Application of bohy5 gene or protein coded thereby in promoting synthesis of broccoli radish glucosinolate

Abstract

The application discloses BoHY5 The application discloses a gene or a protein coded by the gene and application of the gene or the protein in promoting synthesis of broccoli radish glucosinolate, and relates to the field of agricultural biotechnology. BoHY5 The gene is related to glucosinolate synthesis, and the total glucosinolate, aliphatic glucosinolate, indole glucosinolate and radish glucosinolate contents of the overexpression lines OE-3 and OE-5 of broccoli are significantly increased compared with those of a wild type WT, so that the gene can be applied to cultivation of a broccoli variety with high radish glucosinolate content and used for improving the quality of the broccoli.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to... BoHY5 Application of genes or their encoded proteins in promoting the synthesis of glucosinolates in broccoli. Background Technology

[0002] With the development of society and economy, the improvement of people's living standards, and the advancement of the "Healthy China Initiative," the efficacy of vegetables in national nutrition and health, especially in disease prevention, has received widespread attention. Broccoli, known as the "crown of vegetables," is considered a traditional Western anti-cancer vegetable. Its main anti-cancer component is sulforaphane, a type of aliphatic glucosinolate. Sulforaphane, a degradation product of sulforaphane, has inhibitory effects on liver cancer, colon cancer, breast cancer, and prostate cancer, and can significantly reduce the risk of cardiovascular disease and myopia. It is one of the most effective anti-cancer substances found in vegetables to date. Studies show that sulforaphane is abundant in cruciferous vegetables, especially broccoli. With the development of molecular design breeding technology, by cloning key genes of sulforaphane in broccoli and analyzing their network regulatory mechanisms, it is possible to precisely create high-sulforaphane broccoli germplasm, accelerate the upgrading of high-quality broccoli varieties, and promote the revitalization of my country's seed industry and the controllability of broccoli germplasm sources. Summary of the Invention

[0003] This invention addresses the aforementioned shortcomings of the prior art by providing... BoHY5 The application of genes or their encoded proteins in promoting the synthesis of glucosinolates in broccoli and enhancing the anti-cancer effects of broccoli.

[0004] The technical solution provided by this invention is as follows:

[0005] This invention provides BoHY5 Application of genes or their encoded proteins in promoting the synthesis of glucosinolates in broccoli.

[0006] The BoHY5 The 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.

[0007] In the aforementioned application, the organ from which broccoli sulforaphane is synthesized is at least one of the stem, leaf, flower, and bud.

[0008] Meanwhile, the present invention also provides a method for improving the synthesis of glucosinolates in broccoli, which involves using... BoHY5 Gene overexpression.

[0009] The method, preferably, the BoHY5The 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.

[0010] The method described further involves removing the broccoli... BoHY5 Gene overexpression includes the following steps: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] BoHY5 Gene overexpression vectors are introduced into broccoli cells, and transgenic broccoli plants are obtained after culturing. Further methods include cultivating homozygous varieties, such as through self-pollination or hybridization.

[0011] The method, wherein the containing BoHY5 The gene overexpression vector is equipped with a base sequence as shown in SEQ ID NO. 1. BoHY5 Plant expression vectors for sequences in which genes are overexpressed.

[0012] Preferably, the plant expression vector is pCBSG015 (Basta).

[0013] BoHY5 The total glucosinolate, aliphatic glucosinolate, indole glucosinolate, and sulforaphane content of the gene-overexpressing mutant broccoli were significantly higher than those of the wild type broccoli.

[0014] This invention discovered broccoli through preliminary research. BoHY5 Genes related to broccoli glucosinolate synthesis, broccoli BoHY5 The total glucosinolate, aliphatic glucosinolate, indole glucosinolate, and glucoraphane content of the overexpression mutants OE-3 and OE-5 were significantly increased compared with the wild-type WT. This gene can be used to breed broccoli varieties with high glucoraphane content for the purpose of improving the quality of broccoli. Attached Figure Description

[0015] Figure 1 for BoHY5 Agarose gel electrophoresis image of the gene.

[0016] Figure 2 for BoHY5 Overexpression of broccoli plants BoHY5 Level of expression.

[0017] Figure 3 The spectrum is for the vector pCBSG015 (Basta).

[0018] Figure 4 For wild-type broccoli plants WT and BoHY5 Comparison of total glucosinolates, aliphatic glucosinolates, indole glucosinolates, and sulforaphane content in the flower heads of overexpression plants (OE-3 and OE-5); "**" indicates P<0.01.

[0019] Figure 5 BoMYB28 positively regulates sulforaphane accumulation in broccoli.

[0020] Figure 6 To display BoHY5 and BoMYB28 Results of yeast one-hybrid experiments on promoter interaction.

[0021] Figure 7 To display BoHY5 and BoMYB28 Results of dual-luciferase assay for promoter interaction. Detailed Implementation

[0022] Example 1: Discovery of genes related to broccoli glucosinolate synthesis

[0023] Based on the author's previous research, broccoli BoMYB28 It is a major transcription factor regulating the biosynthesis of aliphatic glucosinolates, and corresponding research results show... BoMYB28 Positively regulating sulforaphane synthesis in broccoli. Based on a previously constructed broccoli yeast library, the inventors successfully screened a yeast strain that can interact with... BoMYB28 promoter-bound transcription factors BoHY5 Further verification was achieved through yeast one-hybrid point-to-point experiments and dual-luciferase reporter gene assays. BoHY 5 and BoMYB28 Gene promoters interact in vitro.

[0024] Example 2: BoHY5 Cloning of genes

[0025] Specific primers are designed using gene sequence information to amplify and clone genes. BoHY5 Full-length sequence. Primer sequences are as follows:

[0026] BoHY5-F: 5'-TCGGAGGAGAGAGTCAAAGGA-3';

[0027] BoHY5-R: 5'-TCTCTTGCTTGCTGTGCTGA-3'.

[0028] Total RNA was extracted from broccoli leaves using the SteadyPure Plant RNA Extraction Kit. The RNA was then reverse transcribed into cDNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Kit (Nanjing Novizan Biotechnology Co., Ltd.). The experimental procedures were performed according to the instruction manual.

[0029] PCR reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 2 min, 35 cycles; 72℃ for 10 min. PCR amplification products were identified by 1.0% agarose gel electrophoresis. Figure 1 Afterwards, the DNA was recovered and purified using a general DNA purification and recovery kit (Shanghai Huiling Biotechnology Co., Ltd.). The DNA was then sent to Qingke Biotechnology (Changsha) Co., Ltd. for sequencing. Sequence alignment analysis revealed that its amino acid sequence was identical to that in the broccoli database. BoHY5 The CDS is 495 bp in length, and its gene sequence is shown in SEQ ID No. 1; it is speculated to encode 164 amino acids, and its amino acid sequence is shown in SEQ ID No. 2.

[0030] Example 3: BoHY5 Transformation of broccoli with gene overexpression vector

[0031] The construction of the vector pCBSG015 (Basta), its spectrum is as follows: Figure 3 As shown, the construction process is as follows: Primers are designed to amplify the desired target gene fragment from the target organism using PCR technology. The pCBSG015 (Basta) vector backbone is linearized by restriction endonuclease digestion. The target gene fragment and the linearized vector backbone are ligated using T4 DNA ligase to form a recombinant vector. The recombinant vector is transformed into Escherichia coli DH5α strain. Positive clones are selected through antibiotic screening and PCR verification. Sequencing analysis of the positive clones confirms that the target gene has been correctly inserted into the vector.

[0032] Using our laboratory's broccoli genetic transformation technology, we conducted an overexpression genetic transformation experiment of the BoHY5 gene using broccoli Bop15-80 material. The experimental steps are as follows: Prepare blank MS medium; take an appropriate amount of seeds, wrap them in gauze, soak them in warm water for 30 minutes, and rinse them in cool water for 30 minutes; disinfect the seeds with alcohol and mercuric chloride, wash them with pure water, and then gently place them on MS medium with tweezers. The seedling age is calculated from the time the seeds show white sprouts after sowing. Generally, the hypocotyl of a healthy seedling (when the cotyledons are just emerging) at 10 days of age is used as the explant. Cut the hypocotyl of the broccoli seedling 0.5 cm, make a "trapezoidal" oblique cut, and place it in the pre-medium for 3 days of light culture. After 3 days, the correctly identified positive Agrobacterium with OD600 in the range of 0.5~0.6 is used in (liquid pre-medium + 100 μM MS + 1 mM MES (medium-ethyl ether) was resuspended and incubated at 28°C for 1-3 hours before being used to infect hypocotyls. After infection, the hypocotyls were cultured in the dark for 2 days in pre-medium medium, then transferred to sterile medium for 3-5 days, followed by differentiation and propagation culture. After the transformed seedlings rooted, they were transferred to seedling trays containing sterile substrate and covered to harden off. After one week, the tray covers were opened daily for ventilation, and after 15 days, the tray covers could be completely opened.

[0033] When the transformed seedlings reached the two-leaf-one-heart stage, total RNA was extracted from broccoli leaves using the SteadyPure Plant RNA Extraction Kit. The expression level of the BoHY5 gene in the transformed seedlings was then quantitatively detected using the SYBR method. The expression level assay structure is shown below. Figure 2 As shown, this indicates that the BoHY5 gene is upregulated in overexpressing plants.

[0034] Once the seedlings show buds, the bulbs are harvested, and the thioglycoside content of the buds is determined. Accurately weigh 0.1 g of the lyophilized sample into a 15 mL centrifuge tube, repeating the process three times. Add 50 μL of 5 μmol / mL 2-propenylthioglycoside as an internal standard, and add 5 mL of 70% methanol solution. Extract at 75℃ for 20 min (mixing every 2-3 min). After cooling, add 2 mL of barium acetate, centrifuge at 8000 rpm, 18℃ 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 incubate at 75℃ for 15 min (mixing every 2-3 min). After cooling, add 0.5 mL of barium acetate, centrifuge at 8000 rpm, 18℃ for 10 min. Finally, dilute the supernatant to 12 mL with 70% methanol solution. mL, place on ice and wait for filtration; Extraction column preparation: Fix the SPE solid phase extraction column (2.5 mL disposable syringe) onto the iron stand, place a small white adsorption disc (filter paper cut into a suitable size disc) in it, take an appropriate amount of DEAE Sephadex A25 solution, and add 9 mL of extract to the self-made DEAE Sephadex A25 chromatography column in portions; after the liquid has flowed out, add 2 mL of 0.02 mol / L sodium acetate, and after the liquid has flowed out again, add 900 μL of 0.5 mg / mL sulfatase, and seal the column; react at 37℃ for 16 h, and then elute with 3 mL of ultrapure water; store at 4℃ (or test within one week in a -20℃ refrigerator) (thin side of the sample vial on the outside, thick side on the inside).

[0035] The results are as follows Figure 4 As shown in the figure. The results indicate that the total glucosinolate, glucoraphane, aliphatic glucosinolate, and indole glucosinolate contents in the OE-3 and OE-5 lines were all higher than those in the wild type, suggesting that BoHY5 positively regulates glucoraphane synthesis in broccoli.

[0036] Further hybridization and glucosinolate content identification (methods as described above) were used to obtain stably heritable broccoli varieties. BoHY5 Overexpressing plants.

[0037] Previous genetic transformation experiments and identification of glucosinolate content in buds revealed that the sulforaphane content in the leaves of BoMYB28-overexpressing broccoli lines (OE-2# and OE-61#) was significantly higher than that of the wild type, while the BoMYB28 RNAi-inhibited expression lines (RNAi-10# and RNAi-11#) were significantly lower than that of the wild type, indicating that BoMYB28 positively regulates sulforaphane synthesis in broccoli. Figure 5 ).

[0038] Yeast one-hybrid site-to-site assays and luciferase complementation assays showed that the BoMYB28 promoter interacts with the transcription factor BoHY5. Figure 6 , Figure 7 ).

Claims

1. The application of the BoHY5 gene or its encoded protein in promoting the synthesis of glucosinolates in broccoli; the amino acid sequence of the encoded protein is shown in SEQ ID NO.2; the nucleotide sequence of the BoHY5 gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The organs in which bromelain is synthesized are at least one of the stem, leaves, flowers, and buds.

3. A method for improving the synthesis of glucosinolates in broccoli, characterized in that, The BoHY5 gene was overexpressed in broccoli; the nucleotide sequence of the BoHY5 gene is shown in SEQ ID NO.

1.

4. The method as described in claim 3, characterized in that, The amino acid sequence of the protein encoded by the BoHY5 gene is shown in SEQ ID NO.

2.

5. The method as described in claim 3, characterized in that, Overexpressing the BoHY5 gene in broccoli involves the following steps: introducing an overexpression vector containing the BoHY5 gene into broccoli cells, and culturing the resulting transgenic broccoli plants.

6. The method as described in claim 5, characterized in that, The gene overexpression vector containing the BoHY5 gene is a plant expression vector containing a sequence for overexpressing the BoHY5 gene with a base sequence as shown in SEQ ID NO.

1.

7. The method as described in claim 5, characterized in that, This further includes breeding homozygous varieties.

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