BcTEM1 gene of brassica rapa and expression protein and application thereof

Abstract

The application discloses a Chinese cabbage transcription factor BcTEM1 gene, an expression protein thereof and application, and relates to the technical field of plant genetic engineering.The Chinese cabbage flowering regulation transcription factor BcTEM1 gene disclosed by the application has a nucleotide sequence as shown in SEQ ID NO.1.A silencing vector containing a VIGS silencing fragment of the Chinese cabbage transcription factor BcTEM1 gene is constructed, and is transformed into Chinese cabbage.The results show that the silencing plants all show obvious early flowering, the flowering time is advanced by about five days compared with the control plants;the BcTEM1 gene expression of the silencing plants is significantly inhibited, and the BcFT and BcAP3 expression levels are both significantly up-regulated.Compared with the control group, the relative LUC activity of a treatment group in which pBcFT-LUC and 35S:BcTEM1-GFP bacterial liquid are simultaneously injected is significantly reduced, which indicates that BcTEM1 inhibits the expression of the BcFT gene.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to the BcTEM1 gene of non-heading Chinese cabbage, its expressed protein, and its applications. Background Technology

[0002] Non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) is an important vegetable crop belonging to the Brassicaceae family and the Brassica genus, and is widely cultivated in my country. Different varieties of non-heading Chinese cabbage exhibit significant differences in flowering time; for example, 'May Slow' is a late-flowering variety (flowering in May), while 'Sijiu Caixin' is an early-flowering variety (flowering in January). Currently, my country cannot achieve year-round supply of non-heading Chinese cabbage; therefore, researching the flowering time regulation mechanism of non-heading Chinese cabbage is of significant practical importance for variety improvement.

[0003] Flowering is a crucial developmental process for plants, ensuring species continuation. Plant flowering is regulated by a combination of external and internal factors. Arabidopsis thaliana exhibits six flowering regulatory pathways, including the photoperiod pathway, vernalization pathway, gibberellin pathway, age pathway, autonomous pathway, and temperature pathway. TEM1, belonging to the RAV transcription factor family, is a flowering repressor gene that plays a role in both the photoperiod and gibberellin pathways. GA3OX1 and GA3OX2 are two GA4 biosynthetic genes involved in gibberellin-regulated flowering. TEM1 directly inhibits the expression of GA3OX1 and GA3OX2, reducing gibberellin levels in plants, thereby decreasing the expression of SOC1 and SPL15, ultimately inhibiting flowering. In the photoperiod pathway, TEM1 regulates flowering through antagonism with CO2. Currently, the function of the TEM1 gene in model plants such as Arabidopsis thaliana is extensively studied, but the role of the TEM1 gene in flowering regulation in non-heading Chinese cabbage remains unexplored. Therefore, further in-depth research is warranted on the function of the transcription factor BcTEM1 gene in flowering regulation in non-heading Chinese cabbage. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, the technical problem this invention aims to solve is to provide the BcTEM1 gene, a transcription factor from non-heading Chinese cabbage. Another technical problem this invention aims to solve is to provide the expression protein of the BcTEM1 gene from non-heading Chinese cabbage. A further technical problem this invention aims to solve is to provide the application of the BcTEM1 gene from non-heading Chinese cabbage in regulating plant flowering time, thereby achieving variety improvement of non-heading Chinese cabbage.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] The nucleotide sequence of the transcription factor BcTEM1 gene in non-heading Chinese cabbage is shown in SEQ ID NO.1.

[0007] The amino acid sequence of the BcTEM1 gene transcription factor expressed in non-heading Chinese cabbage is shown in SEQ ID NO.2.

[0008] Application of the transcription factor BcTEM1 gene in non-heading Chinese cabbage in regulating flowering time.

[0009] Application of the transcription factor BcTEM1 gene in non-heading Chinese cabbage in regulating the expression of downstream genes.

[0010] One method to promote early flowering in plants involves silencing the expression of the BcTEM1 gene, including:

[0011] 1) Construct a silencing vector containing the VIGS silencing fragment of the BcTEM1 gene, a transcription factor from non-heading Chinese cabbage;

[0012] 2) The constructed silencing vector containing the VIGS silencing fragment of the BcTEM1 gene transcription factor from non-heading Chinese cabbage was transformed into non-heading Chinese cabbage.

[0013] 3) Cultivate, screen, and obtain non-heading Chinese cabbage plants with earlier flowering time.

[0014] The VIGS silencing fragment of the non-heading Chinese cabbage transcription factor BcTEM1 gene has the nucleotide sequence shown in SEQ ID NO. 3.

[0015] The method for constructing the silencing vector is as follows: the VIGS silencing fragment of the non-heading Chinese cabbage transcription factor gene BcTEM1 is ligated with a 40bp reverse complementary sequence to obtain an 80bp hairpin structure fragment; the 80bp hairpin structure fragment is inserted into the pTY-s vector to construct the pTY-s-BcTEM1 vector, which is a silencing vector containing the VIGS silencing fragment of the non-heading Chinese cabbage transcription factor BcTEM1 gene.

[0016] The nucleotide sequence of the 80bp hairpin structure fragment is shown in SEQ ID NO.4.

[0017] Application of the VIGS silencing fragment of the transcription factor gene BcTEM1 in non-heading Chinese cabbage in regulating the expression of downstream gene BcFT.

[0018] Compared with the prior art, the advantages of the present invention are as follows:

[0019] This invention discloses the BcTEM1 gene, a transcription factor regulating flowering in non-heading Chinese cabbage, whose nucleotide sequence is shown in SEQ ID NO.1 and whose amino acid sequence of the expressed protein is shown in SEQ ID NO.2. This invention constructs a silencing vector containing a VIGS silencing fragment of the BcTEM1 gene from non-heading Chinese cabbage, transforms it into non-heading Chinese cabbage, and cultivates, screens, and obtains non-heading Chinese cabbage plants with advanced flowering time. The results show that all silenced non-heading Chinese cabbage plants exhibited significantly earlier flowering, approximately five days earlier than the control group; BcTEM1 gene expression was significantly inhibited in the silenced plants, while the expression levels of BcFT and BcAP3 were significantly upregulated. Compared with the control group, the treatment group simultaneously injected with pBcFT-LUC and 35S:BcTEM1-GFP bacterial solution showed significantly reduced relative LUC activity; the results indicate that BcTEM1 inhibits BcFT gene expression. Therefore, the non-heading Chinese cabbage transcription factor BcTEM1 gene provided by this invention will play an important role in the regulation of plant flowering period, provide an important candidate gene for the breeding of non-heading Chinese cabbage, and provide important gene resources and theoretical basis for the breeding of new non-heading Chinese cabbage varieties. Attached Figure Description

[0020] Figure 1 Electrophoresis diagram of PCR amplification of the transcription factor BcTEM1 gene in non-heading Chinese cabbage (lane 1 is DNA Marker2000, lane 2 is the PCR amplification product of BcTEM1);

[0021] Figure 2 A graph showing the expression levels of the transcription factor BcTEM1 gene in different tissues of non-heading Chinese cabbage (Root represents the root, Stem represents the stem, Leaf represents the leaf, and Flower represents the flower).

[0022] Figure 3 The structure diagram of the expression vector pCAMBIA1302;

[0023] Figure 4 Subcellular localization map of the transcription factor BcTEM1 gene in non-heading Chinese cabbage (A is the vector structure diagram of 35S:BcTEM1-GFP and 35S:GFP, B is the subcellular localization map; the scale bar in the figure is 50μm).

[0024] Figure 5 A schematic diagram of the structure of the VIGS silencing carrier pTY-s used;

[0025] Figure 6Flowering phenotype of plants with silenced transcription factor BcTEM1 gene in non-heading Chinese cabbage (A) (Control is the control plant, 1 is the silenced plant BcTEM1-pTY-s-1, 2 is the silenced plant BcTEM1-pTY-s-2) and quantitative analysis of downstream genes (B).

[0026] Figure 7 Diagram showing the regulatory relationship between transcription factor BcTEM1 and the BcFT gene (A is the structure of the dual-luciferase experimental vector; B is the diagram showing the inhibition of BcFT expression by transcription factor BcTEM1). Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described can be performed according to the methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition) or conventional methods in the art, or according to the kit and product instructions.

[0028] The material used in this application is the plant material of the late-flowering, non-heading Chinese cabbage variety 'May Slow', which was planted on the campus of Nanjing Forestry University.

[0029] Example 1

[0030] 1. RNA extraction and cDNA synthesis

[0031] Total RNA was extracted from non-heading Chinese cabbage leaves using the RNAsimpleTotal RNA Extraction Kit (Tiangen Biotech Co., Ltd.) according to the manufacturer's instructions. III. cDNA was obtained by reverse transcription using the qPCR first-strand cDNA synthesis kit (Yisheng Biotechnology Co., Ltd.), and stored at -20℃ for later use.

[0032] 2. PCR amplification of the CDS region of BcTEM1

[0033] The nucleotide sequence of the BcTEM1 gene was downloaded from the non-heading Chinese cabbage genome database (http: / / nhccbase.njau.edu.cn / website / ), and primers were designed using Primer 5 software to amplify the BcTEM1 gene.

[0034] Forward primer: 5'-ATGGGGAGAAGAAAAGTAGAGATCA-3',

[0035] Reverse primer: 5'-CTTGAGCAGCGGGAGAGTTT-3'.

[0036] The 50μL PCR amplification system consisted of: 25μL of 2×Phanta Max Master Mix (purchased from Novizan), 1μL of cDNA, 2μL each of forward and reverse primers, and ddH2O added to a final volume of 50μL.

[0037] The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles; 72℃ for 5 min.

[0038] The PCR products were recovered by gel extraction after 1% agarose gel electrophoresis. Figure 1 The pMD19-T vector (purchased from Takara) was ligated into DH5α competent cells, and single clones were selected for colony PCR detection. Positive clones were sequenced (GenScript Biotechnology Co., Ltd.), and the sequencing results were accurate. The sequencing results showed that the nucleotide sequence of the BcTEM1 gene is shown in SEQ ID NO.1, with a length of 1077 bp (terminator removed). The amino acid sequence of its expressed protein is shown in SEQ ID NO.2.

[0039] Example 2

[0040] Root, stem, leaf, and flower tissues of non-heading Chinese cabbage during the flowering period were selected, and the expression of the BcTEM1 gene in different tissues of non-heading Chinese cabbage was detected by real-time quantitative PCR (qPCR). Total RNA extraction and cDNA synthesis methods were the same as in Example 1. The BcActin gene was used as an internal control gene. Primer sequences are shown below:

[0041] qBcTEM1-S: 5'-TTACTCCGTCACCGACAAAAGG-3'

[0042] qBcTEM1-A: 5'-CTCGAAGCAAACCACATCACC-3'

[0043] qBcActin-S: 5'-GTTGCTATCCAGGCTGTTCT-3';

[0044] qBcActin-A: 5'-AGCGTGAGGAAGAGCATAAC-3'

[0045] The PCR reaction system consisted of: 10 μL of 2×SYBR Grenn qPCR Master Mix (purchased from Spombio), 0.8 μL each of forward and reverse primers, 1 μL of cDNA, and ddH2O to a total volume of 20 μL.

[0046] The PCR reaction program was: 95℃ for 1 min; 95℃ for 10 s, 60℃ for 30 s, for 40 cycles. Other reaction parameters were set to system defaults. Each reaction was configured with 3 biological replicates, utilizing 2... -ΔΔCT The relative expression levels of genes were calculated using a method, and the graphs were plotted using Excel.

[0047] The results are as follows Figure 2 As shown, the BcTEM1 gene is expressed in different tissues of non-heading Chinese cabbage, with the highest expression level in the flower.

[0048] Example 3

[0049] 1. Construction of a BcTEM1 overexpression vector in non-heading Chinese cabbage

[0050] The pCAMBIA1302 vector (purchased from Zeye Biotechnology) was digested with EcoRI and BamHI (vector structure shown in Figure 1). Figure 3 (As shown).

[0051] The double enzyme digestion reaction system was as follows: 21 μL of pCAMBIA1302 plasmid / recovered fragment, 5 μL of buffer, 2 μL each of EcoRI and BamHI, and 20 μL of H2O.

[0052] The double enzyme digestion reaction procedure is: 37℃ for 4 hours.

[0053] Using the full-length amplified fragment of the BcTEM1 gene as a template, adapter primers with seamless ligation to the expression vector pCAMBIA1302 were designed. The primer sequences are shown below:

[0054] BcTEM1-S-EcoRI:

[0055] 5'-GGAATTCATGGATTACAGCTGTCTAGACGAC-3',

[0056] BcTEM1-A-BamHI:

[0057] 5'-CGGGATCCCAAGACGTTGATTATGGCCTGC-3'.

[0058] EcoRI and BamHI restriction enzyme sites were added to both ends of the BcTEM1 gene amplification primers. The linearized vector fragment after double digestion was ligated to the gene amplification fragment using T4 DNA ligase (purchased from Thermo Fisher Scientific).

[0059] The primers for ligating the BcTEM1 gene were: 1 μL ligase, 2 μL buffer, 3 μL linearized vector, and 14 μL BcTEM1 amplification fragment. The reaction program was: overnight at 4°C. The ligation product was transformed into DH5α competent cells (purchased from Weidi Biotechnology). Single clones were selected for colony PCR detection, and positive clones were sequenced (GenScript Biotechnology Co., Ltd.). The sequencing results were accurate, yielding the pCAMBIA1302-BcTEM1 recombinant vector, and the plasmid was extracted.

[0060] 2. Subcellular localization of BcTEM1

[0061] The recombinant plasmids pCAMBIA1302-BcTEM1 and pCAMBIA1302 were transformed into Agrobacterium GV3101 competent cells (purchased from Weidi Biotechnology) using the freeze-thaw method and cultured in the dark at 28°C for 48 h. The Agrobacterium transformation method was performed according to the manufacturer's instructions. Single clones were selected for colony PCR detection. Positive clones were inoculated into LB liquid medium (containing 50 μg / mL kanamycin and rifampin) and cultured with shaking at 28°C for one day until the bacterial growth rate reached OD. 600 =0.8. Centrifuge at 5000 rpm for 10 min to collect bacterial cells, and resuspend in injection buffer to OD. 600 =0.6, incubate in the dark for 4 hours. The injection buffer contains 1 ml of 10 mM MES solution, 1 ml of 10 mM magnesium chloride solution, 10 μL of 150 μM acetylsylgenone solution, and ddH2O to a total volume of 10 ml. Inject the incubated bacterial solution into tobacco leaves using a sterile syringe, incubate in the dark for 12 hours, and then incubate normally for 48 hours. Take the tobacco leaves that have been injected with the bacterial solution, vacuum transfer them to DAPI dye, and observe them using an Olympus BX71 microscope.

[0062] The results are as follows Figure 4 As shown, BcTEM1 is located in the cell nucleus.

[0063] Example 4

[0064] 1. Constructing a VIGS silencing vector for non-heading Chinese cabbage

[0065] A 40bp specific sequence from the non-conserved region of the BcTEM1 gene was selected as a silencing fragment (nucleotide sequence shown in SEQ ID NO.3). This was then combined with a 40bp reverse complementary sequence to obtain an 80bp sequence forming a hairpin structure, the nucleotide sequence of which is shown in SEQ ID NO.4. This sequence was then inserted into the pTY-s vector. Figure 5The recombinant vector pTY-s-BcTEM1 was obtained. The hairpin fragment synthesis, pTY-s vector construction and sequencing were all completed by Genscript Biotech Co., Ltd. After the pTY-s-BcTEM1 E. coli culture was cultured at 37℃ with shaking for 12 h, the plasmid was extracted and ready to be transformed into non-heading Chinese cabbage.

[0066] 2. Obtaining Silent Plants

[0067] Non-heading Chinese cabbage plants that had grown for one month and exhibited uniform growth were selected as the silencing plant material. The pTY-s-BcTEM1 and pTY-s plasmids were coated with gold powder. The gold powder coating system consisted of 8.5 μL gold powder, 5 μL plasmid, 50 μL 2.5M CaCl2, and 20 μL 0.1M spermidine. After mixing, the mixture was incubated on ice for 20 min, vortexed for 10 s, centrifuged at 10,000 rpm for 5 s, the supernatant was discarded, and the plants were washed once with anhydrous ethanol. The gold-coated plasmids were then bombarded with the non-heading Chinese cabbage plants using a gene gun (Bio-Rad, PDS1000 / He). The pTY-s plasmid-coated plants served as control plants. Five plants were bombarded each time, with each plasmid replicated four times. Thirty days after bombardment, if the plants developed symptoms (mottled leaves), it indicated that the silencing vector had been successfully transferred into the non-heading Chinese cabbage plants.

[0068] 3. Phenotypic characteristics of silent plants and expression of flowering-related genes

[0069] Total RNA was extracted from diseased leaves (showing mosaic patterns) of non-heading Chinese cabbage plants, and cDNA was obtained by reverse transpiration, following the same method as in Example 1. The expression of BcTEM1, BcFT, and BcAP3 genes in silenced non-heading Chinese cabbage plants was detected by qPCR, following the same method as in Example 2. The flowering time of the silenced non-heading Chinese cabbage plants was also observed and recorded. Primer sequences are shown below:

[0070] qBcTEM1-S: 5'-TTACTCCGTCACCGACAAAAGG-3'

[0071] qBcTEM1-A: 5'-CTCGAAGCAAACCACATCACC-3'

[0072] qBcAP3-S: 5'-TCAGCCCTAACACCACAACG-3'

[0073] qBcAP3-A: 5'-GCTCCTGAATATCAAGCTCGTC-3'

[0074] qBcFT-S: 5'-GTCCTAGCAATCCTCACCTCCG-3'

[0075] qBcFT-A: 5'-CGCCATCCTGGTTCATACACTG-3'

[0076] The results are as follows Figure 6 As shown, both non-heading Chinese cabbage plants exhibited significantly earlier flowering, with flowering occurring approximately five days earlier than the control. Figure 6 A). Compared with the control group, the expression level of the BcTEM1 gene was significantly downregulated in the silent plants, indicating that the expression of the BcTEM1 gene was significantly suppressed in the silent plants. Figure 6 B). The expression levels of BcFT and BcAP3 were significantly upregulated in silent plants, indicating that BcFT and BcAP3 are target genes of BcTEM1. Figure 6 C).

[0077] 4. Dual-luciferase assay

[0078] Non-heading Chinese cabbage leaves were selected as experimental material. DNA was extracted using a plant genomic DNA extraction kit (Tiangen Biotech Co., Ltd.), following the kit's instructions. The nucleotide sequence of the BcFT gene promoter was downloaded from the non-heading Chinese cabbage genome database (http: / / nhccbase.njau.edu.cn / website / ). Forward and reverse primers were designed using Primer 5 software to amplify the BcFT gene promoter. The primer sequences are shown below:

[0079] pBcFT-S:

[0080] 5'-AAAAAAATGATGAATTGAAAAGCTTCGATCCGAACCAAGTTGTGC-3',

[0081] pBcFT-A:

[0082] 5'-CATACAGAGCACATGCCTCGAGCTTTGATCTAAAACAAACAGGTGG-3'.

[0083] The 50μL PCR amplification system consisted of: 25μL of 2×Phanta Max Master Mix (purchased from Novizan), 1μL of DNA, 2μL each of forward and reverse primers, and ddH2O added to a final volume of 50μL.

[0084] The PCR program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 2 min, 35 cycles; 72℃ for 5 min.

[0085] The PCR product and pGreenII 0800(LUC) vector were digested with Hind III and Kpn I, and the recovered product was ligated to obtain the pBcFT-pGreenII 0800(pBcFT-LUC) recombinant vector, following the same method as in Example 3. The recombinant vector was transformed into Agrobacterium GV3101, and the Agrobacterium was resuspended after preparing the injection solution. The resuspended pBcFT-LUC bacterial solution and pCAMBIA1302-BcTEM1(35S:BcTEM1-GFP) bacterial solution were mixed evenly at a volume ratio of 1:1 and injected into tobacco leaves. An equal volume mixture of pBcFT-LUC and pCAMBIA1302(35S:GFP) bacterial solutions was injected into tobacco leaves as a control group. Each bacterial solution mixture was injected into five leaves. After normal incubation in a light incubator for 3 days, the injected tobacco leaves were ground with liquid nitrogen. The Reporter Assay System (purchased from Promega) was used to measure the values ​​of firefly luciferase and Renilla luciferase using a microplate reader. Experimental data were recorded using Excel, and the ratio of firefly luciferase to Renilla luciferase was calculated to measure relative LUC activity.

[0086] The results are as follows Figure 7 As shown, compared with the control group (pBcFT-LUC+35S:GFP), the relative LUC activity of tobacco leaves injected with pBcFT-LUC+35S:BcTEM1-GFP bacterial solution was significantly reduced. The results indicate that BcTEM1 inhibits the expression of the BcFT gene.

[0087] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. Application of silencing the expression of Brassica chinensis transcription factor BcTEM1 gene as shown in SEQ ID NO. 1 in promoting early flowering of Brassica chinensis.

2. A method for promoting early flowering in Brassica campestris, characterized by, The expression of BcTEM1 gene is silenced in Brassica chinensis; the specific steps include: 1) constructing a silencing vector containing a VIGS silencing fragment of Brassica chinensis transcription factor BcTEM1 gene; the construction method of the silencing vector is as follows: a 80 bp hairpin structure fragment is obtained by connecting a VIGS silencing fragment of Brassica chinensis transcription factor BcTEM1 gene with a 40 bp sequence reverse complementary thereto; the 80 bp hairpin structure fragment is inserted into a pTY-s vector to construct a pTY-s-BcTEM1 vector, i.e. a silencing vector containing a VIGS silencing fragment of Brassica chinensis transcription factor BcTEM1 gene; the nucleotide sequence of the 80 bp hairpin structure fragment is as shown in SEQ ID NO. 4; the nucleotide sequence of the VIGS silencing fragment of Brassica chinensis transcription factor BcTEM1 gene is as shown in SEQ ID NO. 3; 2) transforming the constructed silencing vector containing a VIGS silencing fragment of Brassica chinensis transcription factor BcTEM1 gene into Brassica chinensis; 3) cultivating, screening and obtaining Brassica chinensis plants with advanced flowering time.

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