Application of BnaTOE1 gene in regulating flowering time of Brassica napus and its identification using molecular marker primers

Abstract

This invention discloses the application of the BnaTOE1 gene in regulating the flowering time of Brassica napus and its molecular marker primers for identification. The DNA nucleotide sequence of the BnaTOE1 gene is shown in SEQ ID NO.1, with a sequence length of 1254 bp, and the encoded amino acid sequence is shown in SEQ ID NO.2. The molecular marker primers used to identify the flowering time of Brassica napus are shown in SEQ ID NO.15 and SEQ ID NO.16. Functional analysis of genetic transformation in Brassica napus shows that BnaTOE1 participates in regulating the flowering time of Brassica napus. The development of molecular markers is of great significance for promoting the genetic improvement and breeding of Brassica napus.

Description

Technical Field

[0001] This invention relates to the application of the BnaTOE1 gene in regulating the flowering time of Brassica napus and its molecular marker primers for identification, belonging to the field of genetic engineering of Brassica napus. Background Technology

[0002] Brassica napus L. is a major rapeseed variety cultivated in my country, characterized by high yield, strong resistance to adverse conditions, and wide adaptability. The earlier flowering time of Brassica napus implies a relatively shorter growth cycle, which helps complete the entire growth and development process in a shorter period, adapting to special climatic conditions in some regions, increasing the number of cropping cycles, and improving land utilization and agricultural productivity (Cui Y, Xu Z, Xu Q. Elucidation of the relationship between yield and heading date using CRISPR / Cas9 system-induced mutation in the flowering pathway across a large latitudinal gradient. Mol Breed, 2021, 41:23). Therefore, identifying key genes regulating flowering time and improving growth period traits is one of the important goals of genetic breeding of Brassica napus. TOE1 encodes an AP2 family protein transcription factor and is a target gene of miR172, involved in regulating the development of plant vegetative organs (Zhu QH, Upadhyaya NM, Gubler F, Helliwell CA. Overexpression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice (Oryza sativa). BMC Plant Biol, 2009, 9:149). Molecular marker-assisted breeding is an effective tool for crop genetic improvement. The development and application of high-throughput, low-cost molecular markers have greatly improved the efficiency and accuracy of crop molecular breeding, which is beneficial for accelerating the breeding of superior varieties of Brassica napus. Summary of the Invention

[0003] Objectives of the Invention: The first objective of this invention is to provide the application of the BnaTOE1 gene in regulating the flowering time of Brassica napus. The second objective of this invention is to provide a transgenic Brassica napus transformed using the BnaTOE1 gene. The third objective of this invention is to provide a method for transforming Brassica napus using the BnaTOE1 gene. The fourth objective of this invention is to provide a molecular marker primer for identifying the flowering period of Brassica napus. The fifth objective of this invention is to provide a method for identifying the flowering period of Brassica napus using the molecular marker primer.

[0004] Technical solution: The application of the BnaTOE1 gene described in this invention in regulating the flowering time of Brassica napus. This BnaTOE1 gene has the ability to regulate the flowering time of Brassica napus.

[0005] Furthermore, the DNA nucleotide sequence of the BnaTOE1 gene is shown in SEQ ID NO.1, with a sequence length of 1254 bp.

[0006] Furthermore, its encoded amino acid sequence is shown in SEQ ID NO.2.

[0007] The application of the BnaTOE1 gene expression cassette, recombinant vector, recombinant microorganism or transgenic cell line described in this invention in regulating the flowering time of Brassica napus or in the genetic breeding of Brassica napus.

[0008] A transgenic Brassica napus rapeseed is obtained by transforming Brassica napus rapeseed with the BnaTOE1 gene described in this invention.

[0009] A method for cultivating transgenic Brassica napus includes the following steps:

[0010] (1) Cloning a DNA fragment of the BnaTOE1 gene from Brassica napus;

[0011] (2) Plasmids containing DNA fragments of the BnaTOE1 gene from Brassica napus were transformed into Agrobacterium using an electroporation method;

[0012] (3) Agrobacterium carrying the transformation plasmid was transformed into the target Brassica napus using the transgenic method to obtain transgenic Brassica napus.

[0013] Furthermore, in step (1), when cloning the DNA fragment of the BnaTOE1 gene of Brassica napus, the DNA of the BnaTOE1 gene is amplified by RT-PCR using F primer and R primer. The sequence of the F primer is shown in SEQ ID NO.3, and the sequence of the R primer is shown in SEQ ID NO.4.

[0014] The provided method for cultivating transgenic Brassica napus aims to increase the mRNA content in the target Brassica napus, resulting in a flowering time morphology of the transgenic Brassica napus that is significantly different from that of the target Brassica napus.

[0015] Expression vectors carrying the DNA fragment containing BnaTOE1 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 BnaTOE1 DNA fragment included in this invention to cultivate plant germplasm with different flowering times.

[0017] The DNA fragment containing BnaTOE1 was recovered using a DNA recovery kit. The fragment was then ligated into the pMDC83 backbone vector using an enzyme digestion and ligation method to construct an overexpression vector for the DNA fragment, named pMDC83-BnaTOE1.

[0018] The pMDC83-BnaTOE1 vector was introduced into Agrobacterium tumefaciens strain GV3101 using electroporation. Agrobacterium infection-mediated transformation was then used to transform the pMDC83-BnaTOE1 vector into the Brassica napus recipient material J9712, successfully obtaining transgenic plants with significantly increased BnaTOE1 expression compared to the wild type. Observations showed that the flowering time of the BnaTOE1-overexpressing transgenic Brassica napus was significantly delayed compared to the wild-type plants, indicating that BnaTOE1 can regulate the flowering time of plants.

[0019] A molecular marker primer for identifying the flowering time of Brassica napus, the molecular marker primers being 11360-MF and 11623-MR, the sequence of primer 11360-MF being shown in SEQ ID NO.15, and the sequence of primer 11623-MR being shown in SEQ ID NO.16.

[0020] A method for identifying the flowering time of Brassica napus includes the following steps:

[0021] (1) Molecular marker primers 11360-MF and 11623-MR were designed based on the variation sites of the BnaTOE1 gene in early-flowering and late-flowering materials of natural populations of Brassica napus.

[0022] (2) Molecular identification of early-flowering and late-flowering materials of Brassica napus was carried out by PCR amplification using molecular marker primers 11360-MF and 11623-M.

[0023] Furthermore, in step (1), the mutation site of BnaTOE1 is that there is a C / T polymorphism at position 17011360 of the ChrC03 chromosome, and a small fragment sequence AATTTTCCTTGGTA (SEQ ID NO.17) is inserted at position 17011623.

[0024] Furthermore, the sequence of primer 11360-MF for the molecular marker is shown in SEQ ID NO.15, and the sequence of primer 11623-MR is shown in SEQ ID NO.16.

[0025] This invention uses Brassica napus (Brassica oleracea var. nana) as research material. RNA-seq analysis of different tissues at various developmental stages of Brassica napus revealed that the accumulation level of BnaTOE1 decreased with age, indicating that this gene may be involved in regulating the transition from the vegetative to the reproductive stage of Brassica napus. Therefore, the DNA fragment of BnaTOE1 was isolated from Brassica napus, and its function in the flowering time of Brassica napus was identified. It was then transformed into wild-type Brassica napus plants using Agrobacterium-mediated transformation, and its biological function within the plant was studied. Based on the BnaTOE1 variation sites in early-flowering and late-flowering materials of natural Brassica napus populations, molecular marker primers were designed to distinguish between early-flowering and late-flowering materials. These primers clearly differentiated between early-flowering and late-flowering materials of Brassica napus. This provides a good foundation for the application of BnaTOE1 in the genetic breeding of Brassica napus and will be of great significance for accelerating the improvement of plant genetic breeding.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0027] This invention, through analysis of the flowering time of transgenic Brassica napus overexpressing BnaTOE1, discovered a candidate gene, BnaTOE1, capable of regulating the flowering time of Brassica napus. Furthermore, based on the variation sites of the BnaTOE1 sequence in early-flowering and late-flowering materials in natural Brassica napus populations, molecular marker primers were designed. These primers can clearly distinguish between early-flowering and late-flowering materials in Brassica napus, and have significant application value for advancing the breeding improvement of growth period traits in Brassica napus. Attached Figure Description

[0028] Figure 1Phenotypic images of wild-type plant J9712 and Brassica napus overexpressing BnaTOE1 under long-day conditions are shown. In the figure, A is the flowering phenotype, B is the expression level of BnaTOE1, and C is the flowering time (**, p<0.01). In the figure, WT is the wild-type control plant; OE-BnaTOE1-1 and OE-BnaTOE1-2 are two lines of Brassica napus overexpressing BnaTOE1; LD is the long-day condition (22h light / 2h darkness); 42DAG is the seedling age of 42 days.

[0029] Figure 2 Results of using BnaTOE1 as a specific molecular marker to distinguish between early-flowering and late-flowering materials of Brassica napus. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0031] The following embodiments define the present invention and describe the methods for cloning DNA fragment sequences containing BnaTOE1 and verifying the function of BnaTOE1. They also provide a rapid and efficient molecular marker primer for identifying early-flowering and late-flowering materials of Brassica napus and its application. Based on the following description and these embodiments, those skilled in the art can determine the essential characteristics of the present 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.

[0032] Example 1: Molecular cloning of the BnaTOE1 DNA fragment from Brassica napus

[0033] Seedlings of the Brassica napus cultivar “Darmor-bzh” at the three-leaf-one-bud stage were flash-frozen in liquid nitrogen and stored at -70℃ 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 the above-mentioned cDNA as a template, and the designed F primer: 5'-ACTCTTCGCCGTCACTTT-3' (SEQ ID NO.3) and R primer: 5'-ATTCAATCCTGGTGCCTTA-3' (SEQ ID NO.4) as primers, DNA amplification was performed by RT-PCR using Phanta Max Super-Fidelity DNA Polymerase from Nanjing Novizan Biotechnology Co., Ltd. The amplification system was as follows: 1 μL Phanta Max Super-Fidelity DNA Polymerase, 25 μL 2× Phanta Max Buffer, 2 μL F primer (10 μM), 2 μL R primer (10 μM), 1 μL dNTP Mix (10 mM), 1 μL cDNA, 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 1 min 30 s, for a total of 35 cycles; 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-Blunt T vector from Beijing TransGen Biotech Co., Ltd., transformed into E. coli competent cells DH5α, and white colonies were picked for colony PCR identification of positive clones. Positive clones were sent to Yangzhou Qingke Biotechnology Co., Ltd. for sequencing. The plasmid confirmed by sequencing was named BnTOE1-T. The cloned DNA fragment containing the BnaTOE1 gene was 1254 bp in length, with the nucleotide sequence shown in SEQ ID NO.1 and the amino acid sequence shown in SEQ ID NO.2.

[0034] SEQ ID NO.1

[0035]

[0036]

[0037] SEQ ID NO.2

[0038]

[0039]

[0040] Example 2: Construction of BnaTOE1 overexpression vector

[0041] To better analyze the function of the BnaTOE1 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 BnaTOE1-T gene DNA sequence clone vector plasmid, which was verified by sequencing, as a template, DNA amplification was performed by PCR using primers pMDC83-TOE1-F: 5'-GACTAGTCATGCTGCTGGATCTCAACC-3' (SEQ ID NO.5) (sequence-specific primer with adapter Spe I site) and pMDC83-TOE1-R: 5'-GGCGCGCCAAAATTGTGGATAAAAAT-3' (SEQ ID NO.6) (sequence-specific primer with adapter Asc I site). The amplification system was: 20 μL 2×Taq Master Mix (Dye Plus) (Nanjing Novizan Biotechnology Co., Ltd.), 1 μL F primer (10 μM), 1 μL R primer (10 μM), 1 μL template DNA, ddH2O added to 50 μL, amplification conditions: 95℃ for 5 min, 95℃ for 30 s, 59℃ for 30 s, 72℃ for 1 min 30 s, for a total of 35 cycles; 72℃ for 5 min. After PCR, electrophoresis analysis was performed, and the target fragment was recovered using a DNA recovery kit from Wanhe Biotechnology Co., Ltd. The amplified fragment was ligated into the pEASY-Blunt T vector from Beijing TransGen Biotechnology Co., Ltd., transformed into E. coli competent cells DH5α, and white single colonies were picked for colony PCR identification of positive clones. Positive clones were sent to Yangzhou Qingke Biotechnology Co., Ltd. for sequencing. The cloning vector plasmid containing the BnaTOE1 DNA sequence was digested with Spe I and Asc I, and the target DNA fragment was recovered using a DNA recovery kit. This fragment was ligated with the corresponding digested pMDC83 backbone vector to construct the BnaTOE1 overexpression vector, named pMDC83-BnaTOE1.

[0042] Example 3: Genetic transformation of Brassica napus using the pMDC83-BnaTOE1 overexpression vector

[0043] A single colony of Agrobacterium tumefaciens GV3101 was picked and inoculated into 25 mL of YEB medium containing 50 mg / L rifampicin (5 g / L beef extract, 1 g / L yeast extract, 5 g / L peptone, 5 g / L sucrose, 0.04 g / L MgSO4·7H2O, pH 7.4) and incubated overnight. 5 mL of the bacterial culture was then transferred to 100 mL of YEB medium (containing 50 mg / L rifampicin) and incubated 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. Add 4 mL of 10% glycerol to suspend the bacterial cells 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 above-mentioned Agrobacterium tumefaciens GV3101 competent cells at -70℃ for later use.

[0044] The pMDC83-BnaTOE1 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 pMDC83-BnaTOE1 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 select transformants, and incubated at 28°C for 16 h.

[0045] 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. Mol Breed, 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.

[0046] The culture medium formula used is as follows:

[0047] Inoculation medium (M0): MURASHIGE & SKOOG MEDIUM (Duchefa Biochemie) + 3 g / L sucrose + 8 g / L agar (pH = 5.8-6.0).

[0048] 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 acetosyringone AS (pH = 5.8).

[0049] Callus differentiation medium (M2): M1 + 300 mg / L Timentin + 25 mg / L Hygromycin B.

[0050] 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.

[0051] Seedling rooting medium (M4): M0 + 300 mg / L Timentin.

[0052] MURASHIGE & SKOOG MEDIUM is abbreviated as MS medium.

[0053] The specific operating steps are as follows:

[0054] (1) Sterilization:

[0055] 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.

[0056] b. Then disinfect with 2% sodium hypochlorite for 20 minutes;

[0057] c. Finally, rinse the wild-type rapeseed J9712 seeds with sterile water 4-5 times to clean them as thoroughly as possible.

[0058] (2) Sowing:

[0059] a. Using sterile forceps, sow 30 sterilized wild-type rapeseed J9712 seeds onto M0 medium.

[0060] b. Place the inoculated culture jar into an incubator and incubate in the dark at 24°C for 6-7 days.

[0061] (3) Shaking:

[0062] 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.

[0063] (4) Preparation and infection of explants:

[0064] 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.

[0065] b. Measuring the OD of Agrobacterium 600 Value (OD 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.

[0066] 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.

[0067] (5) Transfer the infected explants to M1 medium, with 20-25 explants per dish, and incubate at 24°C in the dark for 36-48 hours.

[0068] (6) Transfer the explants from M1 to M2 medium and then to a light incubator for 3 weeks (24℃, 16h light / 8h dark).

[0069] (7) Transfer the explants to M3 medium and subculture them every 2-3 weeks until green shoots appear;

[0070] (8) Finally, the explants are transferred to M4 medium to root, which takes 2-4 weeks.

[0071] Example 4: Identification of positive transgenic Brassica napus plants

[0072] Genomic DNA was extracted from the rapeseed cultured in Example 3 using a rapid plant DNA extraction method. The specific steps are as follows:

[0073] (1) Take two tender leaves (about 0.2g), cut them into pieces and put them into a 2mL centrifuge tube. Add 250μL DNA buffer (500mM Tris-HCl, 300mM NaCl, 300mM Sucrose, pH=7.5) and two steel balls (6.7mm in diameter). Use a sampler to crush the leaf sample at 50Hz for 180s.

[0074] (2) Incubate the crushed sample at 95°C for 10 min;

[0075] (3) Remove the sample and cool it to room temperature, then centrifuge at 12,000 rpm for 5 min;

[0076] (4) Transfer 50 μL of supernatant to a new 1.5 mL centrifuge tube and dilute it 5 times for later use.

[0077] 1 μL of DNA was used as a template for PCR amplification using primers 35S (5'-TCCCACTATCCTTCGCAAG-3') (SEQ ID NO.7) and GFP (5'-TCAGGGTAACGGGAGAAGC-3') (SEQ ID NO.8). The amplification system consisted of 10 μL 2×TaqMaster Mix (Dye Plus) (Nanjing Novizan Biotechnology Co., Ltd.), 0.5 μL F primer (10 μM), 0.5 μL R primer (10 μM), 1 μL template DNA, and ddH2O added 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 30 s, for a total of 35 cycles; followed by 72℃ for 5 min. Using transgenic Brassica napus DNA as a template, the specific target fragment was amplified, demonstrating that the target vector pMDC83-BnaTOE1 has been integrated into the Brassica napus genome.

[0078] Example 5: Overexpression of BnaTOE1 delayed flowering time in Brassica napus.

[0079] The expression of BnaTOE1 in some transgenic Brassica napus plants was detected using real-time quantitative PCR. The expression levels of BnaTOE1 are as follows:

[0080] RNA was extracted using the Vazyme RNA Isolater Total RNA Extraction Reagent Kit. The expression level of BnaTOE1 was detected by qPCR using the HisScriptIII RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit and the 2×Q3 SYBR qPCR Master mix (Universal) expression assay kit. The upstream primer was qBnaTOE1-F: 5'-ACAGGGCATACAACAAGG-3' (SEQ ID NO. 9), and the downstream primer was qBnaTOE1-R: 5'-TCATACGGATTAGAAGGGA-3' (SEQ ID NO. 10). Actin was used as an internal reference 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). qPCR analysis was performed using a StepOnePlus real-time PCR instrument. The amplification system consisted of: 5 μL 2×Q3 SYBR qPCR Master mix (Nanjing Novizan Biotechnology Co., Ltd.), 0.2 μL F primer (10 μM), 0.2 μL R primer (10 μM), 3 μL cDNA, and ddH2O added to a final volume of 10 μL. The reaction program was: 95℃ for 30 s, 95℃ for 10 s, 60℃ for 30 s, with fluorescence signal acquisition for a total of 40 cycles; from 60℃ to 95℃, fluorescence signal was acquired every 1℃ for 1 s. Each sample was tested in triplicate. After the reaction, the results were analyzed and plotted using the software included with the StepOnePlus Real-Time PCR instrument (StepOne Software v2.3) to calculate the relative expression level of BnaTOE1 in the transgenic Brassica napus.

[0081] The results showed that transgenic plants with significantly increased BnaTOE1 expression levels compared to wild-type Brassica napus were successfully obtained. Figure 1 (B)

[0082] Wild-type plants J9712 and overexpressing plants OE-BnaTOE1 were grown for 14 days under 16h light / 8h darkness conditions, then transferred to 22h light / 2h darkness conditions for 28 days (seedling age 42 days). Phenotypic characteristics were observed, and flowering time was recorded. The phenotypes of wild-type plants J9712 and BnaTOE1 overexpressing Brassica napus under long-day conditions are shown below. Figure 1As shown. The square pots used for planting rapeseed are 13cm × 13cm in size, and each pot is divided into two planting areas, i.e. Figure 1 In section A, the left side represents the wild-type control plant J9712, and the right side represents the transgenic material. Three rapeseed plants were planted in each area. The results showed that the flowering time of the transgenic Brassica napus overexpressing BnaTOE1 was delayed by 5 days compared to the wild-type. Figure 1 (C)

[0083] Example 6: Development and Application of BnaTOE1-Specific Molecular Markers

[0084] Molecular marker primers were designed based on the BnaTOE1 variant sites in early-flowering and late-flowering materials of natural populations of Brassica napus. The specific method is as follows:

[0085] 1. Genomic DNA was extracted from Brassica napus using the CTAB method, and the steps are as follows:

[0086] (1) Take 0.5g of tender leaf tissue and put it into a 2mL clean centrifuge tube. Add liquid nitrogen and grind it into powder.

[0087] (2) Add 450 μL of CTAB extraction buffer preheated at 65℃, place it in a 65℃ water bath for 30 min, and gently mix every 5 min to ensure the solution is fully in contact with the tissue for reaction.

[0088] (3) After the reaction, take out the sample and cool it to room temperature. Then add an equal volume of chloroform / isoamyl alcohol (v / v = 24:1), gently invert and mix for 15 min, and then centrifuge at 15℃ and 10000 rpm for 5 min.

[0089] (4) Aspirate 600 μL of supernatant into a new EP tube and repeat step (3) above;

[0090] (5) Transfer the supernatant to a new tube, add 0.7 times the volume of pre-cooled isopropanol, gently invert and mix to allow the DNA to precipitate and aggregate into a flocculent precipitate, and place in a -20°C freezer for half an hour.

[0091] (6) Centrifuge at 10,000 rpm for 5 minutes at room temperature and discard the supernatant;

[0092] (7) Add 1 mL of 75% anhydrous ethanol and wash twice, then air dry in a fume hood;

[0093] (8) Add sterile ddH2O containing RNase and place the DNA sample in a 37°C water bath for about 30 minutes to accelerate dissolution;

[0094] (9) After the precipitate has completely dissolved, the purity and concentration of the sample are detected by spectrophotometer and agarose electrophoresis.

[0095] CTAB extraction buffer formulation: 2×CTAB: 100mM Tris-HCl (pH=8.0), 20mM EDTA (pH=8.0), 1.4M NaCl, 2% CTAB (w / v).

[0096] 1 μL of DNA was used as a template to amplify the BnaTOE1 sequence of early-flowering and late-flowering materials from natural populations of Brassica napus using PCR. The amplification primers were TOE1(CO3)-F:5'-CAACGTCTCAAAACCGTTAGTTC-3' (SEQ ID NO.13) and TOE1(CO3)-R:5'-CACACATTTCTTTTGTAAGCCTCC-3' (SEQ ID NO.14). The PCR system and amplification conditions were the same as those in Example 1.

[0097] The PCR products were recovered and sequenced using TOE1(C03)-R (SEQ ID NO.14). The differences in the BnaTOE1 sequence between early-flowering and late-flowering materials of Brassica napus were determined by comparing the sequencing peaks. Specifically, a C / T polymorphism was found at position 17011360 of chromosome ChrC03, and a small sequence AATTTTCCTTGGTA (as shown in SEQ ID NO.17) was inserted at position 17011623.

[0098] Molecular marker primers were designed based on the above-mentioned variant sites, with the following sequences: 11360-MF: 5'-TCTTTCTCCTGAGAATGCGGT-3' (SEQ ID NO.15); 11623-MR: 5'-CACTGTCTGGAATTTACCAAGGA-3' (SEQ ID NO.16). PCR amplification was performed on DNA from five early-flowering varieties of Brassica napus (Andor, Okara-B9, Dara, Risalpur, Kurram Agency) and five late-flowering varieties (Ceres, Jumbo, Cobra, CR3252, Zhongshuang9) using the primers described above. The amplification system was as follows: 10 μL 2×Taq Master Mix (Dye Plus) (Nanjing Novizan Biotechnology Co., Ltd.), 0.5 μL F primer (10 μM), 0.5 μL R primer (10 μM), 1 μL template DNA, and ddH2O added to a final volume of 20 μL. The amplification conditions were: 94℃ for 5 min; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, for a total of 35 cycles; 72℃ for 5 min. Subsequent agarose gel electrophoresis analysis revealed that the early-flowering materials showed a clear band between 250 bp and 500 bp, while the late-flowering materials did not exhibit this band. Figure 2The above results indicate that the BnTOE1 specific molecular marker can distinguish between early-flowering and late-flowering materials of Brassica napus.

[0099] This invention isolates the gene BnTOE1, which regulates the flowering time of Brassica napus, and provides a method to influence the flowering time of Brassica napus, as well as a rapid and efficient molecular marker primer for distinguishing between early-flowering and late-flowering materials of Brassica napus. The specific molecular marker primers designed in this invention can clearly distinguish between early-flowering and late-flowering varieties of Brassica napus, which is of great significance for improving the growth period traits and genetic breeding of Brassica napus.

[0100] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present 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 deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. Overexpression BnaTOE1 Use of a gene in delaying flowering time in Brassica napus, BnaTOE1 The DNA nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the encoded amino acid sequence is shown as SEQ ID NO.

2.

2. The method of claim 1 BnaTOE1 Use of the expression cassette, the recombinant vector, the recombinant microorganism or the transgenic cell line in delaying flowering time in Brassica napus or in Brassica napus genetic breeding, said genetic breeding being the breeding of Brassica napus germplasm with delayed flowering time.

3. A method for breeding a transgenic Brassica napus plant, characterized in that, comprising the following steps: (1) DNA fragments of genes cloned from Brassica napus BnaTOE1 and methods of use thereof (2) Using electric shock to treat rapeseed with cabbage-type flowers BnaTOE1 Plasmid transformation of gene DNA fragments into Agrobacterium; (3) using the transgenic method to transform the target Brassica napus with the agrobacterium with the transformation plasmid to obtain the transgenic Brassica napus, i.e. the Brassica napus with delayed flowering time.

4. The method of breeding a transgenic Brassica napus according to claim 3, wherein, In step (1), a DNA fragment of a gene of Brassica napus was cloned using RT-PCR with a F primer and a R primer, wherein the sequence of the F primer is shown in SEQ ID NO. 3 and the sequence of the R primer is shown in SEQ ID NO.

4. BnaTOE1 In step (1), a DNA fragment of a gene of Brassica napus was cloned using RT-PCR with a F primer and a R primer, wherein the sequence of the F primer is shown in SEQ ID NO. 3 and the sequence of the R primer is shown in SEQ ID NO.

4. BnaTOE1 In step (1), a DNA fragment of a gene of Brassica napus was cloned using RT-PCR with a F primer and a R primer, wherein the sequence of the F primer

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