Application of BnWUS gene in regulating cell differentiation and leaf development of Brassica napus

Abstract

The application discloses application of BnWUS in regulating cell differentiation and leaf shape development of Brassica napus. The DNA nucleotide sequence of the BnWUS gene is shown as SEQ ID NO. 1, the sequence length is 951 bp, and the encoded amino acid sequence is shown as SEQ ID NO. 2. Functional analysis of Brassica napus genetic transformation shows that BnWUS is involved in regulating cell differentiation and leaf shape development of Brassica napus, overexpression of BnWUS can realize regulation of the leaf shape of Brassica napus, and the yield per plant is improved, thereby providing a theoretical basis for plant type improvement breeding of Brassica napus.

Description

Technical Field

[0001] This invention relates to the application of the BnWUS gene in regulating cell differentiation and leaf development in Brassica napus and to a method for transforming Brassica napus using the BnWUS gene. Background Technology

[0002] Rapeseed (Brassica napus L.) is the world's third largest oilseed crop and holds a vital position in my country's oilseed industry. Brassica napus, as the main cultivated variety in my country, is the largest source of domestically produced vegetable oil, accounting for approximately 50% of the country's total vegetable oil production (Liu Cheng, Feng Zhongchao, Xiao Tanghua, et al. Current Status, Potential and Countermeasures of Rapeseed Industry Development in my country. Chinese Journal of Oil Crops, 2019, 41:485-489). Therefore, the development of the rapeseed industry is particularly important for ensuring my country's oilseed supply security. Leaves are the main site for photosynthesis and transpiration in plants, playing a crucial role in crop growth, development, yield, and quality. Moderate leaf curling can reduce sunlight radiation and decrease transpiration and water loss under water-scarce conditions, thereby improving plant stress resistance. It can also reduce the planting area per plant, increasing crop planting density and yield per unit area (Yang M, Huang C, Wang M, et al. Fine mapping of anup-curling leaf locus (BnUC1) in Brassica napus. BMC Plant Biol, 2019, 19:324). WUS is a typical transcription factor encoded by the WOX gene family. In addition to containing a conserved homeobox domain, its C-terminus includes an activating acidic domain WUS-box (TLPLFPMH) and a conserved repressive domain (EAR-like motif). This unique protein structure also allows WUS to play different functions in plant growth and development (Ikeda M, Mitsuda N, Ohme-Takagi M. Arabidopsis WUSCHEL is a bifunctional transcription factor that acts as a repressor in stem cell regulation and as an activator in floral patterning. Plant Cell, 2009, 21: 3493-3505). It holds promise for increasing rapeseed yield. Summary of the Invention

[0003] Purpose of the invention: One purpose of this invention is to provide the application of the BnWUS gene of Brassica napus in regulating cell differentiation and leaf development of Brassica napus. Another purpose of this invention is to provide a method for increasing the yield per plant by transforming Brassica napus using the BnWUS gene of Brassica napus.

[0004] Technical Solution: The application of the BnWUS gene from Brassica napus in regulating cell differentiation and / or leaf development in Brassica napus. This BnWUS gene has the ability to regulate cell differentiation and leaf development in Brassica napus.

[0005] This invention relates to the application of the expression cassette, recombinant vector, recombinant microorganism, or transgenic cell line of the BnWUS gene in regulating cell differentiation and / or leaf shape development in Brassica napus. Phenotypes related to cell differentiation and / or leaf shape development include callus size and adventitious bud regeneration, leaf parameters, and / or leaf epidermal cell parameters. Leaf parameters include leaf length, leaf width, and / or leaf length; leaf epidermal cell parameters include the length, width, area, and / or density of leaf epidermal cells. Overexpression of the BnWUS gene leads to more vigorous callus proliferation and larger callus area in Brassica napus, a significant increase in the number of buds formed, smaller leaf width, and increased leaf length / width and leaf length / width ratios, resulting in slender leaves with outward curling. Overexpression of the BnWUS gene also significantly increases the length, width, and area of ​​leaf epidermal cells in Brassica napus, while reducing the cell density on the adaxial surface. Overexpression of the BnWUS gene significantly increased the content of IAA methyl ester (MeIAA), indolebutyric acid (IBA), and zeatin in Brassica napus.

[0006] The application of the BnWUS gene of Brassica napus in the genetic breeding of Brassica napus, as described in this invention, is that overexpression of the BnWUS gene can increase the yield per plant of Brassica napus.

[0007] The application of the expression cassette, recombinant vector, recombinant microorganism or transgenic cell line of the Brassica napus BnWUS gene described in this invention in the genetic breeding of Brassica napus.

[0008] The nucleotide sequence of the BnWUS gene described in this invention is shown in SEQ ID NO.1, with a sequence length of 951 bp. The amino acid sequence encoded by the BnWUS gene is shown in SEQ ID NO.2.

[0009] This invention also includes a method for cultivating transgenic Brassica napus, comprising the following steps:

[0010] (1) Cloning the DNA fragment of the BnWUS gene from Brassica napus;

[0011] (2) The plasmid containing the DNA fragment of the Brassica napus BnWUS gene was transformed into Agrobacterium using an electroporation method;

[0012] (3) Agrobacterium carrying the transformation plasmid was used to transform the target plant to obtain transgenic rapeseed.

[0013] In step (1), when cloning the DNA fragment of the BnWUS gene of Brassica napus, RT-PCR is used with F primer and R primer. The sequence of F primer is shown in SEQ ID NO.3, and the sequence of R primer is shown in SEQ ID NO.4.

[0014] The present invention provides a method for cultivating transgenic Brassica napus, which aims to increase the content of mRNA in the target Brassica napus, resulting in transgenic Brassica napus with cell differentiation and leaf morphological development phenotypes that are significantly different from those of the target Brassica napus.

[0015] Expression vectors carrying the DNA fragment containing BnWUS of this invention can be introduced into plant cells using conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation transformation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition)).

[0016] The host can be transformed using an expression vector containing the DNA fragment of BnWUS included in this invention to cultivate plant germplasm with different cell differentiation capabilities and leaf shapes.

[0017] The DNA fragment containing BnWUS was recovered using a DNA recovery kit. The fragment was then ligated into the pCAMBIA1300-FLAG backbone vector using an enzyme digestion and ligation method to construct an overexpression vector for the DNA fragment, named pCAMBIA1300-FLAG-BnWUS.

[0018] The pCAMBIA1300-FLAG-BnWUS vector was introduced into Agrobacterium tumefaciens strain GV3101 using electroporation. Agrobacterium infection-mediated transformation was then used to transform the pCAMBIA1300-FLAG-BnWUS vector into the Brassica napus recipient material J9712, successfully obtaining transgenic plants with significantly increased BnWUS expression compared to the wild type. Observations revealed that, compared to wild-type plants, the BnWUS-overexpressing transgenic Brassica napus exhibited more vigorous callus proliferation, longer and thinner leaves, and outward leaf curling, indicating that BnWUS can regulate cell differentiation and leaf development in Brassica napus.

[0019] This invention uses Brassica napus (Brassica napus) as the research material, isolates the BnWUS DNA fragment from Brassica napus, identifies the function of BnWUS in regulating cell differentiation and leaf development in Brassica napus, transforms it into wild-type Brassica napus plants using Agrobacterium-mediated transformation, and studies its biological functions within the plant. This provides a solid foundation for the application of BnWUS in the genetic breeding of Brassica napus and will be of great significance for accelerating the improvement of plant genetic breeding.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0021] This invention analyzes the callus differentiation ability and leaf shape of transgenic Brassica napus overexpressing BnWUS, and discovers a gene BnWUS that can regulate cell differentiation and leaf development in Brassica napus. This allows for the regulation of rapeseed leaf shape and increases the yield per plant, providing a theoretical basis for rapeseed plant type improvement breeding. Attached Figure Description

[0022] Figure 1 The figure shows the qPCR analysis results of BnWUS expression level in Brassica napus overexpressing BnWUS. J9712 is the transgenic recipient material of Brassica napus, and OE-BnWUS-3, OE-BnWUS-4, OE-BnWUS-7, and OE-BnWUS-8 are different Brassica napus lines overexpressing BnWUS.

[0023] Figure 2 The images show callus tissues of wild-type J9712 and BnWUS-overexpressing Brassica napus hypocotyls. In the images, A shows the callus phenotype (Bar=1 cm) after 23 days of culture on differentiation medium using hypocotyls of wild-type J9712 and BnWUS-overexpressing line (OE-BnWUS-4) as explants, and B shows the measured callus area of ​​wild-type J9712 and BnWUS-overexpressing line.

[0024] Figure 3This image shows a morphological comparison between wild-type J9712 and BnWUS-overexpressing lines. A represents the phenotypic image of 7-week-old seedlings (Bar = 5 cm), and B represents the phenotypic image of leaves (Bar = 2 cm). C represents the leaf width of wild-type J9712 and BnWUS-overexpressing materials, D represents the leaf length to leaf width ratio of wild-type J9712 and BnWUS-overexpressing materials, and E represents the leaf length to leaf width ratio of wild-type J9712 and BnWUS-overexpressing materials. Leaf length (LL); Leaf width (LW); Bladelength (BL); n = 6; *, p < 0.05; **, p < 0.01.

[0025] Figure 4 Scanning electron microscopy (SEM) images of cells from different locations (middle and edge) of the near- and far-axial surfaces of leaves from BnWUS-overexpressing and control plants are shown, along with statistical analysis of cell length, width, area, and density. In the images, A is a schematic diagram of leaf sampling; B is a scanning electron micrograph of epidermal cells from wild-type J9712 and BnWUS-overexpressing rapeseed leaves (Bar = 20 μm); and C shows the statistical analysis of the length, width, area, and density of leaf epidermal cells. Values ​​are expressed as mean ± standard error, n = 5; *, p < 0.05; **, p < 0.01.

[0026] Figure 5 Figure 1 shows the endogenous hormone content in leaves of BnWUS-overexpressing plants and the control J9712. A represents the content of methyl indoleacetate, B represents the content of indolebutyric acid, and C represents the content of zeatin. Values ​​are expressed as mean ± standard error, n = 3; *, p < 0.05; **, p < 0.01. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] The following embodiments define the present invention and describe the methods for cloning DNA fragment sequences containing BnWUS and verifying the function of BnWUS. Based on the following description and these embodiments, those skilled in the art can determine the essential features of the invention and can make various changes and modifications to adapt it to different uses and conditions without departing from the spirit and scope of the invention.

[0029] Example 1: Molecular cloning of the BnWUS DNA fragment from Brassica napus

[0030] Seedlings of the Brassica napus variety "Darmor-bzh" at the three-leaf-one-bud stage were flash-frozen in liquid nitrogen and stored at -70°C for total RNA extraction. Total RNA was extracted using the Vazyme RNA Isolater Total RNA Extraction Reagent kit. First-strand synthesis of Brassica napus cDNA was performed according to the instructions of the HisScriptIII RT SuperMix for qPCR (+gDNA wiper) from Nanjing Novizan Biotechnology Co., Ltd. Using the first strand of cDNA as a template, and the designed F primer: 5'-CTTGAAATGGAGCAACCGC-3' (SEQ ID NO.3) and R primer: 5'-GATAAAGATGGCGAAAGGGAG-3' (SEQ ID NO.4) as primers, DNA amplification was performed according to the instructions of Vazyme's Phanta Max Super-Fidelity DNA Polymerase. The reaction system consisted of 2×Phanta Max Buffer 25 μL, Primer-F (10 μM) 2 μL, Primer-R (10 μM) 2 μL, dNTP Mix (10 mM) 1 μL, cDNA 1 μL, and ddH2O added to a final volume of 50 μL. The amplification conditions were: 95℃ for 5 min, 95℃ for 30 s, 59℃ for 30 s, 72℃ for 30 s, for a total of 35 cycles; followed by 72℃ for 5 min. After PCR, electrophoresis analysis was performed, and the target amplified fragment was recovered using a DNA recovery kit from Kangwei Century Biotechnology Co., Ltd. The amplified fragment was ligated into the pEASY-BluntT vector from Beijing TransGen Biotech Co., Ltd., transformed into E. coli competent cells DH5α, and white colonies were picked for PCR identification of positive clones. Positive clones were sent to Yangzhou Qingke Biotechnology Co., Ltd. for sequencing. The plasmid confirmed by sequencing was named BnWUS-T. The cloned DNA fragment containing the BnWUS gene was 951 bp in length, with the nucleotide sequence shown in SEQ ID NO.1 and the amino acid sequence shown in SEQ ID NO.2.

[0031] SEQ ID NO.1

[0032] ATGGAGCAACCGCAACATCATCACCATCACCATCAACAAACCGACCAAGAGAGCGGTAACAACAATAAGTCCGGCTCTGGTGGTTACCCATGTCGCCAAACGAGCACAAGATGGACACCAACGACGGAGCAGATCAGAATCCTCAAAGATCTCTACTACAACAACGGAGTTCGGTCACCAACAGCCGACCAGATCCAGAAGATCTCTGCACGGCTGAGACAGTACGGAAAGATCGAGGGTAAGAACGTCTTTTACTGGTTCCAGAACCATAAGGCTCGCGAGCGACAGAAGAAGAGATTCAACGGCACAACCATGACCACACCAACAACTTCATCCCCCAACTCGGTTATGATGGCCAACGACCATTATCATCATAACCATCATCCTCTTCTTCAGCACCATCATCATGGTCACCATCATCATGGTGTTACCATGCATAGGCCTGCTTCGGTCAATATTAAGCTTGACCAAGAAAATCATCTCCTTCATCAGAACATATCTTATCCCAACTTTCAGAACGGGAATTTAAATCATGCAAGTTCAGGCGCTGAATATGGTGCTCTTAATGCTTCTTCTAATGGCTACATGAGTAGCCATCTCTATGGATCTATGGAACAAGATTATTCAGTGAGCTACAACAACGTAAGTGGAGGATGGACAAACATGGATCATAATCATCATTACTCAGCTCCAGCTTACAACTTCTTCGATAGACCAAAGCCTCTGTCTGGACTGGAAGATCATGAAGAAGAAGAATATGGTGGCGATGCTTATCTGGAACATAGTCGTACACTTTCCCTCTTCCCTATGCACGGTGAGGATAACATCAACGGCGGTGGTGGTGCCTTTTTGAAGTACGGACAATCGGACGGTCGTGATTATTATGGTAGAGGCTCTTGTGCTTCTCTCAAGTTATGTTTAAACTCCTACGCAGGCGTCTCACCGGATTAA;

[0033] SEQ ID NO.2

[0034] MEQPQHHHHHHQQTDQESGNNNKSGSGGYPCRQTSTRWTPTTEQIRILKDLYYNNGVRSPTADQIQKISARLRQYGKIEGKNVFYWFQNHKARERQKKRFNGTTMTTPTTSSPNSVMMANDHYHHNHHPLLQHHHHGHHHHGVTMHRPASVNIKLDQEN HLLHQNISYPNFQNGNLNHASSGAEYGALNASSNGYMSSHLYGSMEQDYSVSYNNVSGGWTNMDHNHHYSAPAYNFFDRPKPLSGLEDHEEEEYGGDAYLEHSRTLSLFPMHGEDNINGGGGAFLKYGQSDGRDYYGRGSCASLKLCLNSYAGVSPD*.

[0035] Example 2: Construction of BnWUS overexpression vector

[0036] To better analyze the function of the BnWUS gene, it was overexpressed in Brassica napus, and the function of the gene was studied by observing the phenotype of transgenic Brassica napus. The overexpression vector was constructed as follows: Using the BnWUS-T plasmid, which was verified by sequencing, as a template, primers WUS-F (5'-GGTACCATGGAGCAACCGCAAC-3') (SEQ ID NO.7, sequence-specific primer with adapter Kpn I site) and WUS-R (5'-CTGCAGATCCGGTGAGACGCCT-3') (SEQ ID NO.8, sequence-specific primer with adapter Pst I site) were used for DNA amplification by PCR. The reaction system was: 25 μL of 2×PhantaMax Buffer, 2 μL of Primer-F (10 μM), 2 μL of Primer-R (10 μM), 1 μL of dNTP Mix (10 mM), 1 μL of Template DNA, and ddH2O added to a final volume of 50 μL. The amplification conditions were: 95℃ for 5 min, 95℃ for 30 s, and 59℃ for 30 s. The PCR process was performed at 72℃ for 1 min 30 s, for a total of 35 cycles; then at 72℃ for 5 min. Electrophoresis analysis was performed after PCR, and the target fragment was recovered using a DNA recovery kit from Wanhe Biotechnology Co., Ltd. The amplified fragment was ligated into the pCAMBIA1300-FLAG backbone vector, which had been double-digested with Kpn I and Pst I, to construct an overexpression vector of BnWUS, named pCAMBIA1300-FLAG-BnWUS.

[0037] Example 3: Genetic transformation of Brassica napus using the pCAMBIA1300-FLAG-BnWUS overexpression vector

[0038] A single colony of Agrobacterium tumefaciens GV3101 was picked and inoculated into 25 mL of YEB medium (containing 50 mg / L rifampin) and cultured overnight. 5 mL of the bacterial culture was then transferred to 100 mL of YEB medium (containing 50 mg / L rifampin) and cultured until OD500 reached. 600 = 0.7-0.8. Place the bacterial suspension on ice for 10 min, centrifuge at 5000 rpm at 4℃ for 10 min to collect the bacterial cells, and wash twice with 100 mL of sterile double-distilled water. Resuspend the bacterial cells in 4 mL of 10% glycerol and transfer to a 50 mL centrifuge tube. Centrifuge at 5500 rpm at 4℃ for 10 min to collect the bacterial cells, resuspend the bacterial cells in 500 µL of 10% glycerol, and transfer to a 1.5 mL centrifuge tube. Store the competent cells of Agrobacterium tumefaciens GV3101 strain at -70℃ for later use.

[0039] The pCAMBIA1300-FLAG-BnWUS plasmid was introduced into competent cells of Agrobacterium tumefaciens GV3101 using electroporation. 50 µL of Agrobacterium tumefaciens GV3101 competent cells were mixed with 5 µL of the pCAMBIA1300BnWUS-FLAG-BnWUS recombinant plasmid and transferred to a 0.1 cm electroporation cuvette. Electroporation parameters were 200 Ω, 1.7 kV, 2.5 F. Immediately after electroporation, 500 µL of LB medium was added. After incubation at 28°C and 220 rpm for 1 h, 100 µL of the bacterial culture was spread onto LB medium containing kanamycin resistance to screen transformants, and incubated at 28°C for 16 h.

[0040] The genetic transformation method for Brassica napus was an improved version of the transformation method developed by the State Key Laboratory of Crop Genetic Improvement at Huazhong Agricultural University (Dai C, Li Y, Li L, Du Z, Lin S, Tian X, Li S, Yang B, Yao W, Wang J, Guo L, Lu S. An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus. MolBreed, 2020, 40: 96). Hypocotyls of sterile Brassica napus seedlings were used as explants, and the genetic transformation of exogenous fragments in Brassica napus was achieved using Agrobacterium-mediated transformation.

[0041] The culture medium formula used is as follows:

[0042] YEB medium: 5 g / L beef extract + 1 g / L yeast extract + 5 g / L peptone + 5 g / L sucrose + 4 g / L MgSO₄ 4· 7H2O (pH=7.4).

[0043] Inoculation medium (M0): MURASHIGE & SKOOG MEDIUM (Duchefa Biochemie) + 30 g / L sucrose + 8 g / L agar (pH=5.8-6.0).

[0044] Co-culture medium (M1): M0 + 18 g / L mannitol + 1 mg / L 2,4-dichlorophenoxyacetic acid 2,4-D + 0.3 mg / L kinetin + 100 μM acetylsalicylic acid AS (pH=5.8).

[0045] Callus differentiation medium (M2): M1 + 300 mg / L Timentin + 25 mg / L Hygromycin B.

[0046] Seedling culture medium (M3): MURASHIGE & SKOOG MEDIUM (Duchefa Biochemie) + 10 g / L glucose + 0.25 g / L xylose + 0.6 g / L morpholine ethanesulfonic acid (MES) + 2 mg / L zeatin + 0.1 mg / L indoleacetic acid (IAA) + 300 mg / L timentin + 25 mg / L hygromycin B.

[0047] Seedling rooting medium (M4): M0 + 300 mg / L Timentin.

[0048] MURASHIGE & SKOOG MEDIUM is abbreviated as MS medium.

[0049] The specific steps are as follows:

[0050] (1) Sterilization:

[0051] a. First, soak the seeds of wild-type Brassica napus J9712 in 75% alcohol for 1 minute. Note that the soaking time should not be too long.

[0052] b. Then disinfect with 2% sodium hypochlorite for 20 minutes;

[0053] c. Finally, rinse the wild-type rapeseed J9712 seeds with sterile water 4-5 times to clean them as thoroughly as possible.

[0054] (2) Sowing:

[0055] a. Using sterile forceps, sow 30 sterilized wild-type Brassica napus seeds J9712 onto M0 medium.

[0056] b. Place the inoculated culture jars into an incubator and incubate in the dark at 24°C for 6-7 days.

[0057] (3) Shaking the fungus:

[0058] Five to six days after inoculation, Agrobacterium GV3101 is inoculated into sterile Erlenmeyer flasks or centrifuge tubes containing LB liquid medium and cultured in a shaker at 28°C and 180-220 rpm.

[0059] (4) Explant preparation and infection:

[0060] a. Using sterile forceps and a scalpel, cut seedlings that have grown for 6-7 days after sowing, and cut their hypocotyls into explant segments 0.8-1 cm in length. Placing the hypocotyl in M1 liquid culture medium during cutting will improve the cutting results. Cut quickly and accurately, without dragging.

[0061] b. Measure the OD of Agrobacterium. 600 OD value (in LB medium) 600 =0.3 is preferred). Centrifuge the pre-cultured bacterial solution at 6000 rpm for 10 min, discard the supernatant, and resuspend the bacterial solution in an equal volume of MS liquid medium containing 100 μM acetylsuccinone AS. Repeat this process once more. Finally, take 2 mL of the bacterial solution and dilute it with 20 mL of MS liquid medium containing 100 μM AS.

[0062] c. Place the cut explants in a bacterial suspension of adjusted concentration and immerse for 10 minutes. Note that the immersion time should not be too long, otherwise it will cause the explants to die. Immersing 150-200 explants per 20 mL of bacterial suspension is appropriate.

[0063] (5) Transfer the infected explants to M1 medium, with 20-25 explants per plate, and incubate at 24°C in the dark for 36-48 h;

[0064] (6) Transfer the explants from M1 to M2 medium and then to a light incubator for 3 weeks (24℃, 16 h light / 8 h dark).

[0065] (7) Transfer the explants to M3 medium and subculture them every 2-3 weeks until green shoots appear;

[0066] (8) Finally, the explants are transferred to M4 medium to root, which takes 2-4 weeks.

[0067] Example 4: Molecular identification of transgenic Brassica napus

[0068] Genomic DNA was extracted from the rapeseed cultured in Example 3 using a rapid plant DNA extraction method. The specific steps are as follows:

[0069] (1) Take two tender leaves (about 0.2 g), cut them into pieces and put them into a 2 mL centrifuge tube. Add 250 µL DNA buffer (500 mM Tris-HCl, 300 mM NaCl, 300 mM Sucrose, pH=7.5) and two steel balls (6.7 mm in diameter). Use a sampler to crush the leaf sample at 50 Hz for 180 s.

[0070] (2) Incubate the crushed sample at 95°C for 10 min;

[0071] (3) Remove the sample and cool it to room temperature, then centrifuge at 12,000 rpm for 5 min;

[0072] (4) Transfer 50 µL of supernatant to a new 1.5 mL centrifuge tube and dilute it 5 times for later use.

[0073] Use 1 µL of DNA as a template and perform PCR amplification using primers 1300-35S-F (5'-GAGAGACACGGGGGACG-3') (SEQ ID NO. 5) and 1300-35S-R (5'-ACTCTTCTCCATCCATTTCC-3') (SEQ ID NO. 6) according to the instructions of Vazyme's 2×Taq Master Mix (Dye Plus). The amplification system consisted of 10 μL of 2×Taq Master Mix (Dye Plus), 0.5 μL of Primer-F (10 μM), 0.5 μL of Primer-R (10 μM), 1 μL of Template DNA, and ddH2O to a final volume of 20 μL. The amplification conditions were: 95℃ for 5 min, 95℃ for 30 s, 59℃ for 30 s, 72℃ for 1 min, for a total of 35 cycles; followed by 72℃ for 5 min. Using transgenic rapeseed DNA as a template, a specific target fragment can be amplified, proving that the target vector pCAMBIA1300-FLAG-BnWUS has been integrated into the rapeseed genome.

[0074] The expression of BnWUS in transgenic Brassica napus was detected by RT-PCR. The detection method is as follows: RNA was extracted using the Vazyme RNA Isolater Total RNA Extraction Reagent Kit, and the expression level of BnWUS was detected by qRT-PCR using the HisScriptIII RT SuperMix for qPCR (+gDNA wiper) and 2 × Taq Master Mix (DyePlus). The upstream primer was qBnWUS-F: 5'-CGTACACTTTCCCTCTTCCCTA-3' (SEQ ID NO.9), and the downstream primer was qBnWUS-R: 5'-AGAGCCTCTACCATAATAATCACG-3' (SEQ ID NO.10). BnaActin was used as an internal control gene. The upstream primer used was qBnaActin-F: 5'-TCTTCCTCACGCTATCCTCCG-3' (SEQ ID NO.11), and the downstream primer was qBnaActin-R: 5'-AGCCGTCTCCAGCTCTTGC-3' (SEQ ID NO.12). PCR amplification was performed according to the instructions of Vazyme's 2 × Taq Master Mix (Dye Plus). The amplification system consisted of 10 μL of 2 × Taq Master Mix (Dye Plus), 0.5 μL of Primer-F (10 μM), 0.5 μL of Primer-R (10 μM), 1 μL of Template DNA, and ddH2O to a final volume of 20 μL. The amplification conditions were: 95℃ for 5 min, 95℃ for 30 s, 59℃ for 30 s, 72℃ for 1 min, for a total of 28 cycles; followed by a final 5 min at 72℃. The results are as follows: Figure 1 The results showed that transgenic plants with significantly increased BnaWUS gene expression levels compared to wild-type Brassica napus were successfully obtained. J9712 was the transgenic recipient material of Brassica napus, and OE-BnWUS-3, OE-BnWUS-4, OE-BnWUS-7, and OE-BnWUS-8 were different overexpression lines of BnaWUS in Brassica napus.

[0075] Example 5

[0076] 1. Overexpression of BnWUS can promote callus formation and adventitious shoot regeneration in the hypocotyl of Brassica napus.

[0077] Explants of wild-type Brassica napus plants J9712 and BnaWUS overexpression lines, which were transferred to M3 medium during the aforementioned rapeseed genetic transformation steps, were observed regularly, and callus formation and adventitious shoot regeneration were recorded. Results are as follows: Figure 2 Compared with the wild-type control J9712, the explants of BnWUS-overexpressing lines showed more vigorous callus proliferation, larger callus area, and a significantly increased number of buds, producing numerous dark green, densely clustered adventitious bud structures, exhibiting a typical multi-bud morphology and demonstrating an enhanced bud regeneration phenotype. Most of these adventitious buds were in the meristematic initiation and early leaf primordium development stages, indicating that BnWUS overexpression can strongly induce the establishment of bud meristems and the formation of leaf primordia.

[0078] 2. Overexpression of BnWUS resulted in long, thin, and outward-curling leaves in Brassica napus.

[0079] Phenotypic analysis was performed on transgenic plants overexpressing BnWUS. Seven-week-old wild-type plants J9712 and overexpressing plants OE-BnWUS were selected, and their leaf length (LL), leaf width (LW), and blade length (BL) were measured. The leaf length / leaf width (LL / LW) and blade length / leaf width (BL / LW) ratios were calculated. It was found that the leaf width of the transgenic rapeseed overexpressing BnWUS was significantly lower than that of the wild type, while the leaf length / leaf width and blade length / leaf width ratios were significantly higher than those of the wild type, showing that the leaves were slender and rolled outwards. Figure 3 ).

[0080] To further explain why BnWUS overexpression causes leaf curling in Brassica napus, we sampled and observed different parts (middle and edge) of the axillary and abaxial surfaces of leaves from wild-type plant J9712 and BnWUS-overexpressing plants using scanning electron microscopy. We also performed statistical analysis on cell length, width, area, and density. The specific steps are as follows:

[0081] (1) Preparation of fixative. 0.2 M phosphate-buffered saline (PBS) (pH=7.2): Stock solution A: 0.2 M Na2HPO4 (71.64 g Na2HPO4·12H2O, diluted to 1 L with distilled water); Stock solution B: 0.2 M NaH2PO4 (31.21 g NaH2PO4·2H2O, diluted to 1 L with distilled water). Solution A: Solution B = 72: 28 (v / v), adjust pH to 7.2. Fixative: 2.5% glutaraldehyde (GA) solution + 0.1 M PBS, i.e., 50 mL 0.2 M PBS + 10 mL 25% GA + 40 mL H2O.

[0082] (2) Cut fresh leaves (0.25 cm) 2The sample was completely immersed in the fixative and fixed at 4°C for more than 2 days. It was then dehydrated in a gradient of 30%, 50%, 70%, 80%, 90%, 95%, 100%, and 100% (with anhydrous Na2SO4) ethanol for 10-15 min at 4°C. After drying, it was attached to the sample stage and sputtered with gold. The sample was then observed using a Zeiss Gemini SEM 300 scanning electron microscope.

[0083] Figure 4 The results showed that the length, width, and area of ​​the leaf epidermal cells in BnWUS-overexpressing plants were significantly larger than those in the wild-type J9712. While the cell density on the abaxial plane was not significantly different from the wild-type, the cell density on the adaxial plane was significantly lower. These results indicate that BnWUS overexpression in Brassica napus leads to an increase in leaf epidermal cell area and a decrease in adaxial cell density, thus affecting leaf shape development.

[0084] 3. Overexpression of BnWUS affects the content of endogenous hormones in the leaves of Brassica napus.

[0085] Based on the transcriptome sequencing analysis of leaves from wild-type plant J9712 and overexpressing plant OE-BnWUS, a large number of differentially expressed genes were found to be enriched in plant hormone signal transduction pathways. Simultaneously, the aforementioned morphological analysis showed changes in the size of leaf epidermal cells in BnWUS-overexpressing plants. To further explain the reasons for the changes in leaf morphology caused by BnWUS overexpression in Brassica napus, this invention further determined the endogenous hormone content in the leaves of BnWUS-overexpressing plants and wild-type plant J9712. The specific steps are as follows:

[0086] (1) BnWUS transgenic plants and control plants (J9712) were planted in the experimental field in Yangzhou. The leaves of different lines of 7-week-old seedlings were sampled, and 3 plants of each line were selected for mixed sampling. The samples were frozen and stored in a -80℃ freezer.

[0087] (2) Take 2 mL of the imported EP tube, weigh it accurately, and record the weight of the EP tube;

[0088] (3) Take out the sample frozen at -80℃ and place it in liquid nitrogen. Weigh it quickly (100 mg) (Note: wipe the moisture off the surface of the EP tube). Record the accurate weight (accurate to the thousandths place) and then place it in liquid nitrogen quickly.

[0089] (4) Add alcohol-burned steel balls to each EP tube and grind the sample for 180 s (50 Hz).

[0090] (5) Add 500~1000 μL of a mixed solution of isopropanol, ddH2O and concentrated hydrochloric acid in a volume ratio of 2:1:0.002 (the volume ratio of sample to solvent is 1:10).

[0091] (6) Add 1 mL of dichloromethane (add reagent on ice), and shake at 4°C for 30 min;

[0092] (7) Centrifuge at 13000 g, 4℃ for 5 min, transfer the supernatant to a 2 mL injection bottle, dissolve the residue in 500 μL of dichloromethane, repeat centrifugation, combine the supernatants and then blow with nitrogen; the angle of the nitrogen blowing needle needs to be carefully adjusted throughout the process to prevent the liquid from being blown out.

[0093] (8) Add 400 μL of a 1:1 mixture of methanol and 0.1% formic acid to the sample vial to dissolve the hormone. After dissolving, filter the solution through an organic filter membrane to obtain the extracted hormone.

[0094] The obtained hormone samples were detected by a 600 Plus Qtrap liquid chromatography-mass spectrometry system. For specific methods and procedures, please refer to the literature report (Jiao Q, Chen T, Niu G, et al. N-glycosylation is involved in instomatal development by modulating the release of active abscisic acid and auxin in Arabidopsis. J Exp Bot, 2020, 71(19): 5865-5879).

[0095] Figure 5 The results showed that the contents of IAA methyl ester (MeIAA), indolebutyric acid (IBA), and zeatin in BnWUS-overexpressing plants were significantly higher than those in wild-type plants. This indicates that BnWUS overexpression may lead to changes in leaf shape in Brassica napus by affecting the contents of endogenous auxins and cytokinins. MeIAA, as a product of IAA methylation, can be converted back to IAA under certain conditions; IBA is generated from IAA but has higher stability than IAA; zeatin is the main naturally occurring active component of cytokinins in plants, and its content can reflect the overall cytokinin content in the plant.

[0096] Example 6: Overexpression of BnWUS can increase the yield per plant of Brassica napus.

[0097] Wild-type control rapeseed J9712 and BnWUS overexpression lines were planted in a transgenic experimental field, with row spacing of 30 cm and plant spacing of 20 cm. Two plots were planted, and at maturity, plant height, branching point height, number of branches, main inflorescence axis length, number of siliques on the main inflorescence axis, number of siliques per plant, silique length, number of seeds per silique, and yield per plant were recorded. The results are shown in Table 1. Overexpression of BnWUS significantly increased the yield per plant of rapeseed. Among them, the yield per plant of the overexpression line OE-BnWUS-3 increased by 21% compared with the control J9712, and the yield per plant of OE-BnWUS-7 increased by 12% compared with the control J9712.

[0098] Table 1 shows the agronomic traits of Brassica napus overexpressing BnWUS.

[0099]

[0100] Numerical values ​​are expressed as mean ± standard error, n = 4; *, p < 0.05; **, p < 0.01.

[0101] This invention isolates the gene BnWUS, which regulates cell differentiation and leaf shape development in Brassica napus. This provides theoretical guidance for research on plant leaf shape-related genes and their molecular functions. The leaf shape-related gene isolated in this invention originates from the plant itself, minimizing environmental impact. Utilizing this isolated gene to study the genetic mechanisms of leaf morphology is significant for breeding and improving rapeseed plant type traits. The above embodiments are merely illustrative of the technical solutions of this invention and not intended to limit it. Although the invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; such modifications or substitutions do not cause the essence of the corresponding technical solutions to depart from the spirit and scope of the technical solutions claimed in this invention.

Claims

1. Application of the BnWUS gene in regulating cell differentiation and / or leaf development in Brassica napus.

2. Application of expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic cell lines of the BnWUS gene in regulating cell differentiation and / or leaf development in Brassica napus.

3. The application according to claim 1 or 2, characterized in that, Phenotypes related to cell differentiation and / or leaf shape development include callus size and adventitious bud regeneration, leaf parameters and / or leaf epidermal cell parameters.

4. The application according to claim 3 or as described above, characterized in that, Leaf parameters include leaf length, leaf width, and / or leaf length; leaf epidermal cell parameters include the length, width, area, and / or density of leaf epidermal cells.

5. Application of the BnWUS gene in the genetic breeding of Brassica napus.

6. Application of expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic cell lines of the BnWUS gene in the genetic breeding of Brassica napus.

7. The application according to any one of claims 1-6, characterized in that, The nucleotide sequence of the BnWUS gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the BnWUS gene is shown in SEQ ID NO.

2.

8. A method for cultivating transgenic Brassica napus, characterized in that, Includes the following steps: (1) Cloning the DNA fragment of the BnWUS gene from Brassica napus; (2) The plasmid containing the DNA fragment of the Brassica napus BnWUS gene was transformed into Agrobacterium using an electroporation method; (3) Agrobacterium carrying the transformation plasmid was used to transform the target plant to obtain transgenic rapeseed.

9. The method for cultivating transgenic Brassica napus as described in claim 8, characterized in that, In step (1), when cloning the DNA fragment of the BnWUS gene of Brassica napus, RT-PCR is used with F primer and R primer. The sequence of F primer is shown in SEQ ID NO.3 and the sequence of R primer is shown in SEQ ID NO.

4.

10. The method for cultivating transgenic Brassica napus as described in claim 8, characterized in that, In step (2), primers WUS-F and WUS-R are used to construct a BnWUS gene overexpression vector, and the BnWUS gene overexpression vector is introduced into competent cells of Agrobacterium tumefaciens GV3101 for transformation; wherein, the WUS-F sequence is shown in SEQ ID NO.7 and the WUS-R sequence is shown in SEQ ID NO.8.

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