Application of BoHY5 gene or protein coded by BoHY5 gene in promoting synthesis of glucoraphanin of broccoli

Abstract

The invention discloses an application of a BoHY5 gene or a protein coded by the BoHY5 gene in promoting synthesis of broccoli glucoraphanin, and relates to the technical field of agricultural biology. The broccoli BoHY5 gene is found to be related to glucosinolate synthesis through screening, the content of total glucosinolate, aliphatic glucosinolate, indole glucosinolate and glucoraphanin of broccoli overexpression strains OE-3 and OE-5 is remarkably increased compared with that of wild WT, and the gene can be applied to cultivation of broccoli varieties with high glucoraphanin content and is used for improving the quality of broccoli.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and particularly to BoHY5 the application of a gene or a protein encoded thereby in promoting the synthesis of glucoraphanin in broccoli. Background Art

[0002] With the development of social economy and the improvement of people's living standards, as well as the promotion of the "Healthy China Initiative", the efficacy of vegetables in national nutrition and health, especially in disease prevention, has received extensive attention. Broccoli is known as the "crown of vegetables" and is called a traditional anti-cancer vegetable in the West. The main component responsible for its anti-cancer effect is glucoraphanin in aliphatic glucosinolates. The degradation product of glucoraphanin, sulforaphane, has an inhibitory effect on liver cancer, colon cancer, breast cancer, prostate cancer, etc., and can significantly reduce the risk of cardiovascular and cerebrovascular diseases, myopia, etc. It is one of the best substances for cancer prevention and anti-cancer effects in vegetables so far. Research shows that glucoraphanin is abundantly present in cruciferous vegetables, especially in broccoli with the highest content. With the development of molecular design breeding technology, by cloning the key genes of glucoraphanin in broccoli and analyzing its network regulation mechanism, the precise creation of high-sulforaphane broccoli germplasms can be achieved, accelerating the replacement of high-quality broccoli varieties, and promoting the revitalization of China's seed industry and the controllability of broccoli seed sources. Summary of the Invention

[0003] Aiming at the above deficiencies in the prior art, the present invention provides BoHY5 the application of a gene or a protein encoded thereby in promoting the synthesis of glucoraphanin in broccoli and in improving the anti-cancer effect of broccoli.

[0004] The technical solutions provided by the present invention are as follows: The present invention provides BoHY5 the application of a gene or a protein encoded thereby in promoting the synthesis of glucoraphanin in broccoli.

[0005] The BoHY5 amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; BoHY5 the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] In the above application, the organs for glucoraphanin synthesis in broccoli are at least one of stem, leaf, flower and bud.

[0007] Meanwhile, the present invention also provides a method for improving the synthesis of glucosinolates in broccoli, which is to overexpress the BoHY5 gene in broccoli.

[0008] In the above method, preferably, the BoHY5 amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; theBoHY5 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0009] In the method described above, further, when BoHY5 the gene is overexpressed in broccoli, the following steps are included: introducing an overexpression vector containing BoHY5 the gene into the cells of broccoli, and obtaining transgenic broccoli plants after cultivation. It further includes cultivating a homozygous variety, for example, by self-crossing or hybridization.

[0010] In the method described above, the gene overexpression vector containing BoHY5 the gene is a plant expression vector with a sequence for overexpressing the gene whose base sequence is shown in SEQ ID NO.1. BoHY5

[0011] Preferably, the plant expression vector is pCBSG015 (Basta).

[0012] BoHY5 The contents of total glucosinolates, aliphatic glucosinolates, indole glucosinolates, and glucoraphanin in the mutant broccoli with overexpressed

[0013] gene are all significantly higher than those in wild-type broccoli. BoHY5 Through previous research, the present invention found that the BoHY5 gene in broccoli is related to the synthesis of glucosinolates in broccoli. The contents of total glucosinolates, aliphatic glucosinolates, indole glucosinolates, and glucoraphanin in the overexpressed mutants OE-3 and OE-5 are significantly increased compared with the wild type WT. This gene can be applied to cultivate broccoli varieties with higher glucoraphanin content for improving the quality of broccoli. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 For BoHY5 the agarose gel electrophoresis identification diagram of the

[0015] Figure 2 For BoHY5 the expression level of the overexpressed broccoli plants BoHY5

[0016] Figure 3 For the map of the vector pCBSG015 (Basta).

[0017] Figure 4 For the comparison diagram of the contents of total glucosinolates, aliphatic glucosinolates, indole glucosinolates, and glucoraphanin in the florets of wild-type broccoli plants WT and BoHY5 overexpressed plants (OE-3 and OE-5); "**" indicates P < 0.01.

[0018] Figure 5BoMYB28 positively regulates the accumulation of glucoraphanin in broccoli.

[0019] Figure 6 To show the results of the yeast one-hybrid experiment on the interaction between BoHY5 and BoMYB28 the promoter.

[0020] Figure 7 To show the results of the dual-luciferase experiment on the interaction between BoHY5 and BoMYB28 the promoter. Detailed implementation manners

[0021] Example 1: Mining of genes related to glucosinolate synthesis in broccoli Based on the previous research foundation of the present inventors, broccoli BoMYB28 is the main transcription factor regulating the biosynthesis of aliphatic glucosinolates, and the corresponding research results show that BoMYB28 positively regulates the synthesis of glucoraphanin in broccoli. Based on the previously constructed broccoli yeast library, the present inventors successfully screened out a transcription factor that can bind to BoMYB28 the promoter BoHY5 . Further verification was carried out through yeast one-hybrid point-to-point experiments and dual-luciferase reporter gene experiments BoHY that there is an in vitro interaction between 5 and BoMYB28 the gene promoter.

[0022] Example 2: BoHY5 Cloning of the gene Specific primers were designed using the gene sequence information for amplification to clone the full-length sequence of the gene BoHY5 . The primer sequences are as follows: BoHY5-F: 5’-TCGGAGGAGAGAGTCAAAGGA-3’; BoHY5-R: 5’- TCTCTTGCTTGCTGTGCTGA-3’.

[0023] The total RNA of broccoli leaves was extracted using the SteadyPure Plant RNA Extraction Kit, and the RNA was reverse-transcribed into cDNA (TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR kit (Nanjing Novoprotein Co., Ltd.)), and the experimental steps refer to the instruction manual.

[0024] PCR reaction conditions: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, extension at 72 °C for 2 min, 35 cycles; 72 °C for 10 min. The PCR amplification products were identified by 1.0% agarose gel electrophoresis (Figure 1 After that, recycling and purification were carried out. The specific method refers to the general DNA purification and recovery kit (Shanghai Huiling Biotechnology Co., Ltd.), and it was sent to Tsingke Biotechnology (Changsha) Co., Ltd. for sequencing. Through sequence alignment and analysis, it was found that its amino acid sequence had no difference from that in the broccoli database, and the gene BoHY5 has a full-length CDS of 495 bp, and the gene sequence is shown in SEQ ID No.1; it is speculated that it encodes 164 amino acids, and the amino acid sequence is shown in SEQ ID No.2.

[0025] Example 3: BoHY5 Transformation of broccoli with the gene overexpression vector Construction of the vector pCBSG015 (Basta), and its map is as Figure 3 shown. The construction process is as follows: Design primers to amplify the required target gene fragment from the target organism by PCR technology. Use restriction endonucleases to digest the vector backbone of pCBSG015 (Basta) to linearize it. Connect the target gene fragment with the linearized vector backbone through T4 DNA ligase to form a recombinant vector. Transform the recombinant vector into Escherichia coli DH5α strain. Through antibiotic screening and PCR verification, pick positive clones, and conduct sequencing analysis on the positive clones to confirm that the target gene has been correctly inserted into the vector.

[0026] Adopt the broccoli genetic transformation technology in our laboratory, and use the broccoli Bop15-80 material to carry out the overexpression genetic transformation experiment of the BoHY5 gene. The experimental steps are as follows: Prepare blank MS medium. Wrap an appropriate amount of seeds with gauze, soak them in warm water for 30 min, and then rinse them with cold water for 30 min; Disinfect the seeds with alcohol and mercuric chloride, wash them with pure water, and then gently place the seeds on the MS medium with forceps. Calculate the seedling age starting from when the seeds germinate and show white after sowing. Generally, use the hypocotyls of healthy seedlings with a seedling age of 10 d (when the cotyledons first appear) as explants. Cut the hypocotyls of broccoli seedlings by 0.5 cm, cut them obliquely in a "trapezoid" shape, and place them in the pre-culture medium for light culture for 3 days. After 3 days, resuspend the positive Agrobacterium with an OD600 within the range of 0.5 - 0.6 that has been correctly identified in (liquid pre-culture medium + 100 μM AS + 1 mM MES), and let it stand at 28 °C for 1 - 3 h and then use it to infect the hypocotyls. After the infection is completed, culture it in the dark in the pre-culture medium for 2 days, and then transfer it to the decontamination medium for culture for 3 - 5 days, and then carry out differentiation and proliferation culture. After the transformed seedlings take root, transfer them to a plug tray filled with sterile substrate, cover it and acclimatize the seedlings. After one week, uncover the plug tray cover every day for ventilation, and after 15 days, the plug tray cover can be completely opened.

[0027] When the seedlings to be transformed grow to two leaves and one heart, the total RNA of broccoli leaves is extracted using the SteadyPure Plant RNA Extraction Kit, and the expression level of the BoHY5 gene in the transformed seedlings is detected by fluorescence quantitative detection using the SYBR method. The measurement results of its expression level are as Figure 2 shown, indicating that the gene BoHY5 is up-regulated in the overexpressing plants.

[0028] When the seedlings to be transformed form flower buds, cut the heads and determine the glucosinolate content of the bud parts. Accurately weigh 0.1 g of the freeze-dried sample and place it in a 15 mL centrifuge tube, with three replicates. Add 50 μL of 5 μmol / mL allyl glucosinolate as an internal standard substance, add 5 mL of 70% methanol solution, and extract at 75 °C in a water bath for 20 min (mix well every 2 - 3 min during this period), then take it out and cool; add 2 mL of barium acetate, centrifuge at 8000 r / min and 18 °C for 10 min; transfer the supernatant to a new 15 mL centrifuge tube, add 3 mL of 70% methanol solution to the remaining precipitate, mix well, and then extract in a 75 °C water bath for 15 min (mix well every 2 - 3 min during this period), then take it out and cool, add 0.5 mL of barium acetate, centrifuge at 8000 r / min and 18 °C for 10 min; finally, the supernatant is made up to 12 mL with 70% methanol solution and placed on ice waiting for filtration; Preparation of extraction column: Fix the SPE solid-phase extraction column (2.5 mL disposable syringe) to the iron stand, put in a small white adsorption disc (a disc cut from filter paper of appropriate size), suck an appropriate amount of DEAE Sephadex A25 solution, and add the 9 mL extract to the self-made DEAE Sephadex A25 chromatography column in portions; after the liquid has drained, add 2 mL of 0.02 mol / L sodium acetate, and then add 900 μL of 0.5 mg / mL sulfatase after the liquid has drained, and seal; react at 37 °C for 16 h and then elute with 3 mL of ultrapure water; store at 4 °C (or detect within one week in a -20 °C refrigerator) (with the thin side of the sample vial outside and the thick side inside).

[0029] The results are as Figure 4 shown. The results show that the total glucosinolate content, glucoraphanin, aliphatic glucosinolate and indole glucosinolate contents in the two lines OE-3 and OE-5 are all higher than those of the wild type, indicating that BoHY5 positively regulates the synthesis of glucoraphanin in broccoli.

[0030] Furthermore, through hybridization and glucosinolate content identification (the identification method is as above), stable genetically inherited broccoli BoHY5 overexpressing plants are obtained.

[0031] Through preliminary genetic transformation experiments and glucosinolate content identification in flower buds, it was found that: the sulforaphane content in the leaves of the overexpression lines (OE-2# and OE-61#) of BoMYB28 in broccoli was significantly higher than that of the wild type, and the RNAi suppression expression lines (RNAi-10# and RNAi-11#) of BoMYB28 in broccoli were significantly lower than the wild type, indicating that BoMYB28 positively regulates the synthesis of sulforaphane in broccoli ( Figure 5 ).

[0032] The results of yeast one-hybrid point-to-point experiments and luciferase complementation experiments showed that there was an interaction between the BoMYB28 promoter and the transcription factor BoHY5 ( Figure 6 , Figure 7 ).

Claims

1. BoHY5 Application of the gene or the protein encoded by the gene in promoting the synthesis of glucoraphanin in broccoli.

2. The use according to claim 1, characterized in that The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. The use according to claim 2, characterized in that Said BoHY5 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. The use according to any one of claims 1 to 3, characterized in that The organ for synthesizing glucoraphanin in broccoli is at least one of stems, leaves, flowers and buds.

5. A method for improving the synthesis of broccoli glucosinolates, characterized in that: Use in broccoli BoHY5 Gene overexpression.

6. The method according to claim 5, characterized in that Said BoHY5 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

7. The method according to claim 6, characterized in that Said BoHY5 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

8. The method according to claim 5, characterized in that In broccoli BoHY5 When the gene is overexpressed, the following steps are included: BoHY5 The gene overexpression vector is introduced into the cells of broccoli, and transgenic broccoli plants are obtained after cultivation.

9. The method according to claim 8, characterized in that The said BoHY5 The gene overexpression vector of the gene is a vector with a base sequence as shown in SEQ ID NO.1 BoHY5 Plant expression vector containing the sequence for overexpression of gene.

10. The method according to claim 8, characterized in that Further including breeding homozygous varieties.

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