Use of boSLIM1 protein in increasing sulforaphane content and indole-3-carbinol content in broccoli

Abstract

The application discloses application of BoSLIM1 protein in increasing contents of sulforaphane and indole-3-methanol in broccoli and belongs to the technical field of plant breeding. In order to simultaneously increase the contents of sulforaphane and indole-3-methanol in broccoli, the application provides application of the protein shown in SEQ ID NO. 2 in increasing the contents of sulforaphane and / or indole-3-methanol and / or glucoraphanin and / or glucobrassicin in broccoli. The contents of sulforaphane and indole-3-methanol in broccoli are simultaneously increased, the nutritional value of the broccoli is greatly improved, the needs of modern consumers are met, the people are facilitated to obtain the nutritional components in the vegetables, and thus the health of the people is benefited.

Description

TECHNICAL FIELD

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

[0002] Broccoli (Brassica oleracea var. italica) Brassica oleracea var. italica ), also known as green cabbage, is one of the varieties of Brassica oleracea of Brassicaceae. Broccoli has rich nutritional and medicinal values. In addition to rich protein, vitamins and mineral elements, it also contains glucoraphanin and various indole derivatives.

[0003] As a high-grade health vegetable, how to adopt a suitable cooking method to reduce the loss of nutritional components and increase the generation of active components, and at the same time maintain the good flavor, texture or color of food is a key problem. Food processing technology as a means to protect and improve nutrition, its influence cannot be ignored, which is related to food quality and safety issues. It is reported that endogenous myrosinase in homogenized broccoli can only convert 25% (or less) of glucoraphanin to sulforaphane, which means that direct intake of broccoli may not be enough to produce the expected health benefits, because the glucosinolate is still in its original form. Since the generation of glucosinolate hydrolysis products involves enzymatic reactions, proper heat processing can increase the content of hydrolysis products. Traditional broccoli processing methods include steaming, boiling, frying, stir-frying, etc. Research has found that all of them will cause loss of glucosinolate and sulforaphane to varying degrees. How to simultaneously increase the contents of sulforaphane and indole-3-carbinol in broccoli is a technical problem to be solved. SUMMARY

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

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

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

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

[0008] The application provides application of a recombinant microbial cell containing a gene shown in SEQ ID NO. 1 in increasing contents of sulforaphane, indole-3-methanol, glucoraphanin or glucobrassicin in broccoli.

[0009] The application provides application of broccoli overexpressing a gene shown in SEQ ID NO. 1 in preparation of food rich in sulforaphane or / and indole-3-methanol.

[0010] The application provides application of broccoli overexpressing a gene shown in SEQ ID NO. 1 in food preparation.

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

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

[0013] Step 2: transferring the recombinant vector obtained in step 1 into agrobacterium to obtain a recombinant agrobacterium;

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

[0015] The application provides a biological preparation, and the biological preparation contains the broccoli obtained by the method.

[0016] Further, the biological preparation is any one of a tablet, a capsule, a granule, a powder, and a liquid preparation.

[0017] The application provides a method for increasing contents of sulforaphane and indole-3-methanol or glucoraphanin or glucobrassicin in broccoli, and overexpressing a gene shown in SEQ ID NO. 1 in broccoli.

[0018] Beneficial effects: the empty vector-transferred broccoli hairy roots are used as a control group, overexpression of BoSLIM1 in the hairy roots of broccoli significantly increases contents of the anticancer active substances sulforaphane and indole-3-methanol in leaves to 1.65 times and 1.69 times, simultaneously increases the contents of sulforaphane and indole-3-methanol in broccoli, greatly improves the nutritional value of broccoli, meets the needs of modern consumers, is beneficial to the acquisition of nutritional components in vegetables by people, and is beneficial to the health of the body. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1Figure for obtaining BoSLIMl gene transformed broccoli hairy roots; A: 15-day-old broccoli, B: ATCC15834 Agrobacterium infection, C: broccoli was taken out from vermiculite and lateral roots were cut off, D: 7-day-old broccoli re-cultured in soil with 1:1 ratio of nutrient soil and vermiculite, E: BoSLIMl gene transformed broccoli hairy roots were obtained;

[0020] Figure 2 Figure for analysis results of BoSLIMl gene transformed broccoli hairy roots glucoraphanin and indole-3-carbinol content;

[0021] Figure 3 Figure for analysis results of BoSLIMl gene transformed broccoli hairy roots glucoraphanin and glucobrassicin content;

[0022] Figure 4 Figure for BoSLIMl gene overexpression identification results. DETAILED DESCRIPTION

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

[0024]

[0025] MGGGDLALSV ADIRMENEQP DDLASDTVAE IDVSDEEIDA EDLERRMWKR IRLKRIKER

[0026] QKGDSQGPQAKEPPKKISDQAQRKKMSRAQDGILKYMLKLMEVCKVRGFVYGIIPEKGKP

[0027] VSGSSDNIRAWWKEKVKFDKNGPAAIAKYEEECLAFGKSDGNRNSQFVLQDLQDATLGSL

[0028] LSSLMQHCDPPQRKYPLEKGTPPPWWPTGKEEWWVKLGLPQSQSPPYRKPHDLKKMWKVG

[0029] VLTAVINHMSPDIAKIKRHVRQSKCLQDKMTAKESAIWLAVLNQEESLIQQPSSDNGTSN

[0030] VTETHRRGNNADRRKTVINSDSDYDVDGTEEASGSVSSKDSRRNQVPAATSQQPVRDQDK

[0031] AGKHKRRKRPRIRSGTLNVQDEEQVEAEGRNVLPDMNHVEAPMLDYNINGTTNHHEEGVL

[0032] EPNISLGPEENGLELVVPEFDSNYTYLPPVDGQAMMPVDERPMLYGANPNQELQFGSGYN

[0033] YYNTSAVFVHNQEEDLIHTQIEMNSQAPPHSNGFDGQGGVLQPHGNEEVGVAGRDMPPQF

[0034] QSDQDKLLDSNILSPFNDLPFDSSTFYSGFDSFGAFDDDYSWFGA;

[0035] Using BoSLIM1-upstream primer and BoSLIM1-downstream primer, according to the PCR reaction system in Table 1, the PCR reaction was carried out to amplify the BoSLIM1 gene sequence, and the target band was obtained after electrophoresis.

[0036] BoSLIMl -upstream primer: GAGCTCGGTACCCGGGGATCCATGGGTGGGGGCGATCTA (SEQ ID NO. 3);

[0037] BoSLIMl -downstream primer: GGTCGACTCTAGAGGATCCAGCTCCAAACCATGAGTAGTC (SEQ ID NO. 4);

[0038] PCR program:

[0039] PCR reaction system of Table 1

[0040]

[0041] PCR program: 94 ̊C 10 min→(94 ̊C 30 s→58 ̊C 30 s→72 ̊C 2 min) x 30→72 ̊C 10 min→4 ̊C.

[0042] 2. Recovery of target fragment and vector ligation

[0043] Recovery of target fragment: SPARK easy Gel DNA Extraction Kit was used for recovery and purification, and the operation method was as follows:

[0044] (1) Under the irradiation of ultraviolet lamp, the gel block with target band was cut with a blade and placed in a 1.5 mL EP tube.

[0045] (2) 600 μL of gel dissolving agent was added, heated in a metal bath at 65 °C until completely melted, and inverted every 2 min to accelerate melting.

[0046] (4) After adding 600 μL of rinse solution WB, centrifugation was performed, the centrifuge was set at 13800 x g for 1 min, and the effluent was discarded. (Repeat once)

[0047] (5) The absorption column EC was placed back into the collection tube and centrifuged at a speed of 13800 x g for 2 min.

[0048] (6) The absorption column EC was placed into a new collection tube, 20 μL of RNase Free H2O was added to the middle of the absorption membrane, and after standing at room temperature for 2 min, centrifugation was performed, the centrifuge was set at 13800 x g for 1 min, and the eluted product was the purified target fragment.

[0049] Vector ligation: the pCAMBIA1301-35S-BoSLIM1-EGFP expression vector was constructed using the method of homologous recombination, and the recombination system was as follows:

[0050] Table 2 Construction of pCAMBIA1301-35S-BoSLIM1-EGFP expression vector

[0051]

[0052] 37 ℃ reaction for 30 min.

[0053] Example 2. Obtain recombinant bacteria

[0054] Take 2 μL of the constructed plasmid pCAMBIA1301-35S-BoSLIM1-EGFP, and add it to 50 μL of the melted Agrobacterium tumefaciens competent ATCC15834, respectively. After mixing, insert it into ice. Set the electroporation instrument program: C = 25 μF, PC = 200 Ω, V = 2400 V. Add the ice-bathed mixture to the pre-cooled electroporation cup, cover the cup, wipe dry, and then insert it into the electroporation instrument groove. Start the electric shock, and after completion, quickly transfer it to the EP tube and add 1 mL of TY medium. Culture at 28 ℃ for 3 h, then spread on the medium containing Kana and Rif. Culture for 48 h, and then perform colony PCR identification. The correct one is used for subsequent hairy root transformation.

[0055] Example 3. Obtain transgenic broccoli hairy roots

[0056] I. (1) When broccoli grows to 2 weeks, select seedlings with consistent growth and robustness for infection. The infection process is as follows: make an oblique cut 2 cm below the cotyledon node, scrape the Agrobacterium tumefaciens bacteria on the oblique surface, and then quickly insert it into a flowerpot containing only vermiculite. Suck OD 600 = 0.8 of the Agrobacterium tumefaciens bacteria obtained in Example 2 into the roots, immediately cover it with a transparent plastic cup to keep it moist, and ensure sufficient water.

[0057] (2) After 1 week of growth, a large number of hairy roots appeared at the cut surface. Remove the root system that is not obliquely cut, and re-plant it in mixed soil to obtain 35S::BoSLIM1 hairy roots, as shown in Figure 1 .

[0058] II. Identification

[0059] Green fluorescence detection and gene expression detection were performed on the hairy roots to obtain positive broccoli hairy root chimeras

[0060] Identification primer: BoSLIM1-qpcr-upper: CGTCAGTCTAAGTGTTTGCAG (SEQ ID NO. 5);

[0061] BoSLIM1-qPCR-downstream: AGTTCCGGATCTGATTCTAGG (SEQ ID NO. 6), and the results of the identification are shown in Table 2, and it is confirmed that the broccoli obtained by overexpressing the BoSLIM1 gene is indeed overexpressing the BoSLIM1 gene. Figure 4

[0062] Example 4. Content of anticancer active substances sulforaphane and indole-3-carbinol

[0063] This example takes the empty transgenic broccoli hairy roots as the control group, and the transgenic broccoli hairy roots obtained in Example 3 which overexpress the BoSLIM1 gene as the experimental group, and the fresh broccoli leaves of the control group and the experimental group are taken for subsequent experiments.

[0064] The content of sulforaphane and indole-3-carbinol in the broccoli powder obtained above is determined, which includes the following steps:

[0065] (1) Extraction and determination method of sulforaphane

[0066] 1 g of fresh broccoli leaves of the control group and the experimental group is weighed and placed in an EP tube, respectively, and 1.0 mL of phosphate buffer with pH=6.0 is added. After being ground into a slurry using a grinder, it is water-bathed at 40°C for 2 h, and after cooling to room temperature, 5 mL of ethyl acetate is added for extraction three times. The upper organic phase is transferred to a new centrifuge tube, dried by nitrogen blowing, and then diluted to 0.2 mL with methanol, mixed well, filtered using a needle filter, and the filtrate is detected by Agilent 1100 high performance liquid chromatograph. The standard curve is established using sulforaphane standard, and the content of sulforaphane is calculated according to the standard curve, and the content is expressed in μg / g FW.

[0067] 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.

[0068] (2) Extraction and determination method of indole-3-carbinol

[0069] ​Take 1 g of fresh broccoli leaves of the control group and the experimental group respectively and place them in EP tubes, add 1.5 mL of pre-cooled 80% methanol solution respectively, grind into slurry using a grinder, soak overnight at 4°C, centrifuge at 8000xg for 10 min, take the supernatant; the centrifugal residue is soaked with 0.5 mL of 80% methanol solution for 2 h, centrifuged at 8000xg for 10 min, take out the supernatant after centrifugation, and combine the two supernatants; nitrogen blow at 40°C until no organic phase is left, then add 2 mL of petroleum ether for extraction and decolorization for 3 times, add 1 mol / L citric acid solution to the lower water phase, adjust the pH to 2-3; add 2 mL of ethyl acetate for extraction for 2 times, transfer the upper organic phase to a new EP tube, dry by nitrogen blowing, add 0.2 mL of methanol for dissolution, mix well, filter using a needle filter, and detect the filtrate by Waters 2695 high performance liquid chromatograph; establish a standard curve using indole-3-methanol standard, and calculate the content of indole-3-methanol according to the standard curve, and express the content as μg / g FW.

[0070] The chromatograph parameters are: 2475 fluorescence detector excitation wavelength 275 nm, emission wavelength 340 nm; the chromatograph column is 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).

[0071] Results: The broccoli hairy roots into which the empty vector is transformed are used as the control group, and the overexpression of BoSLIM1 in the broccoli hairy roots significantly increases the contents of the anticancer active substances sulforaphane and indole-3-methanol in the leaves, which are increased by 1.65 times and 1.69 times, respectively, 1.65 times refers to the increase of sulforaphane by 1.65 times, and 1.69 times refers to the increase of indole-3-methanol, and the results are shown in Table 2 and Table 3. Figure 2 and Table 3.

[0072] Example 5. Contents of the precursor substances sulforaphane glucosinolate and glucoraphanin

[0073] Since the aliphatic glucosinolate sulforaphane glucosinolate can be hydrolyzed by myrosinase to generate sulforaphane, and the indole glucosinolate glucoraphanin can be hydrolyzed by myrosinase to generate indole-3-methanol, it can be seen that sulforaphane glucosinolate and glucoraphanin are the precursor substances of sulforaphane and indole-3-methanol, respectively.

[0074] In this example, the transgenic broccoli hairy roots into which the empty vector is transformed are used as the control group, the transgenic broccoli hairy roots obtained in Example 3 which overexpress BoSLIM1 gene are used as the experimental group, and the leaves of the above-mentioned control group and experimental group are cut, and the contents of sulforaphane glucosinolate and glucoraphanin are determined, which specifically includes the following steps:

[0075] (1) Extraction and determination of sulforaphane

[0076] S1: 500 mg of broccoli leaf samples in the control group and the experimental group were weighed and placed in 2 mL EP tubes, frozen with liquid nitrogen, and then crushed with a tissue crusher. The crusher was set to 50 Hz for 5 min. Then, 1 mL of pre-cooled 70% methanol solution was added to each EP tube, and the tubes were shaken and crushed again in the crusher, which was set to 30 Hz for 5 min. Then, the tubes were centrifuged at 15000 x g at 4°C for 10 min. The supernatant was transferred to a new 10 mL EP tube, and the above steps were repeated twice. 4 mL of the supernatant was taken and placed on ice for later use. g

[0077] S2: A chromatography column was prepared in advance. The column was flushed with 5 mL of deionized water. After the deionized water flowed out, the bottom cover was covered, and 500 μL of 0.5 mol / L acetic acid buffer was slowly added. When the acetic acid buffer flowed to 2 mm from the gel surface, the bottom cover was covered.

[0078] S3: The dextran gel DEAE A-25 resin solution was shaken and 1 mL was slowly added to the treated chromatography column in S2. The addition position should be at the same height to ensure the consistency of the gel surface height. The bottom cover of the chromatography column was opened, and the liquid was allowed to flow out. The gel was allowed to sink, and the chromatography column was flushed with 5 mL of deionized water. When the liquid surface was 2 mm from the gel surface, the bottom cover was covered. The supernatant obtained in S1 was added to the corresponding chromatography column, and the addition was slow to prevent the gel surface from floating. Then, 2 mL of pre-cooled 70% methanol solution was used to flush twice, 2 mL of deionized water was used to flush five times, and 2 mL of 20 mmol / L, pH=5 acetic acid buffer was used to flush once. When the liquid surface was 2 mm from the gel surface, the bottom cover was covered. Then, 0.5 mL of sulfatase was added, the bottom cover was covered when the liquid surface was 2 mm from the gel surface, and the top cover was covered. The column was incubated at room temperature overnight.

[0079] S4: 600 μL of deionized water was used to elute the chromatography column in S3 after overnight incubation at room temperature into a 2 mL EP tube, and then freeze-dried. The dried powder was dissolved in 150 μL of deionized water, and then centrifuged and filtered in a filter membrane tube. The centrifuge was set to 4°C, the speed was 13800 x g, the time was 10 min, and the filtrate was collected in an HPLC liner tube. g

[0080] ​​The UPLC ultra-high performance liquid chromatograph is used to detect the content of radish glucosinolate, the detection wavelength is 229 nm, the flow rate of mobile phase is 1.0 mL / min, and the temperature is 25°C; the mobile phase is water (A) and methanol (B), the running time is: 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 chromatogram curve is obtained, and the content of radish glucosinolate is:

[0081]

[0082] 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 radish glucosinolate, A2 is the response coefficient of the standard sample (the standard sample coefficient is 1), C is the internal standard addition amount (nmol), and m is the mass of the plant sample (mg).

[0083] (2) Extraction and determination of brassinolide glucosinolate

[0084] The extraction and determination method of brassinolide glucosinolate is the same as that in the embodiment (1).

[0085] The content of brassinolide glucosinolate is:

[0086]

[0087] 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 radish glucosinolate, A2 is the response coefficient of the standard sample (the standard sample coefficient is 1), C is the internal standard addition amount (nmol), and m is the mass of the plant sample (mg).

[0088] Results: The precursors of sulforaphane and indole-3-carbinol are detected, and it is found that overexpression of BoBoSLIM1 in hairy roots of broccoli significantly increases the content of radish glucosinolate and brassinolide glucosinolate in leaves, which is increased by 1.51 times and 1.62 times, respectively. The content of radish glucosinolate is increased by 1.51 times, and the content of brassinolide glucosinolate is increased by 1.62 times. The results are shown in Table 2 and Table 3. Figure 3 and Table 3.

[0089] Table 3

[0090]

Claims

1. Use of a protein as shown in SEQ ID NO. 2 in increasing sulforaphane, indole-3-carbinol, glucoraphanin content or / and glucobrassicin content in Brassica oleracea.

2. Use of a gene as shown in SEQ ID NO. 1 in increasing sulforaphane, indole-3-carbinol, glucoraphanin content or / and glucobrassicin content in Brassica oleracea.

3. Use of a recombinant vector containing a gene as shown in SEQ ID NO. 1 in increasing sulforaphane, indole-3-carbinol, glucoraphanin content or / and glucobrassicin content in Brassica oleracea.

4. Use of Brassica oleracea overexpressing a gene as shown in SEQ ID NO. 1 in preparing food rich in sulforaphane or / and indole-3-carbinol.

5. Use of Brassica oleracea overexpressing a gene as shown in SEQ ID NO. 1 in food preparation.

6. A method of breeding broccoli plants enriched in sulforaphane and indole-3-carbinol, characterized in that, The specific steps of the method are as follows: Step 1: connecting a gene sequence as shown in SEQ ID NO. 1 with a pCAMBIA1301-35S-EGFP vector to obtain a recombinant vector; Step 2: transferring the recombinant vector obtained in step 1 into Agrobacterium to obtain a recombinant Agrobacterium; Step 3: infecting Brassica oleracea with the recombinant Agrobacterium obtained in step 2 to obtain transgenic Brassica oleracea.

7. A method of increasing the content of sulforaphane, indole-3-carbinol, glucoraphanin or / and glucobrassicin in broccoli, characterized in that, Overexpressing a gene as shown in SEQ ID NO. 1 in Brassica oleracea.

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