Use of BoCBL or BoCGS1 in regulating the content of glucoraphanin in broccoli

Abstract

The application discloses application of BoCBL or BoCGS1 in regulating content of glucoraphanin in broccoli and belongs to the field of genetic engineering. The application finds that overexpression of BoCBL or BoCGS1 in broccoli can significantly increase the content of glucoraphanin in broccoli, the content of GRA in the hairy root of the overexpression CBL and CGS1 is increased by 179% and 120% respectively, and the content of the above-ground part is increased by 146% and 30% respectively. Therefore, the two genes can be used for regulating the content of glucoraphanin in broccoli and cultivating a broccoli strain with high glucoraphanin content.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically involving the application of the broccoli gene BoCBL or BoCGS1 in regulating the glucosinolate content in broccoli. Background Technology

[0002] The CGS1 gene encodes cystathionine gamma synthase. Methionine is an essential amino acid that is metabolized and transformed in organisms through a series of enzymatic reactions. The methionine cycle is a key part of this process, involving the remethylation of methionine to homocysteine, which is then converted to L-cysteine. In the methionine cycle, the cystathionine gamma synthase encoded by the CGS1 gene is a key enzyme catalyzing the combination of cysteine ​​and serine to form cystathionine. This step is a rate-limiting step in methionine biosynthesis and is crucial for maintaining intracellular methionine levels. In addition, CGS1 participates in the transsulfurization pathway. When extracellular cysteine ​​is insufficient, transsulfurization-mediated cysteine ​​biosynthesis can provide sufficient cysteine ​​to support cell growth and proliferation. Since methionine is a building block of proteins and the initiating amino acid for mRNA translation, the function of the CGS1 gene is essential for plant growth and development. The downstream product of methionine, thioadenosylmethionine, is the most important methyl donor in organisms and also plays a role in transsulfurization and transaminopropylation. Its derivative, thiomethylmethionine, is an important sulfur transport molecule closely related to biological sulfur metabolism. Besides directly participating in methionine synthesis, the CGS1 gene also participates in the regulation of epigenetic modifications. Studies have shown that the CGS1 gene regulates DNA and histone methylation modifications through a one-carbon metabolic cycle to maintain genome stability and regulate gene expression. This epigenetic regulatory mechanism further emphasizes the importance of the CGS1 gene in the regulation of plant sulfur metabolism. The CGS1 gene plays a feedback repression role in methionine synthesis. The methionine synthesis pathway in higher plants and microorganisms has been well studied, and the synthesis process is basically the same, except for the difference in the activation of the carboxyl group after the formation of L-homoserine. In plants, L-homoserine undergoes phosphorylation, while in microorganisms it undergoes acetylation or succinylation, resulting in different substrates in the CGS1-catalyzed reaction between bacteria and plants. CGS1 expression is influenced by substrate feedback inhibition; therefore, in genetic engineering regulation, the molecular structure of the CGS1 gene needs to be modified to reduce feedback inhibition. In general, the CGS1 gene plays a crucial role in methionine synthesis and sulfur metabolism, and its normal function is essential for maintaining the physiological functions of organisms and promoting plant growth and development.

[0003] CBL, or cystathionine β-lyase, catalyzes the penultimate reaction in methionine biosynthesis. This step is crucial for methionine synthesis as it acts as a bridge between the precursor and the final product. In the transsulfurization pathway, CBL acts as the rate-limiting enzyme, and its activity directly affects the efficiency of methionine synthesis. When extracellular cysteine ​​is insufficient, transsulfurization-mediated cysteine ​​biosynthesis can provide enough cysteine ​​to support cell growth and proliferation.

[0004] Glucoraphanin, a key aliphatic glucosinolate found in cruciferous vegetables, involves a multi-stage enzymatic reaction and a multi-layered regulatory network in its synthesis. The synthetic pathway begins with the side-chain elongation of methionine, catalyzed by the branched-chain aminotransferase BCAT4 to generate 2-oxo-4-methylthiobutyric acid (OMTB), which is then catalyzed by the methylthioalkylmalonic acid synthase MAM1 to form an extended-chain intermediate. In the core structure construction stage, cytochrome P450 enzymes CYP79F1 and CYP83A1 catalyze the conversion of methionine derivatives into aldoximes and thiohydroxyoxime esters, respectively, completing the assembly of the glucosinolate backbone. Side-chain modification is mediated by the flavin monooxygenase FMOGSOX1 through oxidation, ultimately forming the biologically active glucosinolate. This process is synergistically regulated by the MYB transcription factor family (MYB28, MYB29, MYB76). Overexpression of MYB28 can increase the glucosinolate content in Chinese kale by 97%. Epigenetic mechanisms such as DNA methylation affect gene expression by regulating S-adenosylmethionine metabolism, while histone modifications dynamically regulate the chromatin accessibility of glucosinolate synthesis-related genes through markers such as H3K4me3.

[0005] The metabolic pathway of glucosinolates exhibits a dual characteristic: in plants, myrosinase specifically hydrolyzes glucosinolates to generate sulforaphane (SFN), but the presence of epidermal specific sulfur protein (ESP) promotes the product to be more inactive than sulforaphane; while in the mammalian gut, probiotics such as Bifidobacterium longum achieve glucosinolate conversion through β-glucosidase, and supplementing with specific strains can increase plasma SFN concentration by 2-3 times. Its metabolites exhibit a wide range of health benefits: In anti-cancer activity, SFN inhibits the PI3K / AKT / mTOR pathway, arresting the cell cycle while upregulating the pro-apoptotic factor Bax and downregulating the anti-apoptotic protein Bcl-2, demonstrating dose-dependent inhibitory effects on solid tumors such as breast cancer and liver cancer. Its antioxidant mechanism relies on the Nrf2 / Keap1 signaling axis, activating phase II detoxification enzymes such as superoxide dismutase (SOD) and heme oxygenase-1 (HO-1) to reduce oxidative stress damage. Its anti-inflammatory activity involves inhibiting NF-κB nuclear translocation and reducing the levels of pro-inflammatory factors such as IL-6 and TNF-α, resulting in a 40%-60% reduction in inflammatory markers in a colitis model. Notably, SFN can also penetrate the blood-brain barrier, reducing IL-1β release by inhibiting NLRP3 inflammasome assembly and simultaneously activating brain-derived neurotrophic factor (BDNF) signaling to improve cognitive function in an Alzheimer's disease model. Furthermore, it exhibits broad-spectrum antibacterial activity against both Gram-negative bacteria (such as Escherichia coli) and Gram-positive bacteria (such as Staphylococcus aureus), with a minimum inhibitory concentration (MIC) of up to 0.78 μg / mL. These multi-target mechanisms of action provide a molecular basis for the functional development of cruciferous vegetables, and also highlight the enormous application potential of optimizing glucosinolate synthesis pathways and regulating metabolic transformation through genetic engineering. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a method for regulating the content of glucosinolates in broccoli.

[0007] The technical solution of the present invention is: the application of the broccoli gene BoCBL or BoCGS1 in regulating the glucosinolate content in broccoli, wherein the gene BoCBL is the nucleotide sequence of a protein encoding the amino acid shown in SEQ ID No. 3; and the gene BoCGS1 is the nucleotide sequence of a protein encoding the amino acid shown in SEQ ID No. 4.

[0008] Furthermore, the nucleotide sequence of the gene BoCBL is shown in SEQ ID No. 1; the nucleotide sequence of the gene BoCGS1 is shown in SEQ ID No. 2.

[0009] Furthermore, the regulation method is as follows: overexpressing the broccoli gene BoCBL or BoCGS1 in broccoli, thereby increasing the glucosinolate content in broccoli.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] This invention found that overexpressing BoCBL or BoCGS1 in broccoli can significantly increase the glucoraphane content. The glucoraphane content in the hairy roots of overexpressing CBL and CGS1 increased by 179% and 120%, respectively, while the aboveground parts increased by 146% and 30%, respectively. Therefore, these two genes can be used to regulate the glucoraphane content in broccoli and cultivate broccoli varieties with high glucoraphane content. Attached Figure Description

[0012] Figure 1 Colony PCR agarose gel electrophoresis image.

[0013] Figure 2 Observation of GFP fluorescence in transgenic hairy roots.

[0014] Figure 3 Expression levels of CBL and CGS1 genes in transgenic hairy roots (* indicates P<0.05); where A is the CBL gene and B is the CGS1 gene.

[0015] Figure 4 GRA content in transgenic hairy roots and leaves (* represents P<0.05, ** represents P<0.01); where A represents hairy roots and B represents leaves. Detailed Implementation

[0016] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from commercial channels.

[0017] Example 1: Cloning and vector construction of CGS1 and CBL genes in broccoli

[0018] 1. Broccoli RNA Extraction

[0019] RNA extraction was performed using the SPARKeasy Plant RNAKit rapid extraction kit. The procedure is as follows:

[0020] (1) Take 100mg of plant material and put it into a 1.5mL sterilized EP tube. After being quick-frozen with liquid nitrogen, put it into a homogenizer that has been baked at 180℃. Add 500μL of lysis buffer RLT and grind it. Then, draw the homogenate back into the 1.5mL EP tube and shake it vigorously by hand to ensure that it is fully lysed.

[0021] (2) After adding the lysis buffer to the genomic DNA removal column, centrifuge at a speed of 13800×g for 2 min and discard the effluent.

[0022] (3) Pipette the filtrate into a clean 1.5mL EP tube, add half a volume of anhydrous ethanol, and mix by suction and beating.

[0023] (4) After the mixture is drawn out and added to the adsorption column RA, it is centrifuged. The centrifuge speed is set to 13800×g and the time is 2min. The effluent is discarded.

[0024] (5) Add 700 μL of protein removal solution RW1 and centrifuge. Set the centrifuge speed to 13800×g and the time to 2 min. Discard the effluent.

[0025] (6) Add 500 μL of rinsing buffer RW and centrifuge at 13800 × g for 2 min. Discard the effluent. (Repeat once)

[0026] (7) Take out the adsorption column RA and put it into a clean RNase-free collection tube. Add 20 μL RNase-free H2O to the middle of the adsorption membrane, place it at room temperature for 2 min, and then centrifuge. Set the centrifuge speed to 13800×g and the time to 2 min. Collect the filtrate, i.e. RNA, and store it at -20℃.

[0027] 2. Obtaining broccoli cDNA

[0028] RNA concentration was determined using a nanometer, and reverse transcription was performed using the SPARKscript I RT Plus Kit (With gDNA Eraser). The procedure is as follows:

[0029] (1) Place a sterile PCR tube on ice, add 1 mg Total RNA and 1 μL gDNA Braser to the tube, and supplement with RNase Free H2O to 10 μL. Mix well and place in a PCR instrument set to 42℃ for 5 min.

[0030] (2) Add 10 μL of 2×SPARKscript I RT Plus Master Mix to the tube, mix well and place it in a PCR instrument. Set the temperature to 50℃ for 15 min, 85℃ for 5 min to obtain the cDNA product. Store at -20℃.

[0031] 3. Construction of broccoli overexpression vectors pGWB504-CBL and pGWB504-CGS1

[0032] Amplification of the target gene: The gene sequences of BoCBL and BoCGS1 were obtained from NCBI (CDS sequences are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively, and the amino acid sequences of the encoded proteins are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively). Primers were designed to amplify the coding region sequences containing BP adapters without a terminator (-TAA). PCR amplification was performed using High-Fidelity DNA polymerase (NEB, M0491S) according to the manufacturer's instructions. The reaction solution was prepared according to the system in Table 1. After mixing, the mixture was subjected to 35 cycles of pre-denaturation at 98℃ for 30 s, denaturation at 98℃ for 10 s, annealing at 58℃ for 30 s, and extension at 72℃ for 45 s, followed by a final extension at 72℃ for 10 min. PCR results were detected using 1% agarose gel electrophoresis. The target gene PCR product was recovered using a gel extraction kit (Sangon Biotech Co., Ltd.), and the PCR product recovery results were finally detected using 1% agarose gel electrophoresis.

[0033] Table 1 PCR amplification system

[0034]

[0035] Ligation of the target fragments: The target genes CBL and CGS1 were ligated into the entry vector pDONR222 using the Gateway system via the BP reaction. According to Gateway... TM BP Clonase TM II. Enzyme mix (Thermo) was used for cloning and recombination reactions. The BP reaction was performed according to the system in Table 2, and after thorough mixing, the mixture was incubated overnight at room temperature.

[0036] Table 2 BP Reaction System

[0037]

[0038] Transformation and identification of recombinant plasmids: The ligation products of the BP reaction of CBL and CGS1 were transformed into *E. coli* DH5α competent cells (CAT#: DL1001, Weidi Biotechnology) using a heat shock method. The heat-shocked transformation mixture was plated on LB solid medium (containing 50 mg / mL kanamycin) and incubated upside down at 37°C for 16-18 h. Plump single colonies carrying the recombinant plasmids were picked for PCR identification. Correctly identified colonies were inoculated into LB liquid medium (containing 50 mg / mL kanamycin) and incubated with shaking at 37°C for 16-18 h. The recombinant plasmids were extracted from the bacterial culture and identified by restriction endonuclease BsrGI. The correct recombinant plasmids were sent to the sequencing center of Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After correct sequencing, pDONR222-CBL(-TAA) and pDONR222-CGS1(-TAA) glycerol bacteria (bacterial culture: 50% glycerol = 1:1) were stored at -80°C for later use. The ligated pDONR222-CBL(-TAA) and pDONR222-CGS1(-TAA) plasmids were used to replace the target gene into the pGWB405 vector via an LR reaction. (According to Gateway) TM LR Clonase TM II. The substitution reaction was performed using Enzyme Mix (Thermo). The reaction solution was prepared according to the components in Table 3, thoroughly mixed, and reacted at room temperature for 6 hours. Then, 2.5 μL of the ligation product was transformed into *E. coli*, and single colonies were picked for colony PCR. Bacteria with bands were cultured in LB broth at 37°C and 180 rpm for 16 hours. Plasmids were extracted and identified by enzyme digestion. Figure 1 After confirming that the sequencing is correct, save the successfully constructed vectors pDONR222-CBL and pDONR222-CGS1 for later use.

[0039] Table 3 Reaction Liquid System

[0040]

[0041] Example 2: Transformation with Agrobacterium rhizogenes

[0042] 1. Electroporation of GFP expression vectors into Agrobacterium rhizogenes: Add 2 μL of the constructed plasmids pGWB405-CBL, pGWB405-CGS1, and pGWB405 to 50 μL of thawed Agrobacterium rhizogenes competent cells ATCC15834. Mix thoroughly by pipetting and then place on ice. Set the electroporation program to: C = 25 μF, PC = 200 ohms, V = 2400 V. Add the ice-bathed mixture to a pre-chilled electroporation cuvette, cover, dry, and insert into the electroporator groove. Start electroporation. After completion, quickly transfer to an EP tube and add 1 mL of TY medium. Incubate at 28℃ for 3 h, then plate onto plates containing 50 mg / L Kan and 50 mg / L Rif. Incubate for 48 h. Perform colony PCR identification; those with correct bands can be used for infection.

[0043] 2. Induction of hairy roots in broccoli: Take an appropriate amount of high-quality broccoli seeds, vernalize them at 4℃ for 3 days, and plant them in a 1:1 mixture of soil and vermiculite. Place them in a culture room with a photoperiod of 16 hours of light / 8 hours of darkness, maintaining a temperature of 23℃. When the broccoli has grown for 2 weeks, select healthy seedlings with consistent growth for infection. Make a slanted cut 2 cm below the cotyledon node, scrape off the Agrobacterium rhizogenes bacteria from the cut surface, and quickly insert it into a flowerpot containing only vermiculite. Inject 4 mL of Agrobacterium rhizogenes bacterial solution (OD600 = 0.8) into the roots, and immediately cover with a transparent plastic cup. After 1 week of growth, a large number of hairy roots appear at the cut surface. Cut off the roots that are not cut at the slanted surface and replant in a 1:1 mixture of soil and vermiculite.

[0044] 3. GFP identification

[0045] After inducing the formation of hairy roots, the portion of the hairy roots near the root tip was cut with surgical scissors and prepared into slides. Under a fluorescence microscope at 488 nm, blue fluorescence and luminescence detection were performed. Positive hairy roots showed strong green fluorescence. Figure 2 ).

[0046] 4. Analysis of qRT-PCR

[0047] Reverse transcription was performed based on the measured RNA concentration, with the total RNA amount controlled at 1 mg, to obtain a quantitative concentration of cDNA.

[0048] The cDNA template was diluted 5-fold to obtain the template for real-time quantitative PCR. Real-time PCR was performed using the SYBR Green assay. The internal control gene was ACTIN2. The expression level of the internal control gene was adjusted to 1 using Microsoft Excel software. -ΔΔct The method calculates the relative expression level of genes.

[0049] The results are as follows Figure 3As shown, the expression levels of CBL and CGS1 in transgenic hairy roots were verified by RT-qPCR analysis. The expression levels of CBL and CGS1 were upregulated by 2.83-fold and 9.14-fold, respectively, proving that these two genes were overexpressed in transgenic hairy roots.

[0050] Example 3: Identification of Glucosamine (GRA) Content in Transgenic Material

[0051] Leaves and hairy roots of the transgenic hairy-root chimera broccoli were placed in 2 mL centrifuge tubes and pre-cooled in liquid nitrogen. The centrifuge tubes were then evenly placed in a ball mill and the milling frequency was adjusted to 20 rpm. To prevent the sample from overheating during shaking, a method of shaking for 30 seconds followed by freezing in liquid nitrogen for 30 seconds was used. This process was repeated three times until the sample was completely pulverized. 1 mL of 70% methanol (chromatographic grade) was quickly added, and the mixture was then placed in an 80°C water bath for 10 min, inverting the tubes frequently to mix. 20 μL of 5 mmol·L⁻¹ mol / L precipitate was then added. -1 Benzylglucosinolate (Phytoplan) was used as an internal standard. The mixture was shaken for 1 min, incubated in a constant temperature water bath at 80℃ for 10 min, shaken for 5 min, and centrifuged at 4℃ for 10 min (5000 rpm). The supernatant was collected. Extraction was repeated twice with 70% methanol. All supernatants were combined and purified using DEAE Sephadex A25 (Amersham Biosciences). Sulfatase (from Helix Pomati 100KU, Sigma) was added, and the mixture was reacted at 25℃ for 12–14 h. Elution was performed with 3 mL of ultrapure water, and the mixture was freeze-dried for approximately 10 hours until a powder was obtained. The powder was then dissolved in 150 μL of ultrapure water, centrifuged for 30 min (16000 g), and the supernatant was used for glucosinolate determination. Ultra-high performance liquid chromatography (UPLC) is a novel chromatographic analysis technique using 1.7 μm ultrafine chromatographic column packing as its core technology. This technique has many advantages compared to high-performance liquid chromatography (HPLC). GRA content was calculated using benzyl glucosinolate as an internal standard (added during sample extraction) and the relative peak area (ratio of the peak area of ​​the identifiable substance to the peak area of ​​the internal standard) at a scanning wavelength of 229 nm. The injection volume was 2 μL, the chromatographic column was a BEH C18 (2.1 mm × 50 mm, 1.7 μm; Waters), mobile phase A was ultrapure water, and mobile phase B was methanol (chromatographic grade). UPLC elution conditions were as follows:

[0052]

[0053] The results are as follows Figure 4As shown, UPLC analysis of GRA in the hairy roots and leaves of transgenic broccoli revealed that overexpression of CBL and CGS1 significantly increased GRA content. The GRA content in the hairy roots of CBL and CGS1 overexpressing genes increased by 179% and 120%, respectively, while the aboveground parts showed increases of 146% and 30%, respectively.

Claims

1. Broccoli genes BoCBL or BoCGS1 The gene is used to increase the glucosinolate content in broccoli. BoCBL The nucleotide sequence is the nucleotide sequence of the protein encoding the amino acid shown in SEQ ID No. 3; the gene BoCGS1 The nucleotide sequence is the nucleotide sequence of the protein encoding the amino acid shown in SEQ ID No.

4.

2. The application according to claim 1, characterized in that, The gene BoCBL The nucleotide sequence is shown in SEQ ID No. 1; the gene BoCGS1 The nucleotide sequence is shown in SEQ ID No.

2.

3. The application according to claim 1 or 2, characterized in that, The method of addition is as follows: overexpressing the broccoli gene in broccoli. BoCBL or BoCGS1 This increases the glucosinolate content in broccoli.

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