BcTCP22 gene of brassica rapa transcription factor and expression protein and application thereof
By constructing a silencing vector for the transcription factor BcTCP22 gene in non-heading Chinese cabbage, the flowering time of the plant was regulated, which solved the uncertainty of flowering time regulation in non-heading Chinese cabbage, achieved earlier flowering time and regulation of gene expression, and promoted the breeding process.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANJING FORESTRY UNIV
- Filing Date
- 2024-12-10
- Publication Date
- 2026-04-28
AI Technical Summary
In the existing technology, the function of the BcTCP22 gene in the regulation of flowering time in non-heading Chinese cabbage is unclear, making it difficult to effectively regulate the flowering time of plants.
A silencing vector containing the VIGS silencing fragment of the BcTCP22 gene transcription factor from non-heading Chinese cabbage was constructed and transformed into non-heading Chinese cabbage. The silencing vector was used to regulate the flowering time of the plant and promote early flowering.
By silencing the BcTCP22 gene, the flowering time was advanced by ten days, the expression level of BcTCP22 was downregulated by 50%, and the expression level of BcFT was upregulated by three times, which significantly accelerated the breeding process and improved economic benefits.
Smart Images
Figure CN119570809B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering technology, and more specifically, relates to the BcTCP22 gene of non-heading Chinese cabbage transcription factor, its expressed protein, and its applications. Background Technology
[0002] Non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) belongs to the genus Brassica in the family Brassicaceae. It is an important leafy vegetable in my country due to its high nutritional value and short growth cycle. However, the flowering time varies greatly among different varieties of non-heading Chinese cabbage, posing a significant challenge to the breeding of new varieties. Therefore, studying the flowering time regulation mechanism of non-heading Chinese cabbage is of great practical significance for variety improvement.
[0003] Flowering is a crucial process in plants transitioning from vegetative to reproductive growth. The diversity of flowering time ensures that plants can be widely distributed across different geographical regions. Day length and temperature are the two most important environmental factors influencing flowering. The effect of day length on flowering is mainly regulated by CO, FKF1, and GI. GI and FKF1 form a complex that targets CDF1, directly promoting FT expression and ultimately promoting flowering. The TCP gene family is involved in various biological processes, including circadian rhythm clocks, leaf size and shape, and the regulation of flower development and flowering. Under long-day conditions, the Arabidopsis tcp22-1 mutant plants flower earlier. Arabidopsis TCP22 interacts with LWD1 to activate the CCA1 promoter and promote CCA1 expression, thereby regulating FT expression and inhibiting flowering. Although the function of Arabidopsis TCP22 has been studied, the function of BcTCP22 in non-heading Chinese cabbage, especially its role in flowering regulation, remains unclear. 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 BcTCP22 transcription factor gene from non-heading Chinese cabbage. Another technical problem this invention aims to solve is to provide the expression protein of the BcTCP22 transcription factor gene from non-heading Chinese cabbage. A further technical problem this invention aims to solve is to provide the application of the BcTCP22 transcription factor gene from non-heading Chinese cabbage in regulating plant flowering time.
[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 BcTCP22 gene in non-heading Chinese cabbage is shown in SEQ ID NO.1.
[0007] The amino acid sequence of the protein expressed by the transcription factor BcTCP22 gene in non-heading Chinese cabbage is shown in SEQ ID NO.2.
[0008] Application of the transcription factor BcTCP22 gene in regulating flowering time in non-heading Chinese cabbage.
[0009] The VIGS silencing fragment of the transcription factor BcTCP22 gene in non-heading Chinese cabbage has the nucleotide sequence shown in SEQ ID NO.3.
[0010] Application of the VIGS silencing fragment of the transcription factor BcTCP22 gene in non-heading Chinese cabbage in regulating flowering time.
[0011] The regulation of plant flowering time aims to promote earlier flowering, including:
[0012] 1) Construct a silencing vector containing the VIGS silencing fragment of the BcTCP22 gene, a transcription factor from non-heading Chinese cabbage;
[0013] 2) The constructed silencing vector containing the VIGS silencing fragment of the non-heading Chinese cabbage transcription factor BcTCP22 gene was transformed into non-heading Chinese cabbage;
[0014] 3) Cultivate, screen, and obtain non-heading Chinese cabbage plants with earlier flowering time.
[0015] The method for constructing the silencing vector is as follows: the VIGS silencing fragment of the non-heading Chinese cabbage transcription factor BcTCP22 gene is directly ligated to a 40bp reverse complementary sequence to obtain an 80bp hairpin structure fragment; the 80bp hairpin structure is inserted into the pTY-s vector to construct the pTY-s-BcTCP22 vector, which is a silencing vector containing the VIGS silencing fragment of the non-heading Chinese cabbage transcription factor BcTCP22 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 BcTCP22 gene 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 BcTCP22 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 BcTCP22 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, compared with the control group, the flowering time of BcTCP22 gene-silenced plants is ten days earlier; the expression level of BcTCP22 in BcTCP22 gene-silenced plants is downregulated by more than 50%, while the expression level of BcFT is upregulated three-fold. This is beneficial for accelerating the breeding process, improving economic benefits, providing an important candidate gene for non-heading Chinese cabbage breeding, and has important reference significance for the improvement of flowering time in cruciferous plants. Attached Figure Description
[0020] Figure 1 Electrophoresis diagram of PCR amplification of the transcription factor BcTCP22 gene in non-heading Chinese cabbage;
[0021] Figure 2 This is a diagram showing the expression pattern of the transcription factor BcTCP22 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 BcTCP22 gene in non-heading Chinese cabbage (A is a schematic diagram of the structure of 35S:BcTCP22-GFP and 35S:GFP, B is a subcellular localization map of transient expression of the BcTCP22-GFP fusion protein in tobacco leaves; the scale bar in the figure is 50 μm).
[0024] Figure 5 The vector structure diagram of the VIGS silencing vector pTY-s used;
[0025] Figure 6 Flowering phenotype of plants with silenced transcription factor BcTCP22 gene in non-heading Chinese cabbage (A) (Control is the control plant, 1 is the silenced plant BcTCP22-pTY-s-1, 2 is the silenced plant BcTCP22-pTY-s-2) and quantitative analysis of downstream genes (B). Detailed Implementation
[0026] 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.
[0027] The material used in this application is non-heading Chinese cabbage, which was grown on the campus of Nanjing Forestry University.
[0028] Example 1
[0029] 1. RNA extraction and cDNA synthesis
[0030] Total RNA was extracted from non-heading Chinese cabbage leaves using the RNAsimpleTotal RNA Extraction Kit (purchased from TIANGEN). cDNA was synthesized by reverse transcription using the HiScript III 1st Strand cDNA Synthesis Kit (purchased from Vazyme) and stored at -20℃ for later use.
[0031] 2. Cloning of the BcTCP22 gene
[0032] The nucleotide sequence of the BcTCP22 gene was downloaded from the non-heading Chinese cabbage genome database (http: / / nhccbase.njau.edu.cn / website / ), and primers for amplifying the BcTCP22 gene were designed using Primer 5 software.
[0033] Forward primer: 5'-ATGTCGAACGACGACGGGAC-3',
[0034] Reverse primer: 5'-ACGTGAGTTATCCTCCTCCTCCC-3'.
[0035] The PCR reaction mixture consisted of: 12.5 μL of high-fidelity enzyme (purchased from Vazyme), 1 μL of cDNA, 1 μL each of forward and reverse primers, and 9.5 μL of deionized water. The mixture was added on ice and mixed thoroughly.
[0036] The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 59℃ annealing for 15 s, 72℃ extension for 40 s, 35 cycles; 72℃ for 8 min.
[0037] The amplified products were subjected to 1% agarose gel electrophoresis. Figure 1The nucleotide sequence of the BcTCP22 gene, a transcription factor from non-heading Chinese cabbage, was obtained by gel extraction, recovery, and sequencing. The sequencing results showed that the nucleotide sequence of the BcTCP22 gene, as shown in SEQ ID NO.1, is 708 bp in length (with the terminator removed), encoding a protein of 236 amino acids, the amino acid sequence of which is shown in SEQ ID NO.2.
[0038] Example 2
[0039] The expression pattern of the BcTCP22 gene in different tissues of non-heading Chinese cabbage was detected using real-time quantitative PCR (qPCR). Total RNA was extracted and cDNA synthesized from root, stem, leaf, and flower tissues of non-heading Chinese cabbage, following the same methods as in Example 1. Based on the nucleotide sequence of the cloned BcTCP22 gene, quantitative primers for the BcTCP22 gene were designed using Primer5 software. The BcActin gene was used as an internal reference gene.
[0040] qBcTCP22-S: 5'-ATAGCCGCCACGGGAACG-3'
[0041] qBcTCP22-A: 5'-CCAAAACCCACCACCACTCATC-3'
[0042] qBcActin-S: 5'-GTTGCTATCCAGGCTGTTCT-3
[0043] qBcActin-A: 5'-AGCGTGAGGAAGAGCATAAC-3'
[0044] The PCR reaction system consisted of: 10 μL SYBR Green, 0.6 μL forward primer, 0.6 μL reverse primer, 7.8 μL deionized water, and 1 μL cDNA.
[0045] The PCR reaction program was: 95℃ for 3 min; 95℃ for 10 s, 60℃ for 30 s, 72℃ for 30 s, for 40 cycles. Using 2... -ΔΔCT The formula calculates the relative expression level of genes.
[0046] The results are as follows Figure 2 As shown, the BcTCP22 gene in non-heading Chinese cabbage is expressed at the highest level in the flower.
[0047] Example 3
[0048] 1. Construct an overexpression vector for the BcTCP22 gene.
[0049] The pCAMBIA1302 vector (purchased from Zeye Biotechnology) was digested with Xba I and Bam HI (vector structure as shown in the image). Figure 3(As shown).
[0050] The double enzyme digestion reaction system consisted of: 20 μL of pCAMBIA1302 plasmid, 5 μL of buffer, 2 μL each of Xba I and Bam HI, and 21 μL of deionized water.
[0051] The double enzyme digestion reaction procedure is: 37℃ for 4 hours.
[0052] Using the full-length amplified fragment of the BcTCP22 gene as a template, adapter primers with seamless ligation to the expression vector pCAMBIA1302 were designed. The primer sequences are shown below:
[0053] BcTCP22-GFP-S:
[0054] 5'-GAGAACACGGGGGACTCTAGAATGTCGAACGACGACGGGAC-3';
[0055] BcTCP22-GFP-A:
[0056] 5'-GCCCTTGCTCACCATGGATCCACGTGAGTTATCCTCCTCCTCCC-3'.
[0057] The linearized vector fragment digested by double enzymes and the above-mentioned gene amplification fragment were ligated at 37°C for 30 minutes using homologous recombinase (purchased from Vazyme).
[0058] The ligation reaction system consisted of: 1 μL linearized vector, 2 μL BcTCP22 amplified fragment, 1 μL Exnase II, 2 μL 5×CEII Buffer, and 4 μL deionized water. The reaction program was 37℃ for 3 min.
[0059] Immediately after the reaction, the cells were placed on ice. The recombinant product was transferred into DH5α competent cells (purchased from Weidi Biotechnology), single colonies were selected, and the cells were shaken in LB medium containing kanamycin. After PCR detection confirmed the results, sequencing was performed to obtain the recombinant plasmid pCAMBIA1302-BcTCP22.
[0060] 2. Subcellular localization of BcTCP22
[0061] The pCAMBIA1302-BcTCP22 and pCAMBIA1302 plasmids were transformed into Agrobacterium GV3101 competent cells (purchased from Weidi Biotechnology) using the freeze-thaw method. The Agrobacterium transformation method was performed according to the manufacturer's instructions. Positive clones detected by PCR were cultured in LB medium containing 50 μg / mL kanamycin and 50 μg / mL rifampin at 28°C until the bacterial culture reached OD500. 600=0.7. Centrifuge at 4000 rpm for 12 min to collect bacterial cells, and resuspend in the solution to OD. 600 =0.6, and allowed to stand in the dark for 3 hours. Following the transient expression technique for tobacco, the bacterial solution after standing was injected into tobacco leaves for subcellular localization observation.
[0062] The results are as follows Figure 4 As shown, laser confocal microscopy results indicate that BcTCP22 is located in the cell nucleus, consistent with typical transcription factor characteristics.
[0063] Example 4
[0064] 1. Constructing a silencing vector for the BcTCP22 gene
[0065] Using the cloned BcTCP22 gene nucleotide sequence as a template, a 40bp sequence was selected from its non-conserved region as a VIGS silencing fragment. The selected silencing fragment was subjected to BLAST in the non-heading Chinese cabbage genome database to confirm that it specifically aligned to BcTCP22. The 40bp silencing fragment (nucleotide sequence shown in SEQ ID NO.3) was directly ligated to its reverse complementary 40bp sequence to obtain an 80bp hairpin structure, the nucleotide sequence of which is shown in SEQ ID NO.4. The above 80bp fragment was synthesized and inserted into the pTY-s vector (…). Figure 5 The synthesis of the recombinant plasmid pTY-s-BcTCP22.80bp fragment and the construction of the pTY-s vector were completed by Genscript Biotech Co., Ltd.
[0066] 2. Obtaining Silent Plants
[0067] The pTY-s-BcTCP22 and pTY-s plasmids were coated with gold powder. The coating system was as follows: 10 μL plasmid, 17 μL gold powder, 40 μL 0.1 M spermidine, and 100 μL 2.5 M CaCl2. The mixture was pipetted and incubated on ice for 35 min, then vortexed for 25 s, centrifuged at 9000 rpm for 1 min, and the supernatant was discarded. The plasmids were washed twice with anhydrous ethanol. One-month-old non-heading Chinese cabbage plants were bombarded with the gold-coated pTY-s and pTY-s-BcTCP22 plasmids using a gene gun (Bio-Rad, PDS1000 / He). Plants bombarded with the pTY-s plasmid served as a control. Six plants were bombarded each time, and each plasmid was bombarded five times. Forty days after bombardment, if the plants developed disease (showing mosaic patterns), it indicated that the silencing vector had been transferred into the non-heading Chinese cabbage plants.
[0068] 3. Quantitative analysis of silencing plant phenotypes and downstream genes
[0069] Total RNA was extracted and cDNA synthesized from diseased leaves (mosaic leaves) of infected plants, following the same method as in Example 1. BcFT quantitative primers were designed to detect the expression of BcTCP22 and BcFT genes in silent non-heading Chinese cabbage plants, following the same method as in Example 2. The flowering time of silent plants was statistically observed. Primer sequences are shown below:
[0070] qBcFT-S: 5'-GTCCTAGCAATCCTCACCTCCG-3'
[0071] qBcFT-A: 5'-CGCCATCCTGGTTCATACACTG-3'
[0072] The results are as follows Figure 6 As shown, compared with the control group, plants with the BcTCP22 gene silenced flowered ten days earlier. Figure 6 A). Compared with control plants, BcTCP22 expression was downregulated by more than 50% in plants with silenced BcTCP22 gene, while BcFT expression was upregulated three-fold. Figure 6 B). The results showed that the expression of the BcTCP22 gene was successfully suppressed in BcTCP22-silenced plants, and that BcFT is a downstream regulatory gene of BcTCP22.
[0073] 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 BcTCP22 gene, a transcription factor in non-heading Chinese cabbage, with the nucleotide sequence shown in SEQ ID NO.1, in promoting early flowering in non-heading Chinese cabbage plants.
2. Application of the VIGS silencing fragment of the BcTCP22 gene, a transcription factor from non-heading Chinese cabbage, in promoting early flowering in non-heading Chinese cabbage plants; the nucleotide sequence of the BcTCP22 gene is shown in SEQ ID NO.1; the specific steps include: 1) Construct a silencing vector containing the VIGS silencing fragment of the non-heading Chinese cabbage transcription factor BcTCP22 gene; the silencing vector is constructed as follows: the VIGS silencing fragment of the non-heading Chinese cabbage transcription factor BcTCP22 gene is directly ligated to a 40bp reverse complementary sequence to obtain an 80bp hairpin structure fragment; the 80bp hairpin structure is inserted into the pTY-s vector to construct the pTY-s-BcTCP22 vector, which is the silencing vector containing the VIGS silencing fragment of the non-heading Chinese cabbage transcription factor BcTCP22 gene; the nucleotide sequence of the VIGS silencing fragment of the non-heading Chinese cabbage transcription factor BcTCP22 gene is shown in SEQ ID NO. 3; the nucleotide sequence of the 80bp hairpin structure fragment is shown in SEQ ID NO. 4; 2) The constructed silencing vector containing the VIGS silencing fragment of the non-heading Chinese cabbage transcription factor BcTCP22 gene was transformed into non-heading Chinese cabbage plants; 3) Cultivate, screen, and obtain non-heading Chinese cabbage plants with earlier flowering time.