Application of BoSLIM1 protein in increasing sulforaphane content and indole-3-methanol content in broccoli

Abstract

The invention discloses application of BoSLIM1 protein in increasing the content of sulforaphane and the content of indole-3-methanol in broccoli, and belongs to the technical field of plant breeding. The invention aims to improve the content of sulforaphane and indole-3-methanol in broccoli at the same time. The invention provides an application of a protein shown as SEQ ID NO.2 in increasing the content of sulforaphane or / and the content of indole-3-methanol or the content of glucoraphanin or / and the content of brassica glucosinolate in broccoli. The content of sulforaphane and indole-3-carbinol in the broccoli is increased at the same time, the nutritional value of the broccoli is greatly increased, the requirements of modern consumers are met, and people can obtain nutritional ingredients in vegetables easily, so that the broccoli is beneficial to body health.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and particularly relates to application of BoSLIM1 protein in increasing the content of sulforaphane and indole-3-carbinol in broccoli. Background Art

[0002] Broccoli (Brassica oleracea var. italica), also known as broccoli, is a variety of Brassica oleracea in the family Cruciferae. Broccoli has rich nutritional and medicinal value. In addition to rich protein, vitamins, and mineral elements, it also contains glucoraphanin and a variety of indole derivatives.

[0003] As a high-end health vegetable, how to use appropriate cooking methods to reduce the loss of its nutrients and increase the production of active ingredients while maintaining the good flavor, texture or color of the food is a key issue. As a means of protecting and improving nutrition, the impact of food processing technology cannot be ignored, which is related to food quality and safety issues. It is reported that the endogenous myrosinase in homogenized broccoli can only convert 25% (or less) of glucoraphanin into sulforaphane, which means that direct intake of broccoli may not be enough to produce the expected health benefits because the glucosinolates are still in their original form. Since the production of glucosinolate hydrolysates involves enzymatic reactions, appropriate thermal processing can increase the content of hydrolysates. Traditional broccoli processing methods include steaming, boiling, frying, and stir-frying. Studies have found that all of them will cause glucosinolates and sulforaphane to lose to varying degrees. How to simultaneously increase the content of sulforaphane and indole-3-carbinol in broccoli is a technical problem that needs to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to simultaneously increase the content of sulforaphane and indole-3-carbinol in broccoli.

[0005] The present invention provides an application of a protein shown in SEQ ID NO.2 in increasing the content of sulforaphane and / or the content of indole-3-carbinol or the content of glucoraphanin or / and the content of brassica glucosinolate in broccoli.

[0006] The present invention provides an application of a gene shown in SEQ ID NO.1 in increasing the content of sulforaphane and / or the content of indole-3-carbinol or the content of glucoraphanin or / and the content of brassica glucosinolate in broccoli.

[0007] The present invention provides an application of a recombinant vector containing a gene shown in SEQ ID NO.1 in increasing the content of sulforaphane or / and the content of indole-3-carbinol or the content of glucoraphanin or / and the content of brassica glucosinolate in broccoli.

[0008] The present invention provides an application of a recombinant microbial cell containing a gene shown in SEQ ID NO.1 in increasing the content of sulforaphane or / and the content of indole-3-carbinol or the content of glucoraphanin or / and the content of brassica glucosinolate in broccoli.

[0009] The present invention provides use of broccoli overexpressing a gene shown in SEQ ID NO.1 in preparing a drug rich in sulforaphane and / or indole-3-carbinol.

[0010] The present invention provides use of broccoli overexpressing a gene shown in SEQ ID NO.1 in preparing anticancer drugs or in food production.

[0011] The present invention provides a method for breeding broccoli rich in sulforaphane and indole-3-carbinol, and the specific steps of the method are as follows:

[0012] Step 1: connect the gene sequence shown in SEQ ID NO.1 with the pCAMBIA1301-35S-EGFP vector to obtain a recombinant vector;

[0013] Step 2: Transform the recombinant vector obtained in step 1 into Agrobacterium to obtain recombinant Agrobacterium;

[0014] Step 3: Infect broccoli with the recombinant Agrobacterium obtained in step 2 to obtain transgenic broccoli.

[0015] The present invention provides a biological preparation, characterized in that the biological preparation contains broccoli obtained by the above method.

[0016] It is further defined that the biological preparation is any one of tablets, capsules, granules, powders, and liquid preparations.

[0017] The present invention provides a method for increasing the content of sulforaphane and indole-3-carbinol or glucoraphanin or / and glucosinolate in broccoli, and the gene shown in SEQ ID NO.1 is overexpressed in broccoli.

[0018] Beneficial effects: The unloaded broccoli hairy roots were used as the control group. Overexpression of BoSLIM1 in the hairy roots of broccoli significantly increased the content of anticancer active substances sulforaphane and indole-3-carbinol in the leaves by 1.65 times and 1.69 times. The content of sulforaphane and indole-3-carbinol in broccoli was increased at the same time, which greatly improved the nutritional value of broccoli, met the needs of modern consumers, and facilitated people's acquisition of nutrients in vegetables, thus benefiting their health. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Diagram of the process of obtaining transgenic BoSLIM1 broccoli hairy roots; A: 15-day-old broccoli, B: ATCC15834 Agrobacterium infection; C: broccoli was taken out of vermiculite and the lateral roots were cut off, D: broccoli was cultured in a soil with a ratio of 1:1 of nutrient soil and vermiculite for another 7 days, E: Obtaining transgenic BoSLIM1 broccoli hairy roots;

[0020] Figure 2 This is the analysis result of sulforaphane and indole-3-carbinol content in the hairy roots of broccoli with BoSLIM1 gene;

[0021] Figure 3 This is the analysis result of glucoraphanin and brassica glucosinolate content in the hairy roots of transgenic BoSLIM1 broccoli;

[0022] Figure 4 This is the result of identification of overexpression of BoSLIM1 gene. DETAILED DESCRIPTION

[0023] Example 1. BoSLIM1 gene amplification and construction of recombinant vector

[0024] 1. Target gene name: BoSLIM1 (Broccoli sulfate restriction 1) Gene sequence: SEQ ID NO.1

[0025] ATGGGTGGGGGCGATCTAGCTTTGTCCGTTGCTGACATCAGGATGGAGAATGAGCAGC

[0026] CTGATGACTTGGCTAGCGATACCGTCGCTGAGATCGATGTGAGTGACGAAGAGATTGAT

[0027] GCAGAAGACTTAGAGAGACGTATGTGGAAAGACCGAGTCAGGCTCAAAAGAATCAAA

[0028] GAGCGTCAAAAAGGAGATTCTCAAGGACCTCAGGCGAAGGAGCCTCCCAAGAAGATC

[0029] TCTGACCAAGCGCAGAGGAAGAAGATGTCTAGAGCACAAGACGGTATACTCAAGTACA

[0030] TGCTGAAGCTAATGGAAGTCTGCAAAGTACGCGGCTTTGTGTACGGCATCATACCTGAA

[0031] AAAGGTAAGCCGGTGAGCGGCTCCTCCGACAATATAAGAGCTTGGTGGAAAGAGAAG

[0032] GTGAAGTTTGATAAAAACGGTCCTGCAGCGATTGCTAAATACGAAGAGGAGTGTTTGG

[0033] CGTTTGGGAAGTCAGACGGGAATAGAAACTCGCAGTTTGTGCTTCAAGACTTGCAGGA

[0034] CGCTACTCTAGGGTCTTTGTTGTCTTCTTTGATGCAGCATTGTGATCCTCCGCAGAGGAA

[0035] GTATCCGTTGGAGAAAGGGACGCCTCCTCCCTGGTGGCCGACGGGGAAGGAAGAGTG

[0036] GTGGGTGAAGCTCGGCTTGCCGCAGAGCCAGAGTCCTCCTTACAGGAAGCCTCATGAT

[0037] CTCAAGAAGATGTGGAAAGTTGGGGTGTTGACGGCTGTGATTAACCATATGTCTCCTGA

[0038] TATTGCTAAGATCAAGAGGCATGTTCGTCAGTCTAAGTGTTTGCAGGACAAGATGACGG

[0039] CGAAAGAGAGCGCGATTTGGTTGGCGGTTTTGAACCAGGAGGAGTCGCTTATTCAGCA

[0040] GCCGAGTAGTGATAATGGGACGTCTAATGTAACTGAGACGCACCGGAGGGGGAATAAC

[0041] GCTGACAGGAGGAAGACTGTGATTAATAGTGATAGTGATTATGATGTTGATGGGACTGA

[0042] GGAGGCTTCAGGCTCGGTTTCTTCTAAAGACAGTAGAAGAAATCAAGTTCCAGCAGCC

[0043] ACTTCACAACAACCAGTAAGAGATCAAGATAAAGCTGGGAAACATAAGAGAAGGAAA

[0044] AGACCTAGAATCAGATCCGGAACTCTTAATGTACAAGATGAAGAACAAGTAGAAGCTG

[0045] AAGGGAGAAATGTTTTACCTGATATGAATCATGTTGAGGCTCCTATGCTAGATTATAACA

[0046] TCAACGGTACTACTAATCACCATGAGGAAGGTGTTTTAGAACCAAACATCTCCTTAGGA

[0047] CCAGAAGAGAACGGTCTGGAACTGGTGGTTCCTGAGTTTGATAGCAACTATACTTATCT

[0048] TCCTCCTGTTGATGGACAAGCTATGATGCCTGTAGACGAAAGACCAATGCTTTACGGAG

[0049] CAAACCCCAACCAGGAGCTGCAATTTGGGTCAGGTTATAACTACTATAATACTTCTGCA

[0050] GTGTTTGTGCATAATCAGGAAGAAGACCTTATCCATACACAGATAGAGATGAACTCACA

[0051] AGCACCGCCTCACAGCAACGGGTTCGATGGCCAAGGAGGAGTACTTCAACCCCATGGG

[0052] AATGAAGAGGTGGGTGTAGCAGGAAGAGATATGCCTCCTCAGTTTCAGAGTGACCAAG

[0053] ATAAACTCTTGGACAGTAACATCCTATCTCCATTCAATGACTTGCCGTTTGATAGCAGCA

[0054] CCTTTTACTCTGGGTTTGATTCCTTTGGTGCATTTGATGATGACTACTCATGGTTTGGAGCTTAG;

[0055] Protein sequence of BoSLIM1 (Broccoli Sulfate Limitation 1): SEQ ID NO.2

[0056] MGGGDLALSVADIRMENEQPDDLASDTVAEIDVSDEEIDAEDLERRMWKDRVRLKRIKER

[0057] QKGDSQGPQAKEPPKKISDQAQRKKMSRAQDGILKYMLKLMEVCKVRGFVYGIIPEKGKP

[0058] VSGSSDNIRAWWKEKVKFDKNGPAAIAKYEEECLAFGKSDGNRNSQFVLQDLQDATLGSL

[0059] LSSLMQHCDPPQRKYPLEKGTPPPWWPTGKEEWWVKLGLPQSQSPPYRKPHDLKKMWKVG

[0060] VLTAVINHMSPDIAKIKRHVRQSKCLQDKMTAKESAIWLAVLNQEESLIQQPSSDNGTSN

[0061] VTETHRRGNNADRRKTVINSDSDYDVDGTEEASGSVSSKDSRRNQVPAATSQQPVRDQDK

[0062] AGKHKRRKRPRIRSGTLNVQDEEQVEAEGRNVLPDMNHVEAPMLDYNINGTTNHHEEGVL

[0063] EPNISLGPEENGLELVVPEFDSNYTYLPPVDGQAMMPVDERPMLYGANPNQELQFGSGYN

[0064] YYNTSAVFVHNQEEDLIHTQIEMNSQAPPHSNGFDGQGGVLQPHGNEEVGVAGRDMPPQF

[0065] QSDQDKLLDSNILSPFNDLPFDSSTFYSGFDSFGAFDDDYSWFGA;

[0066] Using 7-day-old broccoli seedlings as a template, using BoSLIM1-upstream primers and BoSLIM1-downstream primers, according to the PCR reaction system in Table 1, a PCR reaction was performed to amplify the BoSLIM1 gene sequence, and the target band was obtained after electrophoresis.

[0067] BoSLIM1-upstream primer: GAGCTCGGTACCCGGGGATCCATGGGTGGGGGCGATCTA (SEQ ID NO. 3);

[0068] BoSLIM1-downstream primer: GGTGTCGACTCTAGAGGATCCAGCTCCAAACCATGAGTAGTC (SEQ ID NO. 4);

[0069] PCR procedure:

[0070] Table 1 PCR reaction system

[0071] system volume dNTP Mix 2μl 10×Ex Taq Buffer 2μl Ex Taq Enzyme 0.3μl template 1μl Upstream primer 1μL Downstream primer 1μL <![CDATA[RNase Free H2O]]> Up to 20μl

[0072] PCR program: 94°C 10min→(94°C 30s→58°C 30s→72°C 2min)×30→72°C 10min→4°C. 2. Recovery of target fragment and vector connection

[0073] Recovery of target fragment: recovery and purification using SPARK easy Gel DNA Extraction Kit, the operation method is as follows:

[0074] (1) Under UV light, use a razor blade to cut out the gel block with the target band and place it into a 1.5 mL EP tube.

[0075] (2) Add 600 μL of sol and heat in a 65°C metal bath until completely melted. Invert every 2 minutes to accelerate melting.

[0076] (3) Add the solution to the EC adsorption column, place it at room temperature for 1 min, and then centrifuge it. Set the centrifuge speed to 13,800 × g for 1 min, and discard the effluent.

[0077] (4) Add 600 μL of WB and centrifuge. Set the centrifuge speed to 13800 × g for 1 min and discard the effluent. (Repeat once)

[0078] (5) Place the EC adsorption column back into the collection tube and centrifuge at a speed of 13,800 × g for 2 min.

[0079] (6) Place the adsorption column EC in a new collection tube, add 20 μL RNase Free H2O to the middle of the adsorption membrane, place at room temperature for 2 min, and then centrifuge. Set the centrifuge speed to 13800 × g and the time to 1 min. The eluted product is the purified target fragment.

[0080] Vector connection: The pCAMBIA1301-35S-BoSLIM1-EGFP expression vector was constructed using homologous recombination. The recombination system is as follows:

[0081] Table 2 Construction of pCAMBIA1301-35S-BoSLIM1-EGFP expression vector

[0082] system volume Exnase ΙΙ 2μl 5×CEΙΙBuffer 4μl Insert product 1μl Linearized vector 3μl <![CDATA[ddH2O]]> Up to 20μl

[0083] React at 37°C for 30 min.

[0084] Example 2. Obtaining recombinant bacteria

[0085] Take 2 μL of the constructed plasmid pCAMBIA1301-35S-BoSLIM1-EGFP and add them to the melted 50 μL of Agrobacterium rhizogenes competent ATCC15834. Mix by pipetting and insert into ice. Set the electroporator program: C=25 μF, PC=200Ω, V=2400V. Add the mixture after ice bath into the pre-cooled electroporator cup, cover the cup lid, wipe dry and insert into the groove of the electroporator, start the electric shock, and quickly transfer to the EP tube and add 1 mL of TY medium. After culturing at 28°C for 3 hours, spread on the medium containing Kana and Rif. After culturing for 48 hours, colony PCR identification can be performed and the correct one can be used for subsequent hairy root transformation.

[0086] Example 3. Obtaining transgenic broccoli hairy roots

[0087] 1. (1) When the broccoli was 2 weeks old, the seedlings with the same growth status and strong were selected for infection. The infection process was as follows: an oblique cut was made 2 cm below the cotyledon node, and the root-inducing Agrobacterium cells were scraped from the oblique cut surface and quickly inserted into a flower pot containing only vermiculite. The OD 600 =0.8, the Agrobacterium rhizogenes solution obtained in Example 2 was injected into the roots, and a transparent plastic cup was immediately placed on the roots to keep them moist and ensure sufficient moisture.

[0088] (2) After one week of growth, a large number of hairy roots appeared on the cut surface. The roots of the non-oblique cut surface were cut off and replanted in mixed soil. The transgenic 35S::BoSLIM1 hairy roots were obtained. Figure 1 shown.

[0089] 2. Identification

[0090] Green fluorescence detection and gene expression detection were performed on the hairy roots to obtain positive broccoli hairy root chimera identification primers: BoSLIM1-qpcr-upstream: CGTCAGTCTAAGTGTTTGCAG (SEQ ID NO. 5);

[0091] BoSLIM1-qpcr-downstream: AGTTCCGGATCTGATTCTAGG (SEQ ID NO.6), after identification, the results are as follows Figure 4 As shown, the broccoli overexpressing the BoSLIM1 gene was indeed overexpressing the BoSLIM1 gene.

[0092] Example 4. Content of anticancer active substances sulforaphane and indole-3-carbinol

[0093] In this example, the transgenic broccoli hairy roots transformed with no vector were used as the control group, and the transgenic broccoli hairy roots overexpressing the BoSLIM1 gene obtained in Example 3 were used as the experimental group. Fresh broccoli leaves from the control group and the experimental group were taken for subsequent experiments.

[0094] The sulforaphane and indole-3-carbinol contents of the broccoli powders of the control group and the experimental group obtained above were determined, specifically comprising the following steps:

[0095] (1) Extraction and determination of sulforaphane

[0096] 1 g of fresh broccoli leaves from the control group and the experimental group were weighed and placed in EP tubes, and 1.0 mL of pH = 6.0 phosphate buffer was added respectively. After grinding into slurry using a grinder, the mixture was placed in a water bath at 40°C for 2 h. After cooling to room temperature, 5 mL of ethyl acetate was added for extraction three times. The upper organic phase was transferred to a new centrifuge tube, blown dry with nitrogen, and then diluted to 0.2 mL with methanol. The mixture was mixed and filtered using a syringe filter. The filtrate was detected by Agilent 1100 high performance liquid chromatograph. A standard curve was established using a sulforaphane standard, and the content of sulforaphane was calculated according to the standard curve, and the content was expressed in μg / gFW.

[0097] The chromatographic parameters were as follows: UV detector wavelength was 254 nm; the chromatographic column was Compass C18 (2) reverse phase column (250 mm*4.6 mm, 5 μm), the column temperature was 30°C, the flow rate was 1 mL / min, the injection volume was 10 μL, and the mobile phase was 0.1% phosphoric acid aqueous solution: acetonitrile = 80:20 (V / V) 0 min; 5:95 (V / V) 25 min.

[0098] (2) Extraction and determination of indole-3-carbinol

[0099] Weigh 1g of fresh broccoli leaves from the control group and the experimental group and place them in EP tubes. Add 1.5mL of pre-cooled 80% methanol aqueous solution and grind them into pulp using a grinder. Extract them overnight at 4℃ and centrifuge them at 8000×g for 10min. Take the supernatant. Use 0.5mL of centrifugal residue to remove the supernatant. The mixture was extracted with 80% methanol aqueous solution for 2 h, centrifuged at 8000×g for 10 min, the supernatant was taken out after centrifugation, and the two supernatants were combined; nitrogen was blown at 40°C until there was no organic phase, and 2 mL of petroleum ether was added for extraction and decolorization for 3 times, 1 mol / L citric acid aqueous solution was added to the lower aqueous phase, and the pH was adjusted to 2-3; 2 mL of ethyl acetate was added for extraction twice, the upper organic phase was transferred to a new EP tube, blown dry with nitrogen, and 0.2 mL of methanol was added to dissolve, mixed, filtered with a syringe filter, and the filtrate was detected by a Waters 2695 high performance liquid chromatograph; a standard curve was established using an indole-3-methanol standard, and the content of indole-3-methanol was calculated according to the standard curve, and the content was expressed in μg / g FW.

[0100] The chromatograph parameters were as follows: 2475 fluorescence detector with an excitation wavelength of 275 nm and an emission wavelength of 340 nm; the chromatographic column was a Compass C18(2) reverse phase column (250 mm*4.6 mm, 5 μm), the column temperature was 35°C, the flow rate was 1 mL / min, and the mobile phase was water:methanol = 70:30 (V / V).

[0101] Results: The empty-loaded broccoli hairy roots were used as the control group. Overexpression of BoSLIM1 in the hairy roots of broccoli significantly increased the content of anticancer active substances sulforaphane and indole-3-carbinol in the leaves to 1.65 times and 1.69 times, respectively. 1.65 times refers to the increase of sulforaphane to 1.65 times, and 1.69 times refers to the increase of indole-3-carbinol to 1.69 times. The results are as follows: Figure 2 And as shown in Table 3.

[0102] Example 5. Content of the Precursor Glucoraphanin and Glucoraphanin

[0103] Since the aliphatic glucosinolate glucoraphanin can be hydrolyzed by myrosinase to produce sulforaphane, and the indole glucosinolate brassica glucoside can be hydrolyzed by myrosinase to produce indole-3-methanol, it can be seen that glucoraphanin and brassica glucoside are the precursors of sulforaphane and indole-3-methanol, respectively.

[0104] In this example, the transgenic broccoli hairy roots into which no vector was introduced were used as a control group, and the transgenic broccoli hairy roots overexpressing the BoSLIM1 gene obtained in Example 3 were used as an experimental group. Broccoli leaves from the control group and the experimental group were cut, and the contents of glucoraphanin and brassica glucosinolate were determined, specifically comprising the following steps:

[0105] (1) Extraction and determination of glucoraphanin

[0106] S1: Weigh 500 mg of broccoli leaf samples from the control group and the experimental group and place them in 2 mL EP tubes. After freezing with liquid nitrogen, use a tissue crusher to crush them. The crusher is set to 50 Hz and 5 min. Then add 1 mL of pre-cooled 70% methanol solution to each EP tube, place it in the crusher and shake it again. The crusher is set to 30 Hz and 5 min. Then centrifuge at 4 ° C and 15000 × g for 10 min, transfer the supernatant to a new 10 mL EP tube, and repeat the above steps twice. Take 4 mL of the above supernatant and place it on ice for later use.

[0107] S2: Prepare the chromatography column in advance, rinse the column with 5 mL of deionized water, cover the bottom cover after the deionized water flows out, slowly add 500 μL of 0.5 mol / L acetate buffer, and cover the bottom cover when the acetate buffer liquid level flows to 2 mm from the gel surface;

[0108] S3: Shake the dextran gel DEAE A-25 resin solution evenly, and slowly add 1 mL to the chromatographic column treated in S2 while shaking. The adding position should be at the same height to ensure that the height of the gel surface is consistent; open the bottom cover of the chromatographic column to allow the liquid to flow out and the gel to sink, and then use 5 mL of deionized water to rinse the chromatographic column. When the liquid level is 2 mm away from the gel surface, cover the bottom cover; add the supernatant obtained in S1 to the corresponding chromatographic column slowly to prevent the gel surface from floating; rinse twice with 2 mL of pre-cooled 70% methanol solution, rinse five times with 2 mL of deionized water, and rinse once with 2 mL of 20 mmol / L, pH = 5 acetate buffer, and cover the bottom cover when it is 2 mm away from the gel surface; add 0.5 mL of sulfatase, cover the bottom cover when it is 2 mm away from the gel surface, and cover the top cover, and leave overnight at room temperature;

[0109] S4: 600 μL of deionized water was taken to elute the chromatography column in S3 after overnight at room temperature into a 2 mL EP tube for freeze drying; the dried powder was dissolved in 150 μL of deionized water, and then placed in a filter membrane tube for centrifugal filtration. The centrifuge was set at 4°C, 13800 × g, and 10 min. The filtrate was collected into an HPLC liner tube.

[0110] The content of glucoraphanin was detected by UPLC ultra high performance liquid chromatography, with a detection wavelength of 229 nm, a mobile phase flow rate of 1.0 mL / min, and a temperature of 25°C; the mobile phases were water (A) and methanol (B), and the running time was: 0 min, A 100%; 7 min, A 75%; 8.6 min, A 40%; 9.2 min, 0%; 9.6 min, 0%; 10.6 min, 100%; 13 min, 100%; the chromatographic curve was obtained, and the content of glucoraphanin was:

[0111]

[0112] Note: S1 is the peak area of ​​the measured sample, S2 is the peak area of ​​the standard sample, A1 is the response coefficient of glucoraphanin, A2 is the response coefficient of the standard sample (the standard coefficient is 1), C is the amount of internal standard added (nmol), and m is the mass of the plant sample (mg).

[0113] (2) Extraction and determination of Brassica Glucosinolates

[0114] The extraction and determination methods of Brassica Glucoside are the same as those in Example (1).

[0115] The content of Brassica Glucoside is:

[0116]

[0117] Note: S1 is the peak area of ​​the measured sample, S2 is the peak area of ​​the standard sample, A1 is the response coefficient of brassica glucosinolate, A2 is the response coefficient of the standard sample (the standard coefficient is 1), C is the amount of internal standard added (nmol), and m is the mass of the plant sample (mg).

[0118] Results: The precursors of sulforaphane and indole-3-carbinol were detected and it was found that overexpression of BoBoSLIM1 in the hairy roots of broccoli significantly increased the contents of glucoraphanin and brassica glucosinolate in leaves by 1.51 times and 1.62 times, respectively. The content of glucoraphanin increased by 1.51 times and the content of brassica glucosinolate increased by 1.62 times. Figure 3 And as shown in Table 3.

[0119] Table 3

[0120] Metabolite content Comparison 35S::BoSLIM1 Sulforaphane content (μg / g fresh weight) 78.75±16.95 129.73±7.23 Indole-3-carbinol content (μg / g fresh weight) 81.64±3.60 138.33±7.56 Glucoraphanin content (μmol / g fresh weight) 23.32±0.59 35.27±2.79 Brassica glucosinolate content (μmol / g fresh weight) 6.15±0.30 9.96±0.45

Claims

1. Use of the protein shown in SEQ ID NO.2 in increasing the content of sulforaphane or / and indole-3-carbinol or glucoraphanin or / and brassica glucosinolate in broccoli.

2. Use of the gene shown in SEQ ID NO.1 in increasing the content of sulforaphane and / or indole-3-carbinol or glucoraphanin or / and brassica glucosinolate in broccoli.

3. Use of a recombinant vector containing the gene shown in SEQ ID NO.1 in increasing the content of sulforaphane or / and indole-3-carbinol or glucoraphanin or / and brassica glucosinolate in broccoli.

4. Use of a recombinant microbial cell containing the gene shown in SEQ ID NO. 1 in increasing the content of sulforaphane or / and indole-3-carbinol or glucoraphanin or / and brassica glucosinolate in broccoli.

5. Use of broccoli overexpressing the gene shown in SEQ ID NO.1 in the preparation of a drug rich in sulforaphane and / or indole-3-carbinol.

6. Use of broccoli overexpressing the gene shown in SEQ ID NO.1 in the preparation of anticancer drugs or in food production.

7. A method for breeding broccoli rich in sulforaphane and indole-3-carbinol, characterized in that: The specific steps of the method are as follows: Step 1: connect the gene sequence shown in SEQ ID NO.1 with the pCAMBIA1301-35S-EGFP vector to obtain a recombinant vector; Step 2: Transform the recombinant vector obtained in step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: Infect broccoli with the recombinant Agrobacterium obtained in step 2 to obtain transgenic broccoli.

8. A biological agent, characterized in that: The biological preparation contains broccoli obtained by the method according to claim 7.

9. The biological agent according to claim 8, characterized in that The biological preparation is any one of tablets, capsules, granules, powders and liquid preparations.

10. A method for increasing the content of sulforaphane, indole-3-carbinol, glucoraphanin, and / or glucosinolate in broccoli, characterized in that: The gene shown in SEQ ID NO.1 was overexpressed in broccoli.

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