Broccoli rich in sulforaphane and indole-3-methanol, its preparation method and application

Abstract

This invention relates to broccoli rich in sulforaphane and indole-3-methanol, its preparation method, and its applications, belonging to the field of food technology. To increase the content of sulforaphane and indole-3-methanol in broccoli, this invention provides a broccoli rich in sulforaphane and indole-3-methanol and its preparation method. This invention utilizes expression vectors or recombinant bacteria overexpressing the BoSULTR1;1 and BoSULTR1;2 genes to prepare transgenic broccoli plants. Nutrient content determination results of the above transgenic broccoli plants show that the BoSULTR1;1 and BoSULTR1;2 genes increase the content of sulforaphane and indole-3-methanol in broccoli by increasing the content of the precursor substances sulforaphane and brassinosteroides. This invention achieves the goal of simultaneously increasing the content of sulforaphane and indole-3-methanol in broccoli, significantly improving the nutritional value of broccoli, and can be applied to the production of foods beneficial to health.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food technology, and relates to broccoli and a preparation method and application thereof. BACKGROUND

[0002] Sulforaphane is an isothiocyanate, and research shows that eating vegetables rich in sulforaphane can effectively prevent the growth of tumors, such as colon cancer, prostate cancer and breast cancer; at the same time, sulforaphane can also play a good preventive role on colitis, and appropriate intake of sulforaphane can protect the intestinal barrier and prevent toxins from invading the distal organs. At present, sulforaphane is the best plant active ingredient found to have anticancer effect.

[0003] Indole-3-carbinol (I3C) has a molecular formula of C9H9NO and a molecular weight of 147.18. Research shows that indole-3-carbinol can inhibit the formation of polycyclic aromatic hydrocarbon-induced rat tumor cells, and can inhibit the occurrence of breast cancer, cervical cancer, prostate cancer and colon cancer by inhibiting the binding of carcinogens to DNA, repairing DNA damage, causing cell apoptosis and other pathways.

[0004] Broccoli is one of the Brassica species of the Cruciferae family, and has high nutritional value and is known as the "crown of vegetables". Broccoli is rich in various antioxidant active ingredients and rich in glucosinolates, among which the aliphatic glucosinolate sulforaphane can be hydrolyzed by myrosinase to generate sulforaphane, and the indole glucosinolate, Brassica glucoside and neoglucobrassicin can be hydrolyzed by myrosinase to generate indole-3-carbinol. Both sulforaphane and indole-3-carbinol have the effects of preventing colitis, preventing cancer, resisting cancer, promoting liver detoxification, enhancing the body's immunity and the like, and therefore, it is beneficial for human health to eat appropriate amount of broccoli in daily life.

[0005] Therefore, the field has been devoted to studying how to improve the content of sulforaphane and indole-3-carbinol in broccoli and improve the nutritional ingredients of broccoli food. SUMMARY

[0006] The present application provides a broccoli rich in sulforaphane and indole-3-carbinol and a preparation method and application thereof to improve the content of sulforaphane and indole-3-carbinol in broccoli.

[0007] One of the purposes of the present application is to provide a preparation method of broccoli rich in sulforaphane and indole-3-carbinol, which comprises the following steps:

[0008] S1. Cloning the BoSULTR1;1 and BoSULTR1;2 genes using primer pairs;

[0009] S2. The BoSULTR1;1 gene cloning sequence and the BoSULTR1;2 gene cloning sequence obtained in S1 are connected with a vector respectively to obtain an expression vector;

[0010] S3. The expression vector obtained in S2 is transformed into Agrobacterium tumefaciens respectively to obtain a recombinant bacteria;

[0011] S4. The plant is infected by the recombinant bacteria obtained in S3 to obtain a transgenic plant.

[0012] In a preferred embodiment of the present application, the nucleotide sequence of the BoSULTR1;1 gene in S1 is shown as SEQ ID NO. 1.

[0013] In a preferred embodiment of the present application, the nucleotide sequence of the BoSULTR1;2 gene in S1 is shown as SEQ ID NO. 2.

[0014] In a preferred embodiment of the present application, the nucleotide sequence of the upstream primer of the primer of the BoSULTR1;1 gene in S1 is shown as SEQ ID NO. 3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4.

[0015] In a preferred embodiment of the present application, the nucleotide sequence of the upstream primer of the primer of the BoSULTR1;2 gene in S1 is shown as SEQ ID NO. 5, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 6.

[0016] In a preferred embodiment of the present application, the vector in S2 is pCAM35S.

[0017] In a preferred embodiment of the present application, the infection method of the recombinant bacteria in S4 is Agrobacterium tumefaciens mediated method.

[0018] In a preferred embodiment of the present application, the plant in S4 is broccoli.

[0019] The second object of the present application is to provide a broccoli rich in sulforaphane and indole-3-carbinol, which is obtained by the preparation method.

[0020] The third object of the present application is to provide the application of the broccoli rich in sulforaphane and indole-3-carbinol in food making, which is to make salad by matching the cooked broccoli with other vegetables, or to make broccoli vegetable juice by placing the broccoli in a juicer.

[0021] The present application has the following beneficial effects:

[0022] The present application provides a broccoli rich in sulforaphane and indole-3-carbinol, and the broccoli rich in sulforaphane and indole-3-carbinol is prepared by using BoSULTR1;1 and BoSULTR1;2 genes, and the present application proves that the transgenic broccoli hairy root chimeras, i.e., transgenic broccoli plants, overexpressing BoSULTR1;1 and BoSULTR1;2 genes are successfully prepared by green fluorescence detection.

[0023] The present application determines the content of sulforaphane and indole-3-carbinol in the transgenic broccoli plants, and the results show that the content of sulforaphane in the transgenic broccoli plants overexpressing BoSULTR1;1 gene is increased by 38.4%, and the content of indole-3-carbinol is increased by 64.8%; the content of sulforaphane in the transgenic broccoli plants overexpressing BoSULTR1;2 gene is less increased, and the content of indole-3-carbinol is increased by 45.3%.

[0024] Aliphatic glucosinolate sinigrin can be hydrolyzed by myrosinase to generate sulforaphane, and indole glucosinolate, glucoraphenin and neoglucobrassicin can be hydrolyzed by myrosinase to generate indole-3-carbinol, so sulforaphane and indole-3-carbinol are precursor substances of sinigrin and glucoraphenin, respectively.

[0025] The present application further determines the content of sinigrin and glucoraphenin in the transgenic broccoli plants, and the results show that the content of sinigrin in the transgenic broccoli plants overexpressing BoSULTR1;1 gene is increased by 51.7%, and the content of glucoraphenin is increased by 89.6%; the content of sinigrin in the transgenic broccoli plants overexpressing BoSULTR1;2 gene is increased by 67.6%, and the content of glucoraphenin is increased by 94.8%. It can be seen that overexpression of BoSULTR1;1 gene and BoSULTR1;2 gene further increases the yield of sulforaphane and indole-3-carbinol by increasing the content of precursor substances sinigrin and glucoraphenin.

[0026] In summary, the present application provides a broccoli rich in sulforaphane and indole-3-carbinol, realizes the simultaneous increase of the content of sulforaphane and indole-3-carbinol in broccoli, greatly improves the nutritional value of broccoli, meets the needs of modern consumers, is conducive to the acquisition of nutritional ingredients in vegetables, and thus is beneficial to health. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Figure 2 is a diagram for PCR identification of the cloning of the coding region of BoSULTR1;1 and BoSULTR1;2 genes in Example 1;

[0028] Figure 2This is a PCR identification diagram of the recombinant bacteria containing overexpression of BoSULTR1;1 and BoSULTR1;2 genes in Example 1.

[0029] Figure 3 This is a green fluorescence detection image of the hairy roots of transgenic broccoli in Example 1;

[0030] Figure 4 The graphs show the determination of sulforaphane and indole-3-methanol content in the transgenic broccoli in Example 2. A is the sulforaphane content determination graph, and B is the indole-3-methanol content determination graph.

[0031] Figure 5 The graphs show the determination of glucosinolate and brassinosteroid content in the transgenic broccoli in Example 3. A is the glucosinolate content determination graph, and B is the brassinosteroid content determination graph. Detailed Implementation

[0032] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0034] Example 1: Preparation of Broccoli Rich in Sulforaphane and Indole-3-Methanol

[0035] (1) Cloning of broccoli genes BoSULTR1;1 and BoSULTR1;2

[0036] The primer of BoSULTR1;1 gene and BoSULTR1;2 gene is used in the embodiment, the upstream primer nucleotide sequence of BoSULTR1;1 gene is shown as SEQ ID No. 3, the downstream primer nucleotide sequence is shown as SEQ ID No. 4; the upstream primer nucleotide sequence of BoSULTR1;2 gene is shown as SEQ ID No. 5, the downstream primer nucleotide sequence is shown as SEQ ID No. 6; the cDNA of broccoli cotyledon is used as a template for PCR amplification, the PCR amplification products of BoSULTR1;1 and BoSULTR1;2 genes are obtained respectively, and the PCR verification is carried out on the obtained PCR amplification products, and the results are shown as Figure 1 BoSULTR1;1 and BoSULTR1;2 genes are successfully cloned.

[0037] The amplification system is: dNTP Mix 2 μL, 10×Ex Taq Buffer 2 μL, Ex Taq Enzyme 0.3 μL, cDNA template 1 μL, upstream primer 1 μL, downstream primer 1 μL, RNase Free H2O to 20 μL;

[0038] The amplification program is: 94°C pre-denaturation for 10 min, 94°C denaturation for 30 s-55°C annealing for 30 s-72°C extension for 2 min, 30 cycles, 72°C thorough extension for 10 min.

[0039] (2) Construction of expression vector

[0040] The SPARK easy Gel DNA Extraction Kit kit is used in the embodiment to recover and purify the electrophoresis bands of the PCR amplification products of BoSULTR1;1 gene and BoSULTR1;2 gene obtained in (1), and the purified amplification products are obtained, which specifically include the following steps:

[0041] S1: under the irradiation of ultraviolet lamp, the gel block with the PCR amplification product bands of BoSULTR1;1 and BoSULTR1;2 genes obtained in (1) is cut off by using a blade respectively, and is respectively placed into a 1.5 mL EP tube, and then 600 μL of sol agent is respectively added, and heating is carried out at 65°C in a metal bath until complete melting, and the gel solution containing the amplification product bands of BoSULTR1;1 and BoSULTR1;2 genes is obtained respectively by inverting every 2 min to accelerate melting;

[0042] S2: Add the gel solution obtained in S1 to the adsorption column EC, place at room temperature for 1 min, centrifuge at 13800×g for 1 min, and discard the effluent; then add 600 μL of washing solution WB to the adsorption column EC, centrifuge at 13800×g for 1 min, discard the effluent, and repeat the washing once.

[0043] S3: Place the EC adsorption column after rinsing in S2 back into the collection tube and centrifuge at 13800×g for 2 min; then place the EC adsorption column into a new collection tube, add 20 μL RNase Free H2O to the middle of the adsorption membrane, incubate at room temperature for 2 min, and centrifuge at 13800×g for 1 min. The elution product is the purified amplification product.

[0044] In this embodiment, homologous recombination was used to construct the expression vector. The recombination system consisted of: 2 μL of Exnase II, 4 μL of 5×CEII Buffer, 1 μL of inserted purified amplification product, 3 μL of linearized vector pCAM35S, and 20 μL of ddH2O. The reaction was carried out at 37°C for 30 min to obtain pCAM35S-BoSULTR1; 1-eGFP and...

[0045] pCAM35S-BoSULTR1;2-eGFP expression vector.

[0046] (3) Construction of recombinant bacteria

[0047] In this embodiment, pCAM35S-BoSULTR1;1-eGFP obtained in (2) and

[0048] 2 μL each of pCAM35S-BoSULTR1 and 2-eGFP expression vectors were added to 50 μL of thawed Agrobacterium rhizogenes competent cells ATCC15834. After mixing thoroughly, the mixture was placed on ice. The resulting ice-bath mixture was then added to a pre-chilled electroporation cuvette (electroporator program: C=25 μF, PC=200 Ω, V=2400 V). The cuvette was capped, dried, and inserted into the electroporator groove. Electroporation was started, and after the reaction was complete, the mixture was quickly transferred to an EP tube. 1 mL of TY medium (each liter of TY liquid medium consists of 5 g peptone, 3 g yeast extract, and 10 mL of 1 M calcium chloride aqueous solution) was added. The mixture was incubated at 28 °C for 3 h. Then, it was plated onto a medium containing Kana and Rif (each liter of TY solid medium consists of 5 g peptone, 3 g yeast extract, 15 g agar, and 10 mL of 1 M calcium chloride aqueous solution). After incubation for 48 h, colony PCR identification was performed. The results are as follows: Figure 2 As shown, pCAM35S-BoSULTR1;1-eGFP and [other components] were successfully obtained.

[0049] pCAM35S-BoSULTR1;2-eGFP expression vector recombinant bacteria.

[0050] (4) Preparation of transgenic broccoli hairy roots

[0051] In this example, broccoli seedlings growing for 2 weeks, growing state is consistent and robust, using the recombinant bacteria obtained in (3) to infect, the process is: under the cotyledon node 2 cm oblique cutting, cutting surface scraping Agrobacterium rhizogenes ATCC15834 bacteria and then quickly inserted into the flowerpot containing only vermiculite, OD 600 = 0.8 of Agrobacterium rhizogenes ATCC15834 bacteria liquid into the root, immediately buckle the transparent plastic cup to keep moist, to ensure adequate water; after 1 week of growth, a large number of hairy roots appear at the cutting surface, cut off the root system of non-oblique cutting, reseeded in mixed soil; green fluorescence detection of transgenic broccoli hairy roots, green fluorescence indicates successful transformation, obtain positive transgenic broccoli hairy root chimeras, that is, transgenic broccoli plants overexpressing BoSULTR1;1 and BoSULTR1;2 genes, as shown in Figure 3 .

[0052] Example 2: Determination of sulforaphane and indole-3-carbinol content

[0053] In this example, the transgenic broccoli hairy roots with empty vector were used as the control group, and the positive transgenic broccoli hairy root chimeras (transgenic broccoli plants overexpressing BoSULTR1;1 and BoSULTR1;2 genes) obtained in Example 1 were used as the experimental group. The fresh broccoli hairy roots of the control group and the experimental group were washed clean and cut into 2 mm slices. 75 g of broccoli slices were added to 150 g of water and then placed in a high-speed disperser for shearing treatment. The high-speed disperser speed was adjusted to 13000 rpm and the shearing time was 5 min. Then 100 g of the sheared sample was homogenized for 2 min at a speed of 7 m / s. The broccoli homogenate sample was then placed in a self-sealing bag and incubated in a water bath at 40℃ for 20 min. After incubation, the sample was placed in a freeze dryer at 40℃ and a vacuum degree of 0.009 mbar for 60 h. After freeze-drying, the sample was crushed for 30 s and passed through a 120 mesh sieve to obtain broccoli powder.

[0054] The control group and experimental group broccoli powders obtained above were determined for sulforaphane and indole-3-carbinol content, which included the following steps:

[0055] Sulforaphane and gallic acid standards were purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.

[0056] (1) Extraction and determination method of sulforaphane

[0057] Take 0.3 g of broccoli powder of the control group and the experimental group respectively and place them in EP tubes, then add 1.0 mL of phosphate buffer with pH = 6.0 respectively, grind into slurry using a grinder, then water bath at 40°C for 2 h, cool to room temperature, then extract 3 times with 5 mL of ethyl acetate, transfer the upper organic phase to a new centrifuge tube, dry by nitrogen blowing, then use methanol to make up to 0.2 mL, mix well, filter using a needle filter, and detect the filtrate by an Agilent 1100 high performance liquid chromatograph; establish a standard curve using a sulforaphane standard, and calculate the content of sulforaphane according to the standard curve, with the content expressed in μg / g FW.

[0058] The chromatograph parameters are: the wavelength of the ultraviolet detector is 254 nm; the chromatograph column is a Compass C18(2) reversed-phase chromatograph column (250 mm*4.6 mm, 5 μm), the column temperature is 30°C, the flow rate is 1 mL / min, the injection volume is 10 μL, and the mobile phase is 0.1% phosphoric acid aqueous solution: acetonitrile = 80:20 (V / V) 0 min; 5:95 (V / V) 25 min.

[0059] (2) Extraction and determination method of indole-3-carbinol

[0060] Take 0.2 g of broccoli powder of the control group and the experimental group respectively and place them in EP tubes, then add 1.5 mL of pre-cooled 80% methanol aqueous solution respectively, grind into slurry using a grinder, then extract overnight at 4°C, centrifuge at 8000 g for 10 min, and take the supernatant; the centrifugal residue is extracted with 0.5 mL of 80% methanol aqueous solution for 2 h, centrifuged at 8000 g for 10 min, and the supernatant is taken out after centrifugation, and the two supernatants are combined; dry by nitrogen blowing at 40°C until no organic phase is present, then extract and decolorize 3 times with 2 mL of petroleum ether, add 1 mol / L citric acid aqueous solution to the lower aqueous phase, and adjust the pH to 2-3; then extract 2 times with 2 mL of ethyl acetate, transfer the upper organic phase to a new EP tube, dry by nitrogen blowing, then dissolve with 0.2 mL of methanol, mix well, filter using a needle filter, and detect the filtrate by a Waters 2695 high performance liquid chromatograph; establish a standard curve using an indole-3-carbinol standard, and calculate the content of indole-3-carbinol according to the standard curve, with the content expressed in μg / g FW.

[0061] The chromatograph parameters are: the excitation wavelength of the 2475 fluorescence detector is 275 nm, and the emission wavelength is 340 nm; the chromatograph column is a Compass C18(2) reversed-phase chromatograph column (250 mm*4.6 mm, 5 μm), the column temperature is 35°C, the flow rate is 1 mL / min, and the mobile phase is water:methanol = 70:30 (V / V) 0 min.

[0062] The content of sulforaphane and indole-3-carbinol in the hairy roots of the test broccoli was determined, and the results are shown in Table 1. Figure 4 As shown in Table 1, the content of sulforaphane in the transgenic broccoli hairy roots overexpressing BoSULTR1;1 gene was increased by 38.4% compared with the control group; the content of sulforaphane in the transgenic broccoli hairy roots overexpressing BoSULTR1;2 gene was slightly increased compared with the control group.

[0063] The content of indole-3-carbinol in the transgenic broccoli hairy roots overexpressing BoSULTR1;1 gene was increased by 64.8% compared with the control group; the content of indole-3-carbinol in the transgenic broccoli hairy roots overexpressing BoSULTR1;2 gene was increased by 45.3% compared with the control group.

[0064] Therefore, overexpression of BoSULTR1;1 gene and BoSULTR1;2 gene can increase the content of sulforaphane and indole-3-carbinol in transgenic broccoli.

[0065] Example 3: Determination of the content of glucoraphanin and glucoraphenin

[0066] Since the aliphatic glucosinolate glucoraphanin can be hydrolyzed by myrosinase to generate sulforaphane, and the indole glucosinolate glucoraphenin can be hydrolyzed by myrosinase to generate indole-3-carbinol, it can be seen that glucoraphanin and glucoraphenin are the precursor substances of sulforaphane and indole-3-carbinol, respectively.

[0067] In this example, the transgenic broccoli hairy roots into which no gene was introduced were used as the control group, and the transgenic broccoli hairy roots obtained in Example 1 overexpressing BoSULTR1;1 and BoSULTR1;2 genes were used as the experimental group. The hairy roots of the control group and the experimental group were made into powders, and the specific steps were the same as in Example 2. The content of glucoraphanin and glucoraphenin was determined, which specifically included the following steps:

[0068] (1) Extraction and determination of glucoraphanin

[0069] S1: 500 mg of the broccoli powder sample of the control group and the experimental group was weighed into a 2 mL EP tube, frozen with liquid nitrogen, and then broken with a tissue crusher, with the crusher set at 50 Hz for 5 min. Then, 1 mL of pre-cooled 70% methanol solution was added to each EP tube, and the tubes were again broken with the crusher, with the crusher set at 30 Hz for 5 min. Then, the tubes were centrifuged at 4°C and 15000 x g for 10 min, and the supernatant was transferred to a new 10 mL EP tube. The above steps were repeated twice. Then, 4 mL of the supernatant was taken and placed on ice for standby;

[0070] S2: Prepare the chromatography column in advance, flush the above chromatography 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 acetic acid buffer, and cover the bottom cover when the acetic acid buffer liquid surface flows to 2 mm away from the gel surface;

[0071] S3: Shake the dextran gel DEAE A-25 resin solution again, slowly add 1 mL to the treated chromatography column in S2 while shaking, and ensure that the gel surface is at the same height. Open the bottom cover of the chromatography column to allow the liquid to flow out, and the gel to sink. Then flush the chromatography column with 5 mL of deionized water, and cover the bottom cover when the liquid surface is 2 mm away from the gel surface. Add the supernatant obtained in S1 to the corresponding chromatography column, and slowly add to prevent the gel surface from floating. Then flush with 2 mL of pre-cooled 70% methanol solution for 2 times, 2 mL of deionized water for 5 times, and 2 mL of 20 mmol / L, pH = 5 acetic acid buffer for 1 time, and cover the bottom cover when the liquid surface is 2 mm away from the gel surface. Then add 0.5 mL of sulfatase, cover the bottom cover when the liquid surface is 2 mm away from the gel surface, and cover the top cover, and incubate at room temperature overnight.

[0072] S4: Take 600 μL of deionized water, elute the chromatography column in S3 after incubation at room temperature into a 2 mL EP tube, and freeze-dry. Dissolve the dried powder with 150 μL of deionized water, and centrifuge to filter in a filter membrane tube. Set the temperature of the centrifuge to 4°C, the speed to 13800 x g, and the time to 10 min, and collect the filtrate into an HPLC inner tube.

[0073] The content of sulforaphane was detected by UPLC ultra-high performance liquid chromatography, the detection wavelength was 229 nm, the flow rate of the mobile phase was 1.0 mL / min, and the temperature was 25°C; the mobile phase was water (A) and methanol (B), 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%; and the chromatogram curve was obtained, and the content of sulforaphane was:

[0074]

[0075] 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 sulforaphane, A2 is the response coefficient of the standard sample (the standard sample coefficient is 1), C is the amount of internal standard added (nmol), and m is the mass of the plant sample (mg).

[0076] (2) Extraction and determination of brassinolide glucosinolate

[0077] The extraction and determination method of brassinolide glucosinolate is the same as that in embodiment (1).

[0078] The glucoraphanin content is:

[0079]

[0080] 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).

[0081] In this example, the contents of sinigrin and glucoraphanin in the tested broccoli hairy roots were determined, and the results are shown in Table 1. Figure 5 As shown in Table 1, the content of sinigrin in the transgenic broccoli hairy roots overexpressing the BoSULTR1;1 gene was increased by 51.7% compared with the control group, and the content of sinigrin in the transgenic broccoli hairy roots overexpressing the BoSULTR1;2 gene was increased by 67.6% compared with the control group.

[0082] As shown in Table 1, the content of glucoraphanin in the transgenic broccoli hairy roots overexpressing the BoSULTR1;1 gene was increased by 89.6% compared with the control group, and the content of glucoraphanin in the transgenic broccoli hairy roots overexpressing the BoSULTR1;2 gene was increased by 94.8% compared with the control group.

[0083] Overexpression of the BoSULTR1;1 gene and the BoSULTR1;2 gene can increase the contents of sinigrin and glucoraphanin in the transgenic broccoli. Since sinigrin and glucoraphanin are precursors of sulforaphane and indole-3-carbinol, respectively, it can be seen that the BoSULTR1;1 gene and the BoSULTR1;2 gene can further increase the yields of sulforaphane and indole-3-carbinol after hydrolysis by myrosinase by increasing the contents of the precursors sinigrin and glucoraphanin.

[0084] The content not described in detail in the specification of the present application is known to those skilled in the art. Although the present application has been disclosed with reference to the preferred embodiments above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method of preparing broccoli enriched in sulforaphane and indole-3-carbinol, characterized in that, The method comprises the following steps: S1. Cloning of the gene using primer pair BoSULTR1;1 and BoSULTR1;2 gene; S2. Obtained from S1 BoSULTR1;1 Gene cloning sequence and BoSULTR1;2 Gene clone sequences are ligated into vectors to obtain expression vectors; S3. The expression vectors obtained in S2 are respectively transformed into Agrobacterium rhizogenes to obtain recombinant bacteria; S4. The recombinant bacteria obtained in S3 are used to infect plants to obtain transgenic plants; As described in S1 BoSULTR1;1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. As described in S1 BoSULTR1;2 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

2. The production method according to claim 1, characterized by, S1 in the BoSULTR1;1 The nucleotide sequence of the upstream primer in the primer of the gene is SEQ ID No. 3, and the nucleotide sequence of the downstream primer is SEQ ID No.

4.

3. The production method according to claim 1, characterized by, S1 in the BoSULTR1;2 The nucleotide sequence of the upstream primer in the primer of the gene is SEQ ID No. 5, and the nucleotide sequence of the downstream primer is SEQ ID No.

6.

4. The method of claim 1, wherein, The vector in S2 is pCAM35S.

5. The preparation method according to claim 1, characterized in that, The infection method of the recombinant bacteria in S4 is Agrobacterium rhizogenes mediation.

6. A broccoli enriched with sulforaphane and indole-3-carbinol, characterized in that, The broccoli is obtained by the preparation method in any one of claims 1 to 5.

7. Use of the broccoli enriched with sulforaphane and indole-3-carbinol according to claim 6 in food preparation, characterized by the fact that, The application is to make a salad by matching the cooked broccoli with other vegetables, or to make a broccoli vegetable juice in a juicer.

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