Application of plant nucleoside hydrolase protein and coding gene thereof in regulating and controlling content of plant nicotinamide ribose

Abstract

The invention discloses a plant nucleoside hydrolase protein and application of a coding gene of the plant nucleoside hydrolase protein in regulation and control of the content of nicotinamide ribose in a plant. The plant nucleoside hydrolase protein comprises kale nucleoside hydrolase BolNSH1, kale nucleoside hydrolase BolNSH2 and kale nucleoside hydrolase BolNSH3 and rice nucleoside hydrolase OsNSH1, OsNSH2 and OsNSH3; the amino acid sequences of the kale nucleoside hydrolases BolNSH1, BolNSH2 and BolNSH3 are as shown in SEQ ID NO: 1-3, and the amino acid sequences of the rice nucleoside hydrolases OsNSH1, OsNSH2 and OsNSH3 are as shown in SEQ ID NO: 4-6. The invention discovers 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 for the first time. The gene is subjected to silent expression in kale, the content of nicotinamide ribose in the kale is increased by 1.5-2.5 times, and the value of kale plants is further increased. The new gene and the new method provided by the invention provide a basis for creating kale germplasm with high nicotinamide ribose content, and have wide application prospects.

Description

Technical Field

[0001] The invention relates to application of a plant nucleoside hydrolase protein and a coding gene thereof in regulating the content of nicotinamide ribose in plants, and belongs to the field of genetic engineering. Background Art

[0002] Kale (Brassica oleraceae L.var.acephala DC.) is a herbaceous plant of the genus Brassica in the family Cruciferae. 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, carotenoids, etc.) have important medicinal values, including anti-cancer, anti-aging, improvement of gastric ulcers, protection of the liver, etc. ( et al., 2023). Therefore, increasing the content of important natural products in kale is of great significance to improving its quality.

[0003] Nicotinamide ribose (NR) is a vitamin B3 derivative naturally present in milk and is an important coenzyme of redox reactions in the body - Nicotinamide adenine dinucleotide (NAD + ) precursor. In recent years, more and more studies have shown that NR plays an important role in the treatment of cardiovascular diseases, neurodegenerative diseases, metabolic diseases, life extension and immune regulation. Nicotinamide riboside can activate NMRK2 and SIRTs to maintain NAD + The homeostasis of NR can protect the cardiac function of mice with heart failure and limit adverse remodeling (Mehmel et al., 2020); NR supplementation can treat Alzheimer's disease and Parkinson's disease caused by DNA damage and oxidative stress (Gong et al., 2013; Berven et al., 2023); Nicotinamide riboside can effectively improve the muscle atrophy mouse model by increasing muscle strength, rejuvenating aging muscle stem cells, and reducing inflammation and fibrosis (Zhang et al., 2016); Nicotinamide riboside can effectively improve the muscle atrophy mouse model by increasing NAD + NR levels 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 on SARS-CoV-2 and COVID-19 infection. Nicotinamide riboside is present at low levels in kale. If its degradation pathway can be blocked to further increase the content of nicotinamide riboside and obtain it from food, it will promote the application of nicotinamide riboside.

[0004] In plants, nicotinamide riboside degradation and NAD +The salvage biosynthesis pathway of nicotinamide is closely connected to the nicotinamide riboside. The research on this pathway in plants is mainly carried out by isotope tracing. Ashihara et al. used isotope tracing to speculate that the degradation of nicotinamide riboside to nicotinamide in plants is catalyzed by nucleosidases, but they did not clone the relevant genes (Ashihara et al., 2012). In plants, only five nucleosidases have been reported in Arabidopsis: AtNSH1, AtNSH2, AtNSH3, AtNSH4, and AtNSH5 (Jung et al., 2011). Among them, 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 riboside and their application in increasing the content of nicotinamide riboside in plants have not been reported. Summary of the invention

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

[0006] Technical solution: The present invention provides an application of a plant nucleoside hydrolase protein in regulating the nicotinamide riboside content of a plant, wherein the plant nucleoside hydrolase protein is kale nucleoside hydrolase BolNSH1, BolNSH2 and BolNSH3 and rice nucleoside hydrolase OsNSH1, OsNSH2 and OsNSH3; the amino acid sequences of kale nucleoside hydrolase BolNSH1, BolNSH2 and BolNSH3 are shown in SEQ ID NOs: 1 to 3, and the amino acid sequences of rice nucleoside hydrolase OsNSH1, OsNSH2 and OsNSH3 are shown in SEQ ID NOs: 4 to 6.

[0007] The present invention also provides the use of the gene encoding the plant nucleoside hydrolase protein in regulating the nicotinamide ribose content of plants, the nucleotide sequences of the kale nucleoside hydrolase BolNSH1, BolNSH2 and BolNSH3 genes are shown in SEQ ID NOs: 7 to 9, and the nucleotide sequences of the rice nucleoside hydrolase OsNSH1, OsNSH2 and OsNSH3 are shown in SEQ ID NOs: 10 to 12.

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

[0009] The present invention also provides the use of a silencing vector or a silencing strain of kale nucleoside hydrolase 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 silencing vectors of BolNSH1, BolNSH2 and BolNSH3 genes into Agrobacterium.

[0012] Furthermore, the primer sequences used to construct the BolNSH1 gene silencing vector are shown in SEQ ID NOs: 13-14.

[0013] Furthermore, the primer sequences used to construct the BolNSH2 gene silencing vector are shown in SEQ ID NOs: 15-16.

[0014] Furthermore, the primer sequences used to construct the BolNSH2 gene silencing vector are shown in SEQ ID NOs: 15-16.

[0015] The present invention also provides the use of a silencing vector or a silencing strain of rice nucleoside hydrolase OsNSH1, OsNSH2 and OsNSH3 genes in increasing the nicotinamide ribose content in rice.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: The present invention first discovered 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. The present invention silences the expression of the gene in kale, increases the content of nicotinamide ribose therein by 1.5-2.5 times, and further increases the value of kale plants. The new gene and new method provided by the present invention provide a basis for creating kale germplasm with high nicotinamide ribose content, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the electrophoresis diagram of kale and rice nucleoside hydrolase proteins.

[0018] Figure 2 The standard curve for determining protein concentration by ELISA.

[0019] Figure 3 This is the HPLC analysis chart of kale nucleoside hydrolases BolNSH1, BolNSH2, and BolNSH3 catalyzing nicotinamide riboside in vitro.

[0020] Figure 4This is the HPLC analysis chart of rice nucleoside hydrolases OsNSH1, OsNSH2, and OsNSH3 catalyzing nicotinamide riboside in vitro.

[0021] Figure 5 These are the LC-MS results of kale and rice nucleoside hydrolases catalyzing nicotinamide riboside to produce nicotinamide in vitro.

[0022] Figure 6 These are the results of gene expression levels and NR content detection of BolNSH1, BolNSH2 and BolNSH3 in gene-silenced kale induced by VIGS. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0024] Example 1: Cloning of plant nucleoside hydrolase genes and expression of target proteins

[0025] 1. Cloning of plant nucleoside hydrolase genes

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

[0027] Table 1 Primer sequences for target gene amplification

[0028]

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

[0030] ATGGAGTTTTGCAACGGTGGGATCTCAAACGGCGACGTTTTGGGTCTCTCTTCTAAACGCGAGAAGCTCATTATCGATACAGACCCAGGAATCGATGATAGCATGGCGATAATGATGGCGTTTCAAACACCAGAGCTGGAGATAGTAGGACTCACTACCGTCTTTGGTAATGTTTCTACGCAAGATGCTACCCGCAACGCCTTACTCCTGTGTGAGGTTGCTGGCTTCCCTGATGTTCCTGTGGCAGAAGGAAGCTCCGAACCCTTAAAGGGTGGGATTCCACGTGTTGCTGATTTTGTGCACGGTAAAAACGGACTAGGAGAGGTCTCTGTTCCTCTTCCTAGTAGAAAGAAATGTGACAAAAGTGCAGCTGAGTTTCTAGTTGAGAAGGTCTCCGAGTATCCTGGTGAAGTCACCATCCTCGCCCTTGGACCTCTAACCAACCTTGCAATAGCCATCAAGCGAGATAGTTCATTTGCGAGTAAGGTGAAGAAAATTGTTGTACTCGGTGGAGCTTTCTTTGCTTTGGGAAATGTCAATCCTGCAGCTGAGGCCAATATATATGGTGACCCGGAAGCAGCTGATGTTGTGTTCACGTGTGGTGCTGATATAACTGTTGTTGGGATAAACATCACAACACAACTTAAACTAACAGATGATGACCTCTTAGCCCTGCGTGACTCCAAGGGCAAACACGCTAAGTTGTTAAGTGACATGTGCAAGTTTTATAGAGACTGGCACGTCAAGTCTGATGGTGTTTACGGAGTTTACCTCCATGACCCGGTCAGCTTTGTGGCTGTAGTACGGCCTGATCTATTCACGTATAAGAAAGGCGTTGTTAGGGTGGAGACTCAAGGAATATGTGTTGGCCACACTCTCATGGACCAAGGCTCAAGAGATGGAATGGGAGCAATCCATGGGTGGGATATTCACCAGTATCAGTGGCATGGACGGTAGATGTAGATGGAGTGTTGGAATATATCAAAGGCATGCTGATGAAGCCTTAA

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

[0032]

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

[0034] ATGGTGGGAGAGCAGAGCAAGAAGATTATCATCGATACTGATCCTGGAATCGATGATGCAATGGCGATATTCGTGGCGCTAAACTCACCAGAGGTTGATGTGATTGGACTCACTACTATCTACGGCAACGTTTACACCACTCTCGCCACTCGTAACGCCTTGCATTTGTTGGAGGTTGCGGGTAGGACAGACATTCCTGTAGCTGAAGGAACACATA AAACTATCATGAATGGCACAAAGCTTCGAATAGCTGACTTCGTTCACGGTAAAGATGGGCTTGGCAACCAAAACTTCCCTCCACCTAAAGGGAAGCCAATTGAAAAATCTGCACCTGAGTTCTTAGTTGAACAAGCTAAGCTTCACCCTGGTGAAATCACTGTTGTTGCTTTGGGACCTCTCACAAATATAGCACTGGCTATTCAGCTTGATCCTGAGTTTTCCAAAAATGTTGGACAAATTGTTCTTCTTGGTGGAGCATTTGCAGTAAATGGAAATGTGAATCCAGCATCAGAAGCTAATATTTTTGGTGATCCAGAAGCTGCGGATATTGTGTTCACATGTGGTGCTGATGTGATTGCTGTGGGAATCAACGTGACTCATCAAGTTATTATGACAGCTGATGATAGGGACAAATTGGCAGCATCGAACGGGAAATTAGCTCAATACCTCTGTAAAATCCTTGGTGTGTACTATTCTTATCATCTTGACGCTTATGAGATTGAAGGTGTTTATCTTCATGATCCTACAACGATACTTGCGGCTTTCCTTCCTTCTCTATTCACTTATACTGAAGGAGTTGTTAGAGTGCAAACTAACGGTATCACTAGAGGACTCACTTTACTGTACAACAATCGAAAAAGGTTTGAGGAAGTGACCGAGTGGACTGACAAACCATCGGTAAAAGTGGCAGTGACGGTTGATGCACCAGCGGTCTTGAAGCTCATAATGGATAGGCTTATGGAGTCTTGA

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

[0036] ATGGGGTCGAACGAGCAGATCCACCGGGACAAGCTCATCATCGACACGGATCCCGGCATCGATGATAGCATGACAATCCTTATGGCGTTCAGAGCTCCAACTGTGGAGATCATAGGGCTCACAACCATATTTGGCAACACCACCACAAAGAATGCGACTCAGAATGCGCTCTTGCTGTGTGAGAGAGCAGGACATCCTGAGGTTCCAGTAGCAGAGGGCAGCGCAGAGCCTCTCAAGGGAGGAGAGCCACGTGTTGCTGACTTTGTTCATGGGTCTGATGGCCTGGGGAATTTGTTTCTTCCTGCACCCACTAGTAAGAAGGTTGATGAAAATGCTGCTGAGTTCATGGTTAATAAGGTCTCTCAATTTCCTGGAGAAGTTTCTATACTCGCATTGGGACCCCTGACAAACGTGGCGCTGGCCATCAAAAGGGACCCTTCCTTTGCAAGTAAGGTGAAGAAAATAGTTGTACTGGGTGGTGCTTTCTTTGCGGCTGGAAATGTCAGCCCAGCTGCTGAAGCAAATATATATGGAGATCCAGAAGCAGCTGACATAGTTTTTACCTCAGGAGCAGATGTTGATGTGGTTGGCATTAACATAACAACTCAAGTCTGTTTTACAGATGAGGATCTCCTGGAGCTAAGAAACTCAAAAGGGAAGCATGCACAGTTCTTATGTGACATGTGCCAGTTCTATAGAGACTGGCATGCCGAATCCGATGGCTTTCATGGAATTTTCCTCCATGATCCTGTGAGCTTCACTGCCC TAGTTCACCCTGAGTACTTCACGTTCAAGAAGGGTGTTGTGAGAGTAGAAACCCAGGGCATTTGCACTGGTCACACTTTAATGGACCAAGGATTGAAAAAGTGGAATTCAGAGAATCCATGGTCTGGCTACAAGCCAATCTCAGTTGCATGGACAGTTGATGTGCCAAATGTTCTCGCATTCGTGAAGGAACTCCTCATGGCTCCATGA

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

[0038] ATGACGACGACGAAGAAGAAGCTCGTCATCGACACCGACCCGGGAATCGATGACGCCATGGCCATCTTCGTGGCGCTGAGGTCGCCGGAGGTGGAGCTGCTGGGCCTCACCACCATCTTCGGCAACGTCTACACCACCCTCGCCACCCGCAACGCCCTCCACCTGCTGGAGGCTGTTGGGAGGACTGACATCCCCGTGGCAGAGGGATCCCATGTAACAATCAAGAAAGCCACCAAGCTACGGATTGCGAGCTTTGTTCATGGCTCGGATGGTCTAGGAAATCAGAACTTTCCTCCGCCAACTGGGAAGCCCTTAGATCAATCGGCCGCCGCGTTTCTTGTCGAGCAGGCAAATCTGTACCCTGGACAAGTTACTGTGGTTGCCCTTGGTCCACTTACCAACCTAGCTTTGGCTATAGAACTTGATCCTTCTTTCCCGAAGAAGATTGGGCAGATTGTGATCCTTGGTGGTGCGTATTCAGTTAATGGAAATGTCAACCCTGCAGCTGAGGCAAATATATTTGGTGACCCTGATGCAGCTGATATAGTTTTCACCTCCGGTGCTGATATTTTGGCTGTGGGAATAAATATTACACACCAAGTAGTTCTTTCTGATGCTGACCGGGAAAAGCTTGAACAGTCTGATAGCAAATATGCCCGATACTTGAGCAAGATCCTGGGTCTTTATTATGATTACCACAAGGACGCCTACTTCATAAAAGGAGTGTATCTTCACGATCCAGCAACACTTATTGCTGCGGTAGATCCGTCACTGATGACTTACACGGAAGGTGTAGTGAGGGTGCAGACTGATGGCATCACAAAGGGTCTCACAGTTTTCGACACAACCAAGAAAAGGTATGGGGAGATAACTGCGTGGACCGGTAAGCCAACCGTGAAGGTTGCTGTGACCGTTGATGCTCCTGCCGTTGTGGAGATGATCATGCAGAGGCTTACGACGGATGATTAG

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

[0040] ATGGATCTGCAGGAGGCGGCGATGGAAGCCAGGAACGGCCATCGGATCCCGCCGACGGAGGAGAAGGTCATCATCGACACGGATCCAGGGATCGATGACAGCGTGGCGATCATGATGGCGTTCGAAGCTCCCGGCGTGAAGGTCGTCGGACTCACCACCATCTTCGGCAACTGCACTACCTCCCACGCCACCCGCAATGCCCTCATCCTGTGCGACAGAGCAGGCCGTCCTGAGGTGCCGGTAGCAGAGGGCAGCGCTGAGCCTCTCAAGGGAGGGAAACCTCACGTTGCTGATTTTGTTCATGGATCGGATGGACTTGGTAACACGTCCTTTCCTGATCCTACCACCACCAACAAAGTTGAGCAGTCAGCTGCAGAGTTTCTGGTTGATAAGGTATCAGAATCTCCAGGAGAGATCTCTGTACTCGCCTTGGGCCCTCTCACTAATATCGCATTGGCCATGAAGAAAGACTCCTCCTTTGCAAGCAAGGTTAAGAGGATAGTTGTGCTGGGTGGAGCTTTCTTTGCAGCTGGGAATGCCACCCCTTCAGCTGAAGCAAATATCCATAGCGACCCAGAAGCAGCTGACATAGTTTTCACTTCTGGGGCAGATATCTACGTAGTTGGCCTTAACATAACAACCCAAGTCTACTTCACAGACAAGGATATGTTGGAGCTAAGGAACTCGAAAGGGAAGCACGCACAATTCCTTTGTGACATCTGCAAGTTCTACAGAGACTGGCATGTCCACTCCTACGGCGTCGATGGTCCGCACATGAAACATCTCTCCTTTCCTTCTTACAAGTTCAGGCTTTGGGAAATTAACGTTCCAGCTCACATTTGTGTGATTTCTTCAGCTCTTTTTCTCCATGATCCTGTGAGTTTCACCGCGCTAGTTCACCCCGAGTACTTCACGTTCAAGAAGGGCGTGGTGAGGGTTGAGACCCAGGGCATCTGCAAGGGGCATACTTCCATGGACATGGGACTGAAGAAGTGGAATTCAGACAACCCGTGGACTGGTTACTCTCCGATCTCGGTTGCTTGGACTGTTGATGTTCCTAAGGTTCTGGCATATGCCAAGGAGCTACTCTTCAACGCACAATGA

[0041] 2. Construction of recombinant vector

[0042] The pET29a(+) (laboratory collection) vector was double-digested with Nde I and Xho I, and the purified digestion product was ligated with the recovered target gene fragment and transformed into Escherichia coli DH5a competent cells. The cells were spread on LB solid medium (containing 50 μg / mL of kanamycin), and single clones were picked for colony PCR identification using pET29a-F / pET29a-R primer pair (Table 2). Positive clones were screened, plasmids were extracted and sequenced, and the recombinant vectors were correctly sequenced.

[0043] Table 2 Primer sequences for identification of recombinant vectors

[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 a single clone of recombinant E. coli BL21 (DE3) and place it in 3 mL of LB medium containing kanamycin (Kan) and culture it at 37°C with shaking at 220 rpm overnight. Add 2 mL of activated bacterial solution (concentration of 1×10 6 ~10 7 cfu / ml) were transferred to a 200mL LB medium bottle at a ratio of 1:100 and cultured in a shaking incubator at 37°C at 220rpm. After 2.5-3 hours, 60μL 1M IPTG inducer was added to the 200mL medium and induced at 16°C and 180rpm for 15 hours. The remaining 1mL of bacterial solution was used to preserve the bacteria.

[0049] (2) Place 200 mL of bacterial solution into a 50 mL round-bottom centrifuge tube and centrifuge at 4°C, 12,000 rpm for 1 min. Discard the supernatant and collect the bacterial cells.

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

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

[0052] (5) The bacterial cells were disrupted using an American Branson Sonifier SFX550 ultrasonic disruptor with the following parameters set: PULSE, 30% power, ON 1 s, OFF 2 s, TOTAL 3 min.

[0053] (6) Obtaining crude enzyme solution: The ultrasonically disrupted sample was centrifuged at 4°C, 12,000 rpm, for 30 min. The supernatant was transferred to a clean centrifuge tube. The supernatant was the crude enzyme solution.

[0054] 5. Purification of target protein (the following operations are all carried out in a 4°C refrigerator)

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

[0056] (2) Target protein binding column: After the equilibration buffer has run out, add all the crude enzyme solution to the chromatography column and flow through it 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 the target protein: Add 3 mL of Elution Buffer (50 mM Na3PO4, 300 mM NaCl, 500 mM imidazole, pH 7.4) to the chromatography column to elute the target protein.

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

[0060] (6) Collect the target protein and use SurePAGE TM Precast gel (concentration 4-20%, GenScript Biotech Co., Ltd.) was used for electrophoresis to obtain bands of corresponding sizes, such as Figure 1 As shown in the figure, M represents 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 is the target protein band of BolNSH3, with a size of 34.7 KDa; Figure 1 D is the target protein band of OsNSH1, with a size of 36.1 KDa; Figure 1 E is the target protein band of OsNSH2, with a size of 34.5 KDa; Figure 1F is 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 in a regenerated cellulose dialysis bag (Shanghai Yuanye Biotechnology Co., Ltd.), placed in 300 mL of pre-cooled dialysis solution (10 mM PBS (pH 7.4)), and placed in a refrigerator at 4°C. The dialysis solution was replaced after 2-4 hours, and the dialysis was continued for at least 2 hours or ended after dialysis overnight.

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

[0064] (1) Develop a standard curve

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

[0066] Table 3 Standard protein BSA concentration gradient

[0067]

[0068] (2) Prepare protein samples

[0069] According to the results of protein electrophoresis, 10 mM PBS was used to prepare the target protein at different dilution times, such as: no dilution, 2-fold dilution, 5-fold dilution, and 10-fold dilution.

[0070] (3) Preparation of BCA working solution

[0071] According to the required total volume of BCA working solution, quantitatively take solution A: solution B = 50:1, mix well, and make BCA working solution.

[0072] (4) Determination of protein concentration by ELISA

[0073] Add 20 μL of BSA standard protein and target protein of various concentrations to the ELISA plate in sequence, then add 200 μL BCA working solution, and set up 2 replicates for each sample. Incubate in a 37°C incubator for 30 minutes. After cooling to room temperature, measure A with an ELISA reader. 562 value.

[0074] The standard group of holes A 562 The average value is the ordinate, and the corresponding protein concentration is the abscissa. The standard curve is drawn in Microsoft Excel software ( Figure 2 ). Based on two identical sample dilutions A 562The protein concentration of the diluted sample is calculated on the standard curve by taking the average of the values. The final sample protein concentration is calculated by selecting a sample with an appropriate dilution, and then the original sample protein concentration is calculated by the dilution multiple.

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

[0076] MEFCNGGISNGDVLGLSSKREKLIIDTDPGIDDSMAIMMAFQTPELEIVGLTTVFGNVSTQDATRNALLLCEVAGFPDVPVAEGSSEPLKGGIPRVADFVHGKNGLGEVSVLPSRKKCDKSAAEFLVEKVSEYPGEVTILALGPLTNLAIAIKRDSSFASKVKKIVV LGGAFFALGNVNPAAEANIYGDPEAADVVFTCGADITVVGINITTQLKLTDDDLLALRDSKGKHAKLLSDMCKFYRDWHVKSDGVYGVYLHDPVSFVAVVRPDLFTYKKGVVRVETQGICVGHTLMDQGLKRWNGSNPWVGYSPVSVAWTVDVDGVLEYIKGMLMKP.

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

[0078] MDSTMENCNGGISNGDVSGLSSKREKLIIDTDPGIDDSMAIMMAFQTRELDILGLTTVFGNVQTQDATRNALLLCEIAGFPDLPVAEGSSEPLKGGIPRVADFVHGKNGLGDVSVPSPCRKKCDKSAAEFLVDKVSQYPGEVTVLALGPLTNLAIAIKRDSSFASKVKKI VILGGAFFSLGNVNPAAEANIYNDPEAADVVFTCGADITVVGINITTQLMLSDDLLSLRESKGKHAKLLSDMCKFYRDWHVKSDGVYGVYLHDPVSFVAVVRPDLFTYKKGVVRVETQGICVGHTLMDQGLKRWNGSNPWVGYSPVSVAWTVDVDGVLEYIKGMLMKP.

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

[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:

[0086] MDLQEAAMEARNGHRIPPTEEKVIIDTDPGIDDSVAIMMAFEAPGVKVVGLTTIFGNCTTSHATRNALILCDRAGRPEVPVAEGSAEPLKGGKPHVADFVHGSDGLGNTSFPDPTTTNKVEQSAAEFLVDKVSESPGEISVLALGPLTNIALAMKKDSSFASKVKRIVVLGGAFFAAGNATPSAEANIHSDPEAADIVFTSGADIYVVGLNITTQVYFTDKDMLELRNSKGKHAQFLCDICKFYRDWHVHSYGVDGPHMKHLSFPSYKFRLWEINVPAHICVISSALFLHDPVSFTALVHPEYFTFKKGVVRVETQGICKGHTSMDMGLKKWNSDNPWTGYSPISVAWTVDVPKVLAYAKELLFNAQ

[0087] Among them, the amino acid sequence similarity between BolNSH1 and OsNSH1 is 74%, the amino acid sequence similarity between BolNSH1 and OsNSH3 is 64%, and the amino acid sequence similarity between BolNSH3 and OsNSH2 is 73%. The final concentrations of the protein samples were BolNSH1: 1020.066μg / mL; BolNSH2: 803.750μg / mL; BolNSH3: 862.206μg / mL; OsNSH1: 792.325μg / mL; OsNSH2: 889.981μg / mL; OsNSH3: 732.406μg / mL.

[0088] Example 2: In vitro enzymatic reaction

[0089] 1. In vitro enzymatic reaction

[0090] A 300 μL reaction system was prepared, which contained 10 mM PBS buffer (pH 7.4), 10 mM MgCl2, 1 mM DTT, 100 μM nicotinamide riboside or nicotinic acid riboside, and 10 μM enzyme (the 6 nucleoside hydrolases prepared in Example 1), the reaction temperature was 30°C, and the reaction time was 10 min. After the reaction was completed, an equal volume of chloroform was added, mixed, and centrifuged at 12,000 rpm for 10 min, repeated 3 times to terminate the enzyme reaction and remove the protein.

[0091] 2. HPLC detection of enzyme catalytic reaction results

[0092] The results of in vitro enzymatic catalytic reactions were analyzed using a Waters HPLC system and a ZORBAX SB-Aq C18 analytical 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, the flow rate was 0.5 mL / min, the column oven temperature was 25°C, and the injection volume was 50 μL. Gradient elution conditions: 2 min B to A volume ratio of 1:99, 25 min B to A volume ratio of 30:70, 30 min B to A volume ratio of 100:0, 35 min B to A volume ratio of 100:0, 36 min B to A volume ratio of 1:99, and the program ended at 40 min. The detection wavelength was 254 nm. The HPLC detection results of nicotinamide riboside catalyzed by BolNSH1, BolNSH2 and BolNSH3 are shown in the figure. Figure 3As shown, Boiled means the corresponding protein is boiled as a negative control; BolNSH1 / 2 / 3 means catalytic reaction; NR Standard means nicotinamide riboside standard; NamStandard means nicotinamide standard; the results show that BolNSH1, BolNSH2 and BolNSH3 can catalyze the hydrolysis of nicotinamide riboside; OsNSH1, OsNSH2 and OsNSH3 catalyze the nicotinamide riboside HPLC detection results as shown Figure 4 As shown, the results showed that OsNSH1, OsNSH2 and OsNSH3 could catalyze the hydrolysis of nicotinamide riboside to produce nicotinamide.

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

[0094] The results of in vitro enzymatic catalytic reactions were analyzed by LC-MS using a Waters Xevo TQ mass spectrometer equipped with an ACQUITY UPLC H-Class system and an ACQUITY UPLC BEHHSS T3 (2.10 mm × 100 mm, 1.8 μm; Waters) analytical column. Analytical method: Mobile phase A was 0.1% formic acid in water, mobile phase B was methanol, flow rate was 0.3 mL / min, column oven temperature was 25 °C, and injection volume was 1 μL. Gradient elution conditions: 0 min B to A volume ratio of 1:99, 6 min B to A volume ratio of 1:99, 6.01 min B to A volume ratio of 60:40, 9 min B to A volume ratio of 60:40, 9.1 min B to A volume ratio of 1:99, 13 min B to A volume ratio of 1:99, 13 min program ended. Mass spectrometry detection was performed using positive ions in selected ion monitoring (SIR) mode. The LC-MS results of nicotinamide riboside catalyzed by nucleoside hydrolase to generate nicotinamide are as follows Figure 5 As shown. Among them, A is a nicotinamide standard, B, C, and D are the analysis of the product peaks of nicotinamide riboside catalyzed by kale BolNSH1, BolNSH2, and BolNSH3, respectively, and E, F, and G are the analysis of the product peaks of nicotinamide riboside catalyzed by rice OsNSH1, OsNSH2, and OsNSH3, respectively. It can be seen from the figure that the product peak of nicotinamide riboside catalyzed by nucleoside hydrolase has the same molecular weight as the nicotinamide standard. The above results show that plant nucleoside hydrolases can catalyze nicotinamide riboside to produce nicotinamide.

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

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

[0097] Referring to the construction method of the 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 500 bp fragments of the BolNSH1, BolNSH2 and BolNSH3 genes in kale (nucleotide sequences such as SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9) and ligated into the PCVA vector (provided by the Institute of Vegetable and Floriculture, Chinese Academy of Agricultural Sciences).

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

[0099]

[0100] 2. Construction of transgenic kale

[0101] The construction method of virus-induced gene silencing transgenic cabbage in cabbage was slightly modified according to 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) PCVA vector containing the target gene, PCVA empty vector, and PCVB empty 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 LB liquid medium (containing 50 μg / mL kanamycin and 25 μg / mL rifamycin) and culture at 28°C for 10-12 h;

[0104] (3) Take 200 μL of the above culture solution and add it to 5 ml of LB liquid medium (containing 50 μg / mL kanamycin and 25 μg / mL rifamycin), and culture at 28°C for 6-10 h until the OD 600 is 1.0;

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

[0106] (5) The bacterial pellet was precipitated with 2 mL of infection buffer (containing 5 mg / mL D -glucose, 10 mM MES, 100 μM etosyringone, 2 mM Na3PO4·12H2O), centrifuged at 6000 g for 8 min at 4 °C, and the supernatant was discarded;

[0107] (6) The bacterial pellet was again inoculated with 2 mL of infection buffer (containing 5 mg / mL D -glucose, 10 mM MES, 100 μM etosyringone, 2 mM Na3PO4·12H2O) and then incubate the bacterial solution at room temperature in the dark for 3-4 h;

[0108] (7) PCVA vector bacterial solution containing the target gene and PCVB empty vector bacterial solution were mixed at a ratio of 1:1 (V / V), and PCVA empty vector bacterial solution and 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 kale leaves 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 after 3 weeks.

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

[0111] Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of the BolNSH3 gene in transgenic kale. Total RNA from transgenic kale leaves was extracted, and then reverse transcribed to synthesize cDNA using Vazyme's HIScript IIQ RT SuperMixfor qPCR (+gDNA wiper). The quantitative reagent used was Vazyme's ChamQ BlueUniversal SYBR qPCR Master Mix. Bol-Actin was used as the internal reference gene. The specific primer pairs for BolNSH1, BolNSH2 and BolNSH3 genes based on qRT-PCR are shown in Table 4. Reaction system (15μL): 2×ChamQ Universal SYBRqPCR Master Mix*7.5μL; forward and reverse primers (10μmol / L) 0.3μL each; cDNA 1μL; ddH2O 5.9μL. The specific conditions are as follows: 95℃ pre-denaturation for 6min; 95℃10s, 60℃20s, 40 cycles; 3 replicates for each sample. Exploit 2 -ΔΔCT The relative transcription level of genes was analyzed by the method (Livak et al., 2001). Figure 6 As shown in A, the expression levels of BolNSH1, BolNSH2 and BolNSH3 genes were significantly decreased.

[0112] Table 5 qRT-PCR primer sequences

[0113]

[0114] 4. Detection of Nicotinamide Riboside Content in Genetically Modified Kale

[0115] The leaves of transgenic kale with reduced gene expression were collected, frozen in liquid nitrogen and ground into powder using a mortar. A certain amount of 50% methanol (W:V=1:5) was added to the powder, and nicotinamide riboside was extracted by ultrasound for 30 minutes. The supernatant was collected and finally LC-MS was used to quantitatively analyze the content of nicotinamide riboside in transgenic kale. Figure 6 As shown in B, the nicotinamide riboside content increased by 1.5-2.5 times, greatly improving the edible value of kale.

Claims

1. Use of a plant nucleoside hydrolase protein in regulating the content of nicotinamide riboside in plants, characterized in that: 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 NOs: 1 to 3, and the amino acid sequences of rice nucleoside hydrolases OsNSH1, OsNSH2 and OsNSH3 are shown in SEQ ID NOs: 4 to 6.

2. Use of the gene encoding the plant nucleoside hydrolase protein according to claim 1 in regulating the content of nicotinamide riboside in plants, characterized in that: The nucleotide sequences of the kale nucleoside hydrolase genes BolNSH1, BolNSH2 and BolNSH3 are shown in SEQ ID NOs: 7-9, and the nucleotide sequences of the rice nucleoside hydrolase genes OsNSH1, OsNSH2 and OsNSH3 are shown in SEQ ID NOs: 10-12.

3. The use according to claim 1 or 2, characterized in that: The plants are kale and rice.

4. Application of silencing vectors or silencing strains of kale nucleoside hydrolase BolNSH1, BolNSH2 and BolNSH3 genes in increasing the nicotinamide riboside content in kale.

5. The use according to claim 4, characterized in that: The silencing vector is obtained by constructing the nucleotide sequences of the BolNSH1, BolNSH2 and BolNSH3 genes into the vector PCVA.

6. The use according to claim 4, characterized in that: The silenced strain is obtained by introducing the silenced vectors of BolNSH1, BolNSH2 and BolNSH3 genes into Agrobacterium.

7. The use according to claim 5, characterized in that: The primer sequences used to construct the BolNSH1 gene silencing vector are shown in SEQ ID NOs: 13-14.

8. The use according to claim 5, characterized in that: The primer sequences used to construct the BolNSH2 gene silencing vector are shown in SEQ ID NOs: 15-16.

9. The use according to claim 5, characterized in that: The primer sequences used to construct the BolNSH3 gene silencing vector are shown in SEQ ID NOs: 17-18.

10. Application of silencing vectors or silencing strains of rice nucleoside hydrolase OsNSH1, OsNSH2 and OsNSH3 genes in increasing the nicotinamide riboside content in rice.

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