Non-heading Chinese cabbage transcription factor BcTEM1 gene as well as expression protein and application thereof

Abstract

The invention discloses a non-heading Chinese cabbage transcription factor BcTEM1 gene as well as an expression protein and application thereof, and relates to the technical field of plant genetic engineering. The invention discloses a flowering regulation transcription factor BcTEM1 gene of non-heading Chinese cabbage, and the nucleotide sequence of the gene is as shown in SEQ ID NO. 1. According to the invention, a silence vector containing a VIGS silence segment of a non-heading Chinese cabbage transcription factor BcTEM1 gene is constructed, and the silence vector is transformed into the non-heading Chinese cabbage. Results show that silent plants have obvious flowering advance, and the flowering time is advanced by about five days compared with that of a control group plant; the expression of the BcTEM1 gene in a silent plant is obviously inhibited, and the expression levels of BcFT and BcAP3 are obviously up-regulated. Compared with a control group, the relative LUC activity of a treatment group injected with pBcFT-LUC and 35S: BcTEM1-GFP bacterial solutions at the same time is remarkably reduced, and it is indicated that BcTEM1 inhibits expression of the BcFT gene.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, and more specifically to a non-heading cabbage transcription factor BcTEM1 gene and its expression protein and application. Background Art

[0002] Non-heading cabbage (Brassica campestris ssp.chinensis Makino) is an important vegetable crop of the genus Brassica in the family Cruciferae and is widely planted in my country. The flowering period of different varieties of non-heading cabbage varies greatly, such as 'Wuyueman' is a late flowering variety (flowering in May) and 'Sijiucaixin' is an early flowering variety (flowering in January). At present, my country cannot achieve year-round supply of non-heading cabbage, so studying the flowering period regulation mechanism of non-heading cabbage is of great practical significance for variety improvement.

[0003] Flowering is an important developmental process for plants to ensure species continuation. Flowering of plants is regulated by a variety of external and internal factors. There are six flowering regulation pathways in Arabidopsis, including the photoperiod pathway, vernalization pathway, gibberellin pathway, age pathway, autonomous pathway and temperature pathway. TEM1 belongs to the RAV transcription factor family and is a flowering repressor gene that functions in the photoperiod pathway and gibberellin pathway. GA3OX1 and GA3OX2 are two GA4 biosynthesis genes that are involved in gibberellin-regulated flowering. TEM1 directly inhibits the expression of GA3OX1 and GA3OX2, reduces the plant gibberellin content, thereby reducing the expression of SOC1 and SPL15, and ultimately inhibiting flowering. In the photoperiod pathway, TEM1 regulates flowering by antagonizing CO. At present, the function of TEM1 gene in model plants such as Arabidopsis is more studied, but the role of TEM1 gene in flowering regulation in non-heading cabbage has not been studied. Therefore, the functional study of the non-heading cabbage transcription factor BcTEM1 gene in flowering regulation deserves further exploration. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a non-heading cabbage transcription factor BcTEM1 gene. Another technical problem to be solved by the present invention is to provide an expression protein of the non-heading cabbage transcription factor BcTEM1 gene. Another technical problem to be solved by the present invention is to provide an application of the non-heading cabbage transcription factor BcTEM1 gene in regulating the flowering period of plants, so as to achieve variety improvement of non-heading cabbage.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

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

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

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

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

[0010] A method for promoting early flowering of plants, silencing the expression of BcTEM1 gene in the plants, comprising:

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

[0012] 2) transforming the constructed silencing vector containing the VIGS silencing fragment of the non-heading cabbage transcription factor BcTEM1 gene into non-heading cabbage;

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

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

[0015] The method for constructing the silencing vector comprises the following steps: connecting the VIGS silencing fragment of the non-heading cabbage transcription factor gene BcTEM1 to a 40bp sequence that is reverse complementary to the VIGS silencing fragment to obtain an 80bp hairpin structure fragment; and inserting the 80bp hairpin structure fragment into a pTY-s vector to construct a pTY-s-BcTEM1 vector, i.e., a silencing vector comprising the VIGS silencing fragment of the non-heading cabbage transcription factor BcTEM1 gene.

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

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

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

[0019] The present invention discloses a non-heading cabbage flowering regulating transcription factor BcTEM1 gene, whose nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2. The present invention constructs a silencing vector containing a VIGS silencing fragment of the non-heading cabbage transcription factor BcTEM1 gene, transforms it into non-heading cabbage, and cultivates, screens, and obtains non-heading cabbage plants with early flowering time. The results show that the silenced non-heading cabbage plants all show obvious early flowering, and the flowering time is about five days earlier than that of the control group plants; the expression of the BcTEM1 gene in the silenced plants is significantly inhibited, and the expression levels of BcFT and BcAP3 are significantly upregulated. Compared with the control group, the relative LUC activity of the treatment group injected with pBcFT-LUC and 35S:BcTEM1-GFP bacterial solution at the same time is significantly reduced; the results show that BcTEM1 inhibits the expression of the BcFT gene. Therefore, the non-heading cabbage transcription factor BcTEM1 gene provided by the present invention will play an important role in regulating the flowering period of plants, provide an important candidate gene for non-heading cabbage breeding, and provide an important gene resource and theoretical basis for cultivating new non-heading cabbage varieties. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 2 This is the expression level diagram of the transcription factor BcTEM1 gene in different tissues of non-heading cabbage (Root is root, Stem is stem, Leaf is leaf, Flower is flower);

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

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

[0024] Figure 5 Schematic diagram of the structure of the VIGS silencing vector pTY-s used;

[0025] Figure 6The flowering 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 the quantitative analysis of downstream genes (B);

[0026] Figure 7 This is an analysis diagram of the regulatory relationship between the transcription factor BcTEM1 and the BcFT gene (A is the structure diagram of the dual luciferase experiment vector; B is the diagram of the transcription factor BcTEM1 inhibiting the expression of BcFT). DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described can be carried out with reference to the methods listed in the book "Molecular Cloning Experiment Guide" (3rd Edition) by J. Sambrook or conventional methods in the art, or according to the kit and product instructions.

[0028] The plant material used in this application is the late-flowering non-heading cabbage variety 'Wuyueman', 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 cabbage leaves using RNAsimpleTotal RNA extraction kit (Tiangen Biochemical Technology Co., Ltd.) according to the instructions. cDNA was obtained by reverse transcription using the qPCR First-Strand cDNA Synthesis Kit (Yisheng Biotechnology Co., Ltd.) and stored at -20°C for future use.

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

[0033] The nucleotide sequence of 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 50 μL PCR amplification system was as follows: 25 μL 2×Phanta Max Master Mix (purchased from Novazonics), 1 μL cDNA, 2 μL each of forward primer and reverse primer, and ddH2O was added to make up to 50 μL.

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

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

[0039] Example 2

[0040] The root, stem, leaf and flower tissues of non-heading cabbage in the flowering period were selected, and the expression of BcTEM1 gene in different tissues of non-heading cabbage was detected by real-time fluorescence quantitative PCR (qPCR). The total RNA extraction and cDNA synthesis methods were the same as in Example 1. BcActin gene was used as the internal reference gene. The primer sequences are as follows:

[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 was: 10 μL of 2×SYBR Grenn qPCR Master Mix (purchased from Sibomu Biotechnology), 0.8 μL of forward primer and reverse primer respectively, 1 μL of cDNA, and ddH2O to make up the total volume to 20 μL.

[0046] The PCR reaction program was: 95°C for 1 min; 95°C for 10 s, 60°C for 30 s, and 40 cycles. Other reaction parameters were set according to the system default settings. Three biological replicates were set for each reaction. -ΔΔCT The relative expression of genes was calculated using the Excel method and graphed.

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

[0048] Example 3

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

[0050] The pCAMBIA1302 vector (purchased from Zeye Biotechnology) was double-digested with EcoR I and Bam HI (the vector structure is as follows Figure 3 shown).

[0051] The double enzyme digestion reaction system was: pCAMBIA1302 plasmid / recovered fragment 21 μL, Buffer 5 μL, EcoR I and BamHI 2 μL each, H2O 20 μL.

[0052] The double enzyme digestion reaction procedure was: 37°C for 4h.

[0053] The full-length fragment of the BcTEM1 gene amplification was used as a template to design a primer with a linker that seamlessly connects to the expression vector pCAMBIA1302. The primer sequence is as follows:

[0054] BcTEM1-S-EcoRI:

[0055] 5'-GGAATTCATGGATTACAGCTGTCTAGACGAC-3',

[0056] BcTEM1-A-BamHI:

[0057] 5'-CGGGATCCCAAGACGTTGATTATGGCCTGC-3'.

[0058] Two restriction sites, EcoR I and Bam HI, were added to both ends of the BcTEM1 gene amplification primer, and the linearized vector fragment after double restriction digestion and the gene amplification fragment were connected using T4 DNA ligase (purchased from Thermo Fisher Scientific).

[0059] The primers for connecting the BcTEM1 gene amplification were: 1 μL of ligase, 2 μL of buffer, 3 μL of linearized vector, and 14 μL of BcTEM1 amplified fragment. The reaction procedure was: overnight at 4°C. The ligation product was transferred into DH5α competent medium (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, and the pCAMBIA1302-BcTEM1 recombinant vector was obtained, and the plasmid was extracted.

[0060] 2. Subcellular localization of BcTEM1

[0061] The pCAMBIA1302-BcTEM1 recombinant plasmid and pCAMBIA1302 plasmid were transferred into Agrobacterium GV3101 competent cells (purchased from Weidi Biotechnology) by freeze-thaw method and cultured in a dark incubator at 28°C for 48 hours. The Agrobacterium transformation method was carried out according to the instructions. Single clones were selected for colony PCR detection, and positive clones were inoculated into LB liquid medium (containing 50 μg / mL kanamycin and rifampicin) and cultured at 28°C for one day until the bacterial solution OD 600 = 0.8. The cells were collected by centrifugation at 5000 rpm for 10 min and resuspended in injection buffer to OD 600 =0.6, and stand in the dark for 4 hours. The injection buffer contains 10mM MES solution 1ml, 10mM magnesium chloride solution 1ml, 150μM acetosyringone solution 10μL, and ddH2O to a total volume of 10ml. Use a sterile syringe to inject the bacterial solution into the tobacco leaves after standing, and culture in the dark for 12 hours and then culture normally for 48 hours. Take the tobacco leaves injected with the bacterial solution, vacuum and transfer to DAPI dye, and observe with an Olympus BX71 microscope.

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

[0063] Example 4

[0064] 1. Construction of VIGS silencing vector for non-heading Chinese cabbage

[0065] A 40 bp specific sequence was selected from the non-conserved region of the BcTEM1 gene as a silencing fragment (the nucleotide sequence is shown in SEQ ID NO.3), and a 40 bp sequence complementary to it was added to obtain an 80 bp sequence forming a hairpin structure, the nucleotide sequence of which is shown in SEQ ID NO.4. It was inserted into the pTY-s vector ( Figure 5) to obtain the recombinant vector pTY-s-BcTEM1. The synthesis of the hairpin structure fragment, the construction of the pTY-s vector and the sequencing were all completed by GenScript Biotech Co., Ltd. The Escherichia coli culture of pTY-s-BcTEM1 was shaken and cultured at 37°C for 12 hours, and then the plasmid was extracted to be transferred into non-heading Chinese cabbage.

[0066] 2. Obtaining Silent Plants

[0067] One-month-old non-heading cabbage plants with uniform growth were selected as silencing plant materials. The pTY-s-BcTEM1 and pTY-s plasmids were coated with gold powder. Gold powder coating system: 8.5 μL of gold powder, 5 μL of plasmid, 50 μL of 2.5M CaCl2, and 20 μL of 0.1M spermidine. After mixing, ice bath for 20 minutes, vortex oscillation for 10 seconds, centrifugation at 10000rpm for 5 seconds, pour out the supernatant, wash once with anhydrous ethanol, and bombard the non-heading cabbage plants with the gold powder-coated plasmids using a gene gun (Bio-Rad, PDS1000 / He). Non-heading cabbage plants bombarded with pTY-s plasmids were used as control plants. Five plants were bombarded each time, and each plasmid was repeated four times. After 30 days of bombardment, if the plant becomes ill (mosaic appears), it means that the silencing vector has been transferred into the non-heading cabbage plant.

[0068] 3. Phenotype of silenced plants and expression of flowering-related genes

[0069] The diseased leaves (with mosaic) of the non-heading cabbage plants were taken to extract total RNA, and the cDNA was obtained by inversion, using the same method as in Example 1. The expression of BcTEM1, BcFT and BcAP3 genes in the silenced non-heading cabbage plants was detected by qPCR, using the same method as in Example 2. At the same time, the flowering time of the silenced non-heading cabbage plants was observed and counted. The primer sequences are as follows:

[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 silent non-heading cabbage plants showed obvious early flowering, about five days earlier than the control ( Figure 6 A). Compared with the control group, the expression level of BcTEM1 gene in the silenced plants was significantly downregulated, indicating that the expression of BcTEM1 gene in the silenced plants was significantly inhibited ( Figure 6 B). The expression levels of BcFT and BcAP3 were significantly upregulated in the silenced plants, indicating that BcFT and BcAP3 are target genes of BcTEM1 ( Figure 6 C).

[0077] 4. Dual luciferase assay

[0078] Non-heading cabbage leaves were selected as experimental materials. DNA was extracted using a plant genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.), and the DNA extraction method was carried out according to the kit instructions. The nucleotide sequence of the promoter of the BcFT gene was downloaded from the non-heading cabbage genome database http: / / nhccbase.njau.edu.cn / website / , and the forward primer and reverse primer were designed using Primer 5 software to amplify the promoter of the BcFT gene. The primer sequences are as follows:

[0079] pBcFT-S:

[0080] 5'-AAAAAAATGATGAATTGAAAAGCTTCGATCCGAACCAAGTTGTGC-3',

[0081] pBcFT-A:

[0082] 5'-CATACAGAGCACATGCCTCGAGCTTTGATCTAAAACAAACAGGTGG-3'.

[0083] The 50 μL PCR amplification system was as follows: 25 μL 2×Phanta Max Master Mix (purchased from Novazonics), 1 μL DNA, 2 μL each of forward primer and reverse primer, and ddH2O was added to 50 μL.

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

[0085] The PCR product and the pGreenII 0800 (LUC) vector were double-digested with Hind III and Kpn I, and the products were recovered and ligated to obtain the pBcFT-pGreenII 0800 (pBcFT-LUC) recombinant vector, using the same method as in Example 3. The recombinant vector was transferred into Agrobacterium GV3101, and the Agrobacterium was resuspended after preparing the injection solution. The resuspended pBcFT-LUC bacterial solution and the pCAMBIA1302-BcTEM1 (35S:BcTEM1-GFP) bacterial solution were evenly mixed at a volume ratio of 1:1 and injected into tobacco. Equal volumes of pBcFT-LUC bacterial solution and pCAMBIA1302 (35S:GFP) bacterial solution were mixed and injected into tobacco leaves as a control group. Five leaves were injected with each bacterial solution mixture. After 3 days of normal culture in a light incubator, the injected tobacco leaves were ground with liquid nitrogen. Use Reporter Assay System (purchased from Promega), using a microplate reader to measure the values ​​of firefly luciferase and Renilla luciferase. Using Excel to record the experimental data, the ratio of firefly luciferase / Renilla luciferase was calculated to measure the relative LUC activity.

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

[0087] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A non-heading Chinese cabbage transcription factor BcTEM1 gene, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expressed protein of the non-heading Chinese cabbage transcription factor BcTEM1 gene according to claim 1, whose amino acid sequence is shown as SEQ ID NO.

2.

3. Use of the non-heading cabbage transcription factor BcTEM1 gene according to claim 1 in regulating the flowering time of plants.

4. Use of the non-heading cabbage transcription factor BcTEM1 gene according to claim 1 in regulating the expression of downstream genes.

5. A method for promoting early flowering of plants, characterized in that: Silencing the expression of the BcTEM1 gene in plants.

6. The method according to claim 5, characterized in that include: 1) constructing a silencing vector containing the VIGS silencing fragment of the non-heading Chinese cabbage transcription factor BcTEM1 gene; 2) transforming the constructed silencing vector containing the VIGS silencing fragment of the non-heading cabbage transcription factor BcTEM1 gene into non-heading cabbage; 3) Cultivate, screen and obtain non-heading Chinese cabbage plants with early flowering time.

7. The method according to claim 6, characterized in that The VIGS silencing fragment of the non-heading Chinese cabbage transcription factor BcTEM1 gene has a nucleotide sequence as shown in SEQ ID NO.

3.

8. The use according to claim 6, characterized in that: The method for constructing the silencing vector comprises the following steps: connecting the VIGS silencing fragment of the non-heading cabbage transcription factor gene BcTEM1 to a 40bp sequence that is reverse complementary to the VIGS silencing fragment to obtain an 80bp hairpin structure fragment; and inserting the 80bp hairpin structure fragment into a pTY-s vector to construct a pTY-s-BcTEM1 vector, i.e., a silencing vector comprising the VIGS silencing fragment of the non-heading cabbage transcription factor BcTEM1 gene.

9. The use according to claim 8, characterized in that: The nucleotide sequence of the 80 bp hairpin structure fragment is shown in SEQ ID NO.

4.

10. Use of the VIGS silencing fragment of the non-heading cabbage transcription factor gene BcTEM1 according to claim 7 in regulating the expression level of the downstream gene BcFT.

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