Brassica napus bncdy201 gene and application thereof in regulating flowering period and vegetative growth period of brassicaceae plants

Abstract

This invention relates to a rapeseed BnCDY201 gene and its application in regulating the flowering and vegetative growth stages of cruciferous plants, belonging to the field of plant genetic engineering technology. The nucleotide sequence of the BnCDY201 gene is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. The overexpression vector of the BnCDY201 gene constructed in this invention was transformed into Arabidopsis thaliana. The resulting transgenic plants exhibited significantly delayed flowering and a significantly increased number of rosette leaves, demonstrating the gene's function in prolonging the vegetative growth stage and delaying flowering. This provides a new genetic resource and effective tool for regulating plant flowering and vegetative growth through genetic engineering, and has significant application value in crop breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a rapeseed BnCDY201 gene and its application in regulating the flowering and vegetative growth periods of cruciferous plants. Background Technology

[0002] Rapeseed is an important oilseed crop, and rice-rapeseed rotation is an important agricultural practice. In actual production, the rapeseed crop rotation will directly affect the sowing or transplanting of the next crop. The flowering time can affect the entire growth cycle and yield of rapeseed. Therefore, cultivating early-maturing rapeseed varieties that flower and mature early can solve the crop rotation contradiction of rice-rapeseed rotation, effectively expand the rapeseed planting area and increase the total yield.

[0003] The regulation of plant flowering time is controlled by a complex gene network. For crops that are harvested for seeds or fruits, earlier flowering can shorten their growth period and increase the multiple cropping index. For crops that are harvested for vegetative organs, delayed flowering can prolong the photosynthetic time, thereby increasing yield. In addition, some crops can avoid the effects of adverse weather by shortening their growth period. For example, early maturity of winter rapeseed can avoid rainy weather and improve rapeseed quality. Rapeseed and Arabidopsis belong to the Brassicaceae family, so they have a high degree of conservation in the regulation of flowering pathways. In recent years, some flowering genes have been identified in rapeseed. For example, rapeseed BnaFLC.A2, BnaFLC.A3b and BnaFLC.A10 mainly respond to low-temperature vernalization. When BnaFLC.A10 and BnaFLC.A2 are mutated simultaneously in winter rapeseed, the flowering period is close to that of spring rapeseed Westar (Yin et al.). (Wan et al. 2025) There are four copies of BnaFT in rapeseed. BnaFT.A2 has the greatest impact on flowering time, followed by BnaFT.C6 and BnaFT.A7, while BnaFT.C2 has no effect on flowering (Wan et al. 2024). However, there are still many unknown flowering genes in rapeseed that need to be revealed. Summary of the Invention

[0004] The purpose of this invention is to provide a rapeseed BnCDY201 gene and its application in regulating the flowering and vegetative growth stages of cruciferous plants in order to solve the above-mentioned problems.

[0005] The present invention achieves the above objectives through the following technical solutions: This invention provides a rapeseed BnCDY201 gene, the nucleotide sequence of which is shown in SEQ ID NO.1. The present invention also provides a protein encoded by the rapeseed BnCDY201 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0006] This invention also provides an application of the rapeseed BnCDY201 gene in regulating the flowering and vegetative growth stages of cruciferous plants.

[0007] As a further optimization of the present invention, the overexpression of the rapeseed BnCDY201 gene can delay the flowering period of cruciferous plants and prolong the vegetative growth period of plants.

[0008] As a further optimization of the present invention, the cruciferous plants are Arabidopsis thaliana and rapeseed.

[0009] A recombinant plasmid was obtained by transforming the rapeseed BnCDY201 gene into a vector.

[0010] As a further optimization of the present invention, the vector is pCAMBIA1305-EGFP.

[0011] A method for obtaining transgenic late-flowering and long vegetative growth period cruciferous plant varieties involves introducing the rapeseed BnCDY201 gene as the target gene into the genome of cruciferous plants for overexpression, thereby cultivating transgenic late-flowering and long vegetative growth period plant varieties. The plants mentioned are Arabidopsis thaliana and rapeseed.

[0012] The beneficial effects of this invention are as follows: The overexpression vector of the BnCDY201 gene constructed in this invention was transformed into Arabidopsis thaliana. The resulting transgenic plants showed a significant delay in flowering time and a significant increase in the number of rosette leaves, indicating the function of this gene in prolonging the vegetative growth period and delaying flowering. This provides a new gene resource and effective tool for regulating plant flowering and vegetative growth through genetic engineering, and has important application value in crop breeding. Attached Figure Description

[0013] Figure 1 Electrophoresis diagram of the rapeseed BnCDY201 gene clone; Figure 2 Map of overexpression plasmid vectors; Figure 3 The image shows PCR detection results of transgenic Arabidopsis plants (Control is pCAMBIA1305BnCDY201 plasmid, #1-12 are BnCDY201 gene overexpression lines). Figure 4 The figures show a comparison of flowering phenotypes (A) and rosette leaf count (B) of the BnCDY201 transgenic Arabidopsis thaliana. WT represents wild-type Arabidopsis thaliana, while OE-1 and OE-2 represent Arabidopsis thaliana lines overexpressing the BnCDY201 gene. Detailed Implementation

[0014] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0015] 1. Materials Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products.

[0016] method 2.1 Extraction of total RNA Using rapeseed leaves as experimental material, total RNA was extracted from Brassica napus using a rapid total RNA extraction kit (RNAprep Pure PlantKit, Proha Biotechnology Co., Ltd.). The specific method is as follows: 1) Take 0.5g of rapeseed seedling leaves and place them in a mortar that has been pre-cooled to 80℃. Add liquid nitrogen and grind until pulverized. 2) Take 0.1g of the ground sample and transfer it into a centrifuge tube without RNase. Add 1ml of lysis buffer RL, mix by inverting for 20s, and then incubate at 25℃ for 5min to allow it to fully lyse. 3) Add 200 μl of chloroform, tighten the cap, shake vigorously for 15 seconds, and incubate at 25°C for 3 minutes; 4) Centrifuge at 4℃ and 12000rpm for 20min, transfer 0.5ml of supernatant to an RNase-free centrifuge tube, add an equal volume of isopropanol and mix gently. 5) Transfer the entire solution to the RNA adsorption column, centrifuge at 12,000 rpm for 45 seconds at 25°C, and discard the waste liquid; 6) Add 500 μl of protein-removing RE solution to the RNA adsorption column, centrifuge at 12000 rpm for 45 s at 25°C, and discard the waste liquid. 7) Repeat step 6) once; 8) Reinsert the RNA adsorption column into the collection tube and centrifuge at 12,000 rpm for 2 min at 25°C to remove residual washing solution. 9) Remove the RNA adsorption column, place it at room temperature for 2 minutes, then place it in a new 1.5 ml RNase-free centrifuge tube, add 100 μl RNase-free water, place it at room temperature for 2 minutes, centrifuge at 12000 rpm for 1 minute, and the solution collected in the centrifuge tube is the total RNA from Brassica napus. 10) The total RNA concentration was determined using a micro spectrophotometer.

[0017] 2.2 Obtaining cDNA from RNA reverse transcription cDNA from Brassica napus was prepared using a reverse transcription kit (SweScript AllinOne RT SuperMix, Sewell Biotechnology Co., Ltd.). The following reagents were added to RNase-free PCR tubes, as shown in Table 1: Table 1. Dosage of each component in the reaction solution Mix the above reagents thoroughly, place the PCR tube into the PCR instrument, and perform reverse transcription. The reaction program is shown in Table 2. Table 2 Reaction Procedure Once the reaction is complete, the cDNA of Brassica napus can be obtained and stored at -80℃ for later use.

[0018] 2.3 Cloning of the BnCDY201 gene The CDS sequence of the BnCDY201 gene was obtained from the rapeseed genome database (http: / / cbi.hzau.edu.cn / rape / download_ext / zs11.genome.fa). Using this sequence as a template, cloning-specific primers for the rapeseed BnCDY201 gene were designed using Primer 5 software. The primer sequences are as follows: SEQ ID NO.3: BnCDY201-F: AagtccggagctagctctgaATGGATAATTATGATGGGACTAATCTGG; SEQ ID NO.4: BnCDY201-R1: gcccttgctcaccatggatccGTAACGGTAACTCCAGAGTGACATATC; Take a new PCR tube, use the obtained cDNA as a template, and amplify the gene coding region using the above primers and high-fidelity enzyme PCR mix (KOD One PCR Master Mix, Toyobo Biotechnology Co., Ltd.). The specific reaction system is shown in Table 3. Table 3 PCR amplification system The PCR reaction procedure is shown in Table 4: Table 4 PCR Amplification Program PCR amplification results: as follows Figure 1As shown, the amplified nucleotide sequence after the reaction was 609 bp in length. The specific information is shown in SEQ ID NO.1. The final PCR product was recovered by agarose gel electrophoresis, and the concentration of the recovered product was determined by a micro spectrophotometer.

[0019] 2.4 Obtaining Arabidopsis thaliana with overexpression of the BnCDY201 gene Arabidopsis thaliana belongs to the Brassicaceae family, Angiosperms, and Dicotyledons. Its advantages include small plant size and high seed production. The Arabidopsis genome is the smallest known plant genome. It is a self-pollinating plant with highly homozygous genes. In rapeseed breeding, Arabidopsis thaliana, a model plant in the same family, is often used as the initial research subject because the two share similar morphological and structural characteristics and exhibit similar molecular regulatory mechanisms in many developmental processes.

[0020] The overexpression plasmid vector backbone pCAMBIA1305-EGFP (vector map shown) was modified using restriction endonucleases SpeI and BamH (Baori Biotechnology Co., Ltd.). Figure 2 The enzyme was digested (as shown), the long fragment was recovered by agarose gel electrophoresis, and the concentration of the recovered product was determined by micro-spectrophotometer. The BnCDY201 gene overexpression vector was constructed using a one-step cloning kit (Seamless Assembly cloning kit, Sino-American Taihe Biotechnology Co., Ltd.). The specific method is as follows: take a new PCR tube, add the reagents shown in Table 5 according to the concentration of the recovered product, then cover the tube, vortex the reagents to mix, and incubate at 50°C for 15 min. Table 5. Dosage of each component in the reaction solution The specific method for creating an Escherichia coli host strain containing a BnCDY201 gene overexpression vector is as follows: 1) Take 5 μl of the reaction product and add it to E. coli DH5α competent cells, and mix gently; 2) Ice bath for 30 minutes, heat shock at 42°C for 45 seconds, ice bath for 2 minutes; 3) Add 500 μL of antibiotic-free LB liquid medium, and incubate at 37°C for 45 min at 200 rpm in a shaker. In a clean bench, aspirate the revived bacteria and spread them on LB solid medium containing 50 μg / ml kanamycin. Incubate overnight at 37°C with the medium inverted. Select single colonies for sequencing analysis. The single colony with correct sequencing is the Escherichia coli host bacteria containing the BnCDY201 gene overexpression vector.

[0021] The specific method for creating an Agrobacterium host bacterium containing a BnCDY201 gene overexpression vector is as follows: 1) Use Escherichia coli host bacteria containing the BnCDY201 gene overexpression vector to extract plasmids, take a tube of Agrobacterium GV3101 competent cells, thaw them and place them on ice; 2) Add 1 μl of plasmid DNA to the competent cells, gently pipette to mix, incubate on ice for 5 min, then freeze in liquid nitrogen for 2 min, heat shock at 37°C for 5 min, and incubate on ice for 5 min. 3) Add 500 μl of antibiotic-free LB liquid medium, and revive in a shaker at 28°C for 3 h. In a clean bench, aspirate the revived bacteria and spread them on YEP solid medium containing 50 μg / ml Kana and 50 μg / ml Rif. Incubate upside down at 28°C for 48 h. PCR detection shows that the bacteria carrying the target vector are Agrobacterium host bacteria containing the BnCDY201 gene overexpression vector.

[0022] Plants overexpressing BnCDY201 were obtained by infecting Arabidopsis thaliana. The specific method is as follows: 1) Agrobacterium containing the BnCDY201 gene overexpression vector was inoculated onto YEP solid medium containing 50 μg / ml Kana and 50 μg / ml Rif, and a single colony was picked after 2 days. 2) Inoculate into 2 mL of YEP liquid medium containing 550 μg / ml Kana and 50 μg / ml Rif, and incubate overnight at 28°C and 220 rpm; 3) Inoculate the cultured bacterial solution at a ratio of 1:50 into 100 mL of liquid culture medium with the same resistance, and incubate at 28°C and 220 rpm for about 8 hours until the OD600 value is 0.8-1.0; 4) Centrifuge the above bacterial suspension at 4500 rpm for 5 min at room temperature, discard the supernatant, and resuspend the bacterial pellet in 1 / 2 MS liquid medium containing 5% Sugar and 0.05% Silwet L-77. Adjust the OD600 value of the resuspended suspension to 0.8. 5) Select wild-type Arabidopsis thaliana in full bloom, cut off the pollinated pods, immerse the Arabidopsis thaliana inflorescence in resuspension for 45 seconds, culture in the dark for 24 hours, and then culture normally. 6) Following the above method, a second infection was carried out one week later, and Arabidopsis seeds were harvested 30 days later. The seeds were then screened for green fluorescent protein genes under a stereofluorescence microscope using the green fluorescent protein gene markers. These seeds were the T1 generation seeds. 7) After sowing T1 generation seeds, manage them normally and harvest T2 generation seeds from individual plants. Different individual plants of the T1 generation are different transgenic lines. 8) After sowing T2 generation seeds, manage them normally. Before harvest, check the green fluorescence segregation ratio of the seeds using stereofluorescence assay. Seeds without segregation can be considered homozygous transgenic plants (e.g., Figure 3As shown, Arabidopsis thaliana lines overexpressing the BnCDY201 gene were obtained.

[0023] 2.5 Investigation on the culture and flowering time of Arabidopsis thaliana overexpressing the BnCDY201 gene Seeds of wild-type and two Arabidopsis thaliana lines overexpressing the BnCDY201 gene were selected, disinfected twice with 12% Kao solution for 5 min each time, and rinsed 8-10 times with sterile water for 3 min each time. The seeds were then sown on 1 / 2 MS solid medium and cultured for 10 days under the photoperiod of 14 h light and 10 h dark. The Arabidopsis thaliana seedlings with good growth were then transferred to nutrient soil and cultured under the photoperiod of 14 h light and 10 h dark at 25℃. The number of rosette leaves and flowering time were counted under the same number of growth days.

[0024] like Figure 4 As shown, the wild-type Arabidopsis thaliana WT had the fewest rosette leaves, while the Arabidopsis thaliana lines OE-1 and OE-2, which overexpressed the BnCDY201 gene, had significantly more rosette leaves than the wild-type Arabidopsis thaliana WT. However, no obvious bolting or flowering was observed, indicating that the overexpression of the BnCDY201 gene delayed the flowering time of Arabidopsis thaliana and accumulated more rosette leaves during flowering, indicating that the vegetative growth period was prolonged.

[0025] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the protection scope of the present invention.

Claims

1. A rapeseed BnCDY201 gene, characterized in that, The nucleotide sequence of this gene is shown in SEQ ID NO.

1.

2. The protein encoded by the rapeseed BnCDY201 gene as described in claim 1, characterized in that, The amino acid sequence of the protein encoded by the rapeseed BnCDY201 gene is shown in SEQ ID NO.

2.

3. The application of the rapeseed BnCDY201 gene as described in claim 1 in regulating the flowering and vegetative growth periods of cruciferous plants.

4. The application according to claim 2, characterized in that, Overexpression of the BnCDY201 gene in rapeseed can delay the flowering period of cruciferous plants and prolong their vegetative growth period.

5. The application according to claim 2, characterized in that, The cruciferous plants mentioned are Arabidopsis thaliana and rapeseed.

6. A recombinant plasmid, characterized in that, It was obtained by transforming the rapeseed BnCDY201 gene as described in claim 1 into a vector.

7. A recombinant plasmid according to claim 6, characterized in that, The vector is pCAMBIA1305-EGFP.

8. A method for obtaining a transgenic late-flowering and long vegetative growth period cruciferous plant variety, characterized in that, The rapeseed BnCDY201 gene of claim 1 was introduced into the genome of a cruciferous plant for overexpression, and transgenic late-flowering and long vegetative growth period plant varieties were obtained. The plants mentioned are Arabidopsis thaliana and rapeseed.

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