGCGGTACCCTCGAGTCTAGAAACACAACATCAGCTGCTTC.

4. The application according to claim 1, characterized in that, Used for building BolNSH2 The primer sequences used for the gene silencing vector are: BolNSH2-vigas-F: GCGAGCTCAGATCTGGATCCTCTAGACGATGATAGCATGGCGATAA; BolNSH2-vigas-R: CGGCTAGCGGTACCCTCGAGTCTAGAAACACCACATCAGCTGCTTC.

5. The application according to claim 1, characterized in that, Used for building BolNSH3 The primer sequences used for the gene silencing vector are BolNSH3-vigas-F: GCGAGCTCAGATCTGGATCCTCTAGAGGACTCACTACTATCTACGGC; BolNSH3-vigas-R: CGGCTAGCGGTACCCTCGAGTCTAGAAACTTGATGAGTCACGTTGA.

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