BoNIA2b gene and application thereof

Abstract

The invention provides a BoNIA2b gene, which has a nucleotide sequence as shown in SEQ ID No.1, or a nucleotide sequence which is obtained by substituting, deleting and / or adding one or more nucleotides to the nucleotide sequence as shown in SEQ ID No.1 and expresses proteins with the same function, wherein the nucleotide sequence is obtained by substituting, deleting and / or adding one or more nucleotides to the nucleotide sequence as shown in SEQ ID No.1; the invention also provides an application thereof. Compared with the prior art, the broccoli BoNIA2b gene has the following advantages that functional verification is carried out on the broccoli BoNIA2b gene, it is found that overexpression of the BoNIA2b gene can promote increase of the content of glucoraphanin and sulforaphane in the hairy roots of broccoli, and it is found through further research that the content of glucoraphanin and sulforaphane in the hairy roots of broccoli can be increased; the overexpressed BoNIA2b gene is mainly used for regulating and controlling the expression level of some synthetic genes of glucoraphanin and sulforaphanin of the hairy roots of broccoli so as to regulate and control the synthesis of glucoraphanin and sulforaphanin. Finally, the BoNIA2b gene is proved to positively regulate the synthesis reaction of glucoraphanin and sulforaphane of broccoli.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, specifically to the BoNIA2b gene and its applications. Background Art

[0002] Broccoli (Brassica oleracea var. italica), a widely consumed cruciferous vegetable, has garnered significant attention for its rich nutritional value and numerous health benefits (Zhang and Hamauzu, 2004; Thomas et al., 2018; Li et al., 2022). As a star variety within the Brassica genus, broccoli is one of the vegetables richest in glucosinolates (GLS). Glucoraphanin (GRA), a type of GLS, undergoes hydrolysis with myrosinase to produce the anticancer substance sulforaphane (SF). Previous studies in our laboratory have shown that the SF content in broccoli hairy roots is 13.07 times and 31.28 times higher than that in leaves and young roots, respectively (Zhang, 2015). SF is considered to have significant anti-cancer properties. Studies have shown that these compounds can inhibit the growth and spread of cancer cells through multiple mechanisms, including inducing apoptosis and inhibiting the activation of carcinogens (Shankar et al., 2008; Liand Zhang, 2013).

[0003] Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite (NO3) using NAD(P)H as an electron donor. - +NADH+H + →NO2 - +NAD + Nitrate reductase (NR) can further reduce nitrite to nitric oxide (NO). This enzyme is a key enzyme in plant nitrogen metabolism, found in the cytoplasm of root and leaf cells in higher plants, and also present in algae and fungi. NR has two subtypes, NR1 and NR2. NR1 can be regulated by NIA1, and NR2 by NIA2. However, there are no reports on the discovery and screening of genes in broccoli NIA family members that enable the synthesis of sulforaphane and glucoraphane in broccoli hairy roots. Summary of the Invention

[0004] In view of the aforementioned technical limitations, this application proposes the BoNIA2b gene and its application; it overcomes the deficiencies and defects mentioned in the background art.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] The inventive point of this application is to provide the BoNIA2b gene, wherein the nucleotide sequence of the coding region of the BoNIA2b gene is as shown in SEQ ID No. 1, or the nucleotide sequence shown in SEQ ID No. 1 is substituted, deleted and / or added with one or more nucleotides and expresses the same functional protein.

[0007] The second inventive point of this application is to provide a protein, the gene encoding which is the aforementioned BoNIA2b gene, and the amino acid sequence of which is shown in SEQ ID No. 2.

[0008] The third inventive point of this application is to provide a biological material containing the aforementioned BoNIA2b gene or the aforementioned protein; the biological material is selected from one or more of recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, and engineered bacteria.

[0009] The fourth inventive point of this application is to provide the use of the above-mentioned BoNIA2b gene, the above-mentioned protein, or the above-mentioned biological material in the preparation of a drug for treating cancer.

[0010] Optionally, in the above-described applications, the drug contains one or more of sulforaphane and thiocarbazone.

[0011] The fifth inventive point of this application is to provide a method for amplifying the BoNIA2b gene, which uses cDNA from broccoli hairy roots as a template and performs PCR amplification using amplification primers to obtain the BoNIA2b gene.

[0012] Optionally, in the above amplification method, the upstream primer is shown in SEQ ID No. 3, and the downstream primer is shown in SEQ ID No. 4.

[0013] The sixth inventive point of this application is to provide the use of the above-mentioned BoNIA2b gene, the above-mentioned protein, or the above-mentioned biological material in the synthesis of sulforaphane and / or sulforaphane.

[0014] Optionally, the above-mentioned application promotes the synthesis of sulforaphane and / or glucoraphane in plants by upregulating the expression level of the BoNIA2b gene.

[0015] Optionally, in the above application, the plant is selected as broccoli, preferably broccoli hairy roots.

[0016] Compared with the prior art, this application has the following advantages:

[0017] This invention validated the function of the BoNIA2b gene in broccoli, finding that overexpression of the BoNIA2b gene increased the content of sulforaphane and glucoraphane in the hairy roots of broccoli. Further research revealed that overexpression of the BoNIA2b gene primarily regulates the synthesis of sulforaphane and glucoraphane by controlling the expression levels of the sulforaphane and glucoraphane synthesis genes BoSUR1, BoGSTF11, MYB28, and MYB29 in the hairy roots of broccoli. Ultimately, this demonstrates that the BoNIA2b gene positively regulates the synthesis of sulforaphane and glucoraphane in broccoli, providing an important genetic resource for establishing broccoli hairy root lines high in sulforaphane and glucoraphane. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the pCAMBIA2300-EGFP overexpression vector.

[0019] Figure 2 The results show the PCR amplification of the target gene and the PCR results of the bacterial culture containing the target gene overexpression vector; among them, Figure 2 A represents the amplification of the CDS fragment of the target gene; Figure 2 B is the target gene CDS fragment and the pCAMBIA2300-EGFP vector construct bacterial culture PCR; Figure 3 To verify positive overexpression plants, 'M' stands for Marker.

[0020] Figure 3 The expression of the BoNIA2b gene in overexpressing plants (verification of positive plants).

[0021] Figure 4 The results show the detection level of BoNIA2b gene expression in broccoli roots (2#, 4#, 9#) that are overexpressed.

[0022] Figure 5 The results show the detection of GRA and SF content in the hairy roots of broccoli after overexpression of the BoNIA2b gene.

[0023] Figure 6 The results show the expression of GRA and SF synthesis genes in the overexpression lines. Detailed Implementation

[0024] To make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below. However, it should be understood that the description herein is only used to explain this application and is not intended to limit the scope of this application.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are intended solely for the purpose of describing specific embodiments and are not intended to limit this application. The reagents and instruments used herein are commercially available, and the characterization methods involved can be found in the relevant descriptions in the prior art and will not be further elaborated herein.

[0026] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0027] Example 1

[0028] The BoNIA2b gene has the nucleotide sequence shown in SEQ ID No. 1, or the nucleotide sequence shown in SEQ ID No. 1 with one or more nucleotides substituted, deleted, and / or added, and expresses the same functional protein.

[0029] SEQ ID No. 1:

[0030]

[0031] The protein is also provided, the encoding gene of which is the aforementioned BoNIA2b gene, and the amino acid sequence of the protein is shown in SEQ ID No. 2.

[0032] SEQ ID No. 2:

[0033] 。

[0034] Biological materials containing the aforementioned BoNIA2b gene or the aforementioned protein are also provided; the biological materials are selected from one or more of recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, and engineered bacteria.

[0035] It also provides the application of the aforementioned BoNIA2b gene, the aforementioned protein, or the aforementioned biomaterials in the preparation of drugs for the treatment of cancer.

[0036] The drug contains one or more of sulforaphane and thioglucosinolate.

[0037] A method for amplifying the BoNIA2b gene is also provided, which uses cDNA from broccoli hairy roots as a template and performs PCR amplification using amplification primers to obtain the BoNIA2b gene.

[0038] The amplification primers include the upstream primer shown in SEQ ID No. 3 and the downstream primer shown in SEQ ID No. 4.

[0039] SEQ ID No. 3:

[0040] gagaacacgggggacgagctctATGGCCACCTCCGTCGAT;

[0041] SEQ ID No.4:

[0042] caccatggtgtcgactctagaGAAGATCAGGAGATCTTCCTTCACG.

[0043] The application of the aforementioned BoNIA2b gene, the aforementioned protein, or the aforementioned biological material in the synthesis of sulforaphane and / or sulforaphane is also provided.

[0044] By upregulating the expression level of the BoNIA2b gene in plants, the synthesis of sulforaphane and / or glucoraphane in plants is promoted.

[0045] The plant selected was broccoli, with broccoli hairy roots being the preferred choice.

[0046] Example 2

[0047] The inventors have discovered for the first time that the broccoli BoNIA2b gene affects the synthesis of GRA and SF in broccoli hairy roots. To further clarify the molecular mechanism by which the broccoli BoNIA2b gene regulates the synthesis of GRA and SF in broccoli hairy roots, the broccoli BoNIA2b gene was cloned, an overexpression vector was constructed, and its biological function in the synthesis of GRA and SF was studied. The experiments are as follows:

[0048] 1. Experimental Materials and Methods:

[0049] 1.1 Experimental Materials:

[0050] The experimental material used was broccoli hairy roots, which were induced from aseptic seedlings of broccoli 'Zhongqing 9', and the seeds were provided by the Institute of Vegetables, Chinese Academy of Agricultural Sciences.

[0051] 1.2 Carriers and competent cells:

[0052] Agrobacterium rhizogenes ATCC15834 competent cells were obtained from Shanghai Weidi Biotechnology Co., Ltd., and the pCAMBIA2300-EGFP overexpression vector was donated by the College of Life Sciences of Lanzhou University. The vector map is shown below. Figure 1 As shown, the vector sequence is as shown in SEQ ID No. 9:

[0053]

[0054] 1.3 Experimental Methods:

[0055] 1.3.1 Primer Design:

[0056] Primers for cloning the BoNIA2b gene in broccoli, qRT-PCR primers, and primers for verifying positive plants were designed using NCBI Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). Primer sequences are shown in Table 1.

[0057] Table 1

[0058]

[0059] The underlined sites in the table represent restriction enzyme sites, where GAGCTC and TCTAGA represent SacⅠ and XbaⅠ restriction enzyme sites, respectively.

[0060] 1.3.2 RNA extraction and reverse transcription:

[0061] 1.3.2.1 Extraction of RNA from broccoli:

[0062] Extraction was performed according to the instructions of the RNAprep pure plant total RNA extraction kit (catalog number DP452, purchased from Tiangen Biotech Co., Ltd.).

[0063] 1.3.2.2 Broccoli RNA reverse transcription (cDNA first-strand synthesis):

[0064] The total RNA extracted above was reverse transcribed using the Evo M-MLV RT kit (catalog number AG11603, purchased from Aikerui Biotechnology) following these steps:

[0065] A. Thaw the RNA taken from -80℃ on ice; thaw the reagents and RNase-free ddH2O from the kit taken from -20℃ at room temperature and then quickly place them on ice, gently shaking to mix before use.

[0066] B. Prepare the reaction system according to Table 2:

[0067] Table 2

[0068]

[0069] C. Perform the first step of reverse transcription according to the reaction procedure shown in Table 3:

[0070] Table 3

[0071]

[0072] D. Prepare the reaction system according to Table 4:

[0073] Table 4

[0074]

[0075] E. Perform the second step of reverse transcription according to the reaction procedure shown in Table 5:

[0076] Table 5

[0077]

[0078] F. After the reaction is complete, the concentration and purity of the cDNA are measured and stored at -20°C for later use.

[0079] 1.4 Target gene amplification and overexpression vector construction:

[0080] 1.4.1 PCR amplification of the target gene:

[0081] Using broccoli hairy root cDNA as a template, the overexpression fragment was amplified using Green Taq Mix (catalog number P131, purchased from Novizan) by adding primers with restriction enzyme SacⅠ and XbaⅠ sites and protective bases.

[0082] The system is shown in Table 6 below:

[0083] Table 6

[0084]

[0085] After the operation is complete, gently shake to mix and then briefly centrifuge. Proceed with the reaction according to the procedure shown in Table 7:

[0086] Table 7

[0087]

[0088] The amplified sequence of the broccoli BoNIA2b gene was obtained from the hairy roots of broccoli using PCR, and its nucleotide sequence is shown in SEQ ID No. 1:

[0089] The nucleotide sequence of the broccoli BoNIA2b gene is shown in SEQ ID No. 1:

[0090] BoNIA2b:

[0091]

[0092] 1.4.2 Ligation of the target gene with the cloning vector pCAMBIA2300-EGFP:

[0093] A. After removing the pCAMBIA2300-EGFP vector plasmid from the -80℃ freezer, thaw it on ice;

[0094] B. The specific enzyme digestion system is shown in Table 8:

[0095] Table 8

[0096]

[0097] C. Calculate the volume of the added target fragment (molar ratio of carrier to target fragment = 3:1), and add the components shown in Table 9 to a sterile 1.5 mL centrifuge tube (the entire operation should be performed on ice):

[0098] Table 9

[0099]

[0100] D. React at 37°C for 30 min; then cool to 4°C or immediately place on ice to cool.

[0101] 1.4.3 Transformation of DH5α Escherichia coli competent cells:

[0102] The cells were transformed into DH5α Escherichia coli competent cells using the heat shock method. The target gene sequence was verified by bacterial culture PCR and sequenced (performed by Shanghai Sangon Biotech Co., Ltd.). The pCAMBIA2300-EGFP empty vector plasmid was used as a control. The cells were then transformed into Agrobacterium rhizogenes ATCC15834 competent cells, and 50% glycerol was added at a 1:1 ratio and stored at -80℃.

[0103] 1.5 Induction of hairy roots, verification of positive plants for resistance screening, and qRT-PCR:

[0104] 1.5.1 Inducing hairy roots:

[0105] A. Take broccoli seeds (full seeds from the current year), rinse under running tap water for 30 minutes, disinfect with 75% alcohol for 20 seconds, rinse repeatedly with sterile water 3 times in a clean bench, disinfect with 1% NaClO for 4 minutes, rinse with sterile water 4-5 times, inoculate on culture medium (MS + 30 g / L sucrose + 5 g / L agar, pH 5.8) and culture for 15 days to obtain sterile seedlings;

[0106] B. The ATCC15834 bacterial strain, stored in a laboratory freezer at -80℃, was inoculated in a clean bench into YMB solid medium containing kanamycin sulfate (Yeast extract: 1 g / L; MgSO4·7H2O: 0.2 g / L; K2HPO4: 0.5 g / L; NaCl: 0.1 g / L; Mannitol: 10 g / L; Agar: 15 g / L; pH: 7.0) for activation. The medium was incubated in the dark at 28℃ for 48-72 h. Single colonies were picked and inoculated into YEB liquid medium (Yeast extract: 1 g / L; MgSO4·7H2O: 0.2 g / L; K2HPO4: 0.5 g / L; NaCl: 0.1 g / L; Mannitol: 10 g / L; pH: 7.0) and incubated in the dark at 180 r / min and 28℃ for 24-72 hours. h, when the bacterial culture is in the logarithmic growth phase, take 0.5 mL and add it to 100 mL of YEB liquid medium (Yeast extract: 1 g / L; MgSO4·7H2O: 0.2 g / L; K2HPO4: 0.5 g / L; NaCl: 0.1 g / L; Mannitol: 10 g / L; pH: 7.0), and incubate in the dark at 180 r / min and 28℃ until OD. 600 =0.5-0.6, for use in infection control;

[0107] C. In a clean bench, use sterilized scissors to cut leaves from sterile seedlings cultured for 15 days, cutting them into 0.5 cm pieces. 2 Leaves of approximately 1000 g / L size were treated with an inoculation needle and inoculated into pre-medium (MS + 30 g / L sucrose + 5 g / L agar) and pre-cultured in the dark at 25°C for 4 days for infection. The activated recombinant ATCC15834 bacterial suspension was centrifuged at 8000 rpm for 10 min in an ultracentrifuge, resuspended in MS liquid medium (MS + 30 g / L sucrose + 100 μmol / L AS), and used to infect the pre-cultured leaves. The infection was carried out in a shaker at 110 rpm for 15 min to ensure complete contact between the Agrobacterium and the leaf wound. Excess bacterial suspension was blotted dry with sterile filter paper, and the leaves were inoculated onto co-medium (MS + 30 g / L sucrose + 5 g / L agar + 100 μmol / L AS) and incubated in the dark at 25°C until hairy roots emerged.

[0108] D. Cut off the induced hairy roots and inoculate them onto sterile medium (MS + 30 g / L sucrose + 5 g / L agar + 2 g / L carbenicillin). Substitute every 4 days and observe the sterilization. After complete sterilization, cut off 2-3 cm hairy roots and inoculate them onto MS liquid medium for proliferation culture. Change the medium every 20 days.

[0109] 1.5.3 Identification of transgenic hairy-root positive plants:

[0110] DNA was extracted using a rapid DNA extraction kit (catalog number KG203, purchased from Tiangen Biotech Co., Ltd.), and amplified using primer sequences (SEQ ID No. 3 and SEQ ID No. 4).

[0111] 1.5.4 Determination of GRA and SF physiological indicators in hairy roots overexpressing the target gene:

[0112] The contents of GRA and SF in the hairy roots of broccoli were determined by HPLC in the blank control group and the experimental group, respectively.

[0113] 1.5.5 Quantitative fluorescence analysis of stress-related genes in target gene overexpression lines:

[0114] Hairy roots of overexpressing roots were harvested, and RNA was extracted using the RNAprep pure plant total RNA extraction kit (catalog number DP452, purchased from Tiangen Biotech Co., Ltd.). Reverse transcription was performed using the FastKing one-step genomic cDNA first-strand synthesis premix kit (catalog number KR118, purchased from Tiangen Biotech Co., Ltd.). Finally, the expression levels of GRA and SF-related genes, including BoBCAT4, BoCYP79F1, BoSUR1, BoUGT74B1, BoGSTF11, BoGSTU20, BoSOT17, BoSOT18, BoFMOGS-OX5, BoAOP2, BoTGG1, BoMYB28, and BoMYB29, were detected using the SuperReal fluorescence quantitative premix kit (catalog number FP205, purchased from Tiangen Biotech Co., Ltd.). The specific methods were described in the instruction manual.

[0115] 2. Results and Analysis:

[0116] 2.1 Amplification of the target gene fragment and construction of the overexpression vector:

[0117] Using broccoli hairy root cDNA as a template, primers are shown in Table 1. Full-length gene amplification was performed using Green Taq Mix premix (Novozymes), and fragment size was detected by agarose gel electrophoresis. The sequences were then determined by sequencing. Figure 2 A). The positive plasmid was ligated to the pCAMBIA2300-EGFP vector using a double enzyme digestion method, and then subjected to bacterial PCR (…). Figure 2 B) The sample was then sent for sequencing, and sequencing was successful.

[0118] 2.2 Detection of target gene overexpression:

[0119] Broccoli leaves were infected with the 35s:BoNIA2b overexpression vector constructed in section 2.1. After hairy roots emerged, resistance selection was performed, and the plants were allowed to grow for a period of time for positive verification. All 10 transgenic hairy roots were positive (N represents wild-type hairy roots). Figure 3 Then, bands 2#, 4#, and 9# with brighter bands were selected to detect the expression level of the BoNIA2b gene in transgenic hairy roots of broccoli. The results showed that, compared with the control group, the expression level of the BoNIA2b gene in overexpressed hairy roots of broccoli was significantly increased. Figure 4 ).

[0120] 2.3 Detection of GRA and SF content in hairy roots overexpressing the BoNIA2b gene:

[0121] The GRA and SF contents were measured in plants overexpressing the target gene 35S:BoNIA2b and the control 35S:00. The results showed that ( Figure 5 The levels of GRA and SF in roots overexpressing BoNIA2b were significantly increased, indicating that the BoNIA2b gene in broccoli is involved in the synthesis of GRA and SF in the hairy roots of broccoli.

[0122] 2.4 Expression of GRA and SF synthesis genes in overexpression lines

[0123] The expression levels of BoBCAT4, BoCYP79F1, BoGSTF11, BoGSTU20, BoFMOGS-OX5, BoAOP2, and BoMYB28 genes in 35S:BoNIA2b plants were significantly higher than those in 35S:00 plants. Figure 6 This indicates that overexpression of the BoNIA2b gene will affect the expression of genes related to GRA and SF synthesis.

[0124] In conclusion, broccoli hairy roots overexpressing the BoNIA2b gene showed increased GRA and SF content compared to the control, thus demonstrating that the aforementioned gene positively regulates the synthesis of GRA and SF in broccoli hairy roots.

[0125] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. BoNIA2b A gene characterized by Said BoNIA2b The nucleotide sequence of the gene coding region is as shown in SEQ ID No. 1, or a nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added as shown in SEQ ID No. 1 and the nucleotide sequence expresses a protein with the same function.

2. A protein, characterized in that The protein encoding gene is the one described in claim 1 BoNIA2b The amino acid sequence of the protein is shown in SEQ ID No.

2.

3. A biomaterial, characterized in that: The biological material contains the BoNIA2b The gene or the protein according to claim 2; the biological material is selected from one or more of recombinant DNA, expression cassette, transposon, plasmid vector, viral vector and engineered bacteria.

4. The method according to claim 1 BoNIA2b Use of the gene, the protein according to claim 2 or the biomaterial according to claim 3 in the preparation of a drug for treating cancer.

5. The use according to claim 4, characterized in that: The medicine contains one or more of glucoraphanin and sulforaphane.

6. A BoNIA2b A method for amplifying a gene, characterized in that Using the cDNA of broccoli hairy roots as a template, PCR amplification was performed using amplification primers to obtain the BoNIA2b Gene.

7. The amplification method according to claim 6, characterized in that Among the amplification primers, the upstream primer is shown as SEQ ID No.3, and the downstream primer is shown as SEQ ID No.

4.

8. The method according to claim 1 BoNIA2b Use of the gene, the protein according to claim 2 or the biomaterial according to claim 3 in synthesizing glucoraphanin and / or sulforaphane.

9. The use according to claim 8, characterized in that: By upregulating the plant BoNIA2b The expression level of the gene promotes the plant to synthesize glucoraphanin and / or sulforaphane.

10. The use according to claim 9, characterized in that: The plant is selected to be broccoli, preferably broccoli hairy roots.

Images