Application of BnaTOE1 gene in regulating flowering time of brassica napus and molecular marker primer for identification of BnaTOE1 gene

Abstract

The invention discloses application of a BnaTOE1 gene in regulating flowering time of brassica napus and a molecular marker primer for identification of the BnaTOE1 gene. The DNA nucleotide sequence of the BnaTOE1 gene is as shown in SEQ ID NO.1, the sequence length is 1254bp, the coded amino acid sequence is as shown in SEQ ID NO.2, and molecular marker primers for identifying the flowering phase of the brassica napus are as shown in SEQ ID NO.15 and SEQ ID NO.16. The invention further discloses a molecular marker for identifying the flowering phase of the brassica napus. Functional analysis of brassica napus genetic transformation shows that BnaTOE1 participates in regulating and controlling the flowering time of brassica napus, and development of the molecular marker has important significance in promoting genetic improvement and breeding of brassica napus.

Description

Technical Field

[0001] The invention relates to application of BnaTOE1 gene in regulating flowering time of Brassica napus and molecular marker primers for identification thereof, and belongs to the field of Brassica napus gene engineering. Background Art

[0002] Brassica napus L. is the main rapeseed cultivated variety in my country, with the characteristics of high yield, strong stress resistance and wide adaptability. The early flowering time of Brassica napus means that its growth cycle is relatively short, which helps to complete the entire growth and development process in a shorter time to adapt to the special climatic conditions in some regions, increase the number of rapeseed crops, and improve land utilization and agricultural production efficiency (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, exploring the key genes that regulate flowering time and improving the growth period traits are one of the important goals of genetic breeding of Brassica napus. TOE1 encodes an AP2 family protein transcription factor, is a target gene of miR172, and is 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 and low-cost molecular markers have greatly improved the efficiency and accuracy of crop molecular breeding, which is conducive to accelerating the selection of excellent varieties of Brassica napus. Summary of the invention

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

[0004] Technical solution: Application of the BnaTOE1 gene of the present invention in regulating the flowering time of Brassica napus. The 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, and the sequence length is 1254 bp.

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

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

[0008] A transgenic Brassica napus is disclosed, wherein the BnaTOE1 gene of the invention is used to transform the Brassica napus to obtain the transgenic Brassica napus.

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

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

[0011] (2) using the electroporation method to transform the plasmid carrying the DNA fragment of the Brassica napus BnaTOE1 gene into Agrobacterium;

[0012] (3) The Agrobacterium carrying the transformation plasmid is used to transform the target Brassica napus by a 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, 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.

[0014] The provided method for cultivating transgenic Brassica napus is to increase the content of the mRNA in the target Brassica napus so that the flowering time phenotype of the transgenic Brassica napus is obviously different from that of the target Brassica napus.

[0015] The expression vector carrying the DNA fragment containing BnaTOE1 of the present invention can be introduced into plant cells by using conventional biotechnology methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, and electroporation (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 expression vector comprising the BnaTOE1 DNA fragment of the present invention can be used to transform a host and cultivate plant germplasms with different flowering times.

[0017] The DNA fragment containing BnaTOE1 was recovered using a DNA recovery kit, and the fragment was connected to the pMDC83 backbone vector using an enzyme ligation method to construct an overexpression vector of the DNA fragment, which was named pMDC83-BnaTOE1.

[0018] The pMDC83-BnaTOE1 vector was introduced into Agrobacterium tumefaciens by electroporation, and the Agrobacterium strain was GV3101. The pMDC83-BnaTOE1 was transformed into the Brassica napus receptor material J9712 by Agrobacterium infection-mediated genetic transformation, and transgenic plants with significantly higher expression of BnaTOE1 compared with the wild type were successfully obtained. It was observed that compared with the wild-type plants, the flowering time of transgenic Brassica napus overexpressing BnaTOE1 was significantly delayed, 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 are 11360-MF and 11623-MR. The sequence of primer 11360-MF is shown in SEQ ID NO.15, and the sequence of primer 11623-MR is shown in SEQ ID NO.16.

[0020] A method for identifying the flowering time of Brassica napus, comprising 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) Using molecular marker primers 11360-MF and 11623-M, PCR amplification was used to molecularly identify early-flowering and late-flowering materials of Brassica napus.

[0023] Furthermore, in step (1), the mutation site of BnaTOE1 is a C / T polymorphism at base 17011360 of chromosome ChrC03, and an insertion of a small fragment sequence AATTTCCTTGGTA (SEQ ID NO. 17) at base 17011623.

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

[0025] The present invention uses Brassica napus as research material, and through RNA-seq analysis of various tissues in different developmental stages of Brassica napus in the early stage, it is found that the accumulation level of BnaTOE1 decreases with age, indicating that the gene may be involved in regulating the transition from the vegetative stage to the reproductive stage of Brassica napus. Therefore, the DNA fragment of BnaTOE1 is isolated from Brassica napus, and the function of BnaTOE1 in the flowering time of Brassica napus is identified, and it is transformed into wild-type Brassica napus plants using Agrobacterium-mediated method, and its biological function in the plant is studied; molecular marker primers for identifying early-flowering and late-flowering materials are designed according to the variation sites of BnaTOE1 in the early-flowering materials and late-flowering materials of the natural population of Brassica napus, and the molecular marker primers can clearly distinguish the early-flowering materials and late-flowering materials of Brassica napus. It provides a good foundation for the application of BnaTOE1 in the genetic breeding of Brassica napus, which 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] The present invention analyzes the flowering time of transgenic Brassica napus that overexpresses BnaTOE1, and discovers a candidate gene BnaTOE1 that can regulate the flowering time of Brassica napus. Molecular marker primers are also designed based on the variation sites of the BnaTOE1 sequences of early-flowering materials and late-flowering materials in natural populations of Brassica napus. The molecular marker primers can clearly distinguish between early-flowering materials and late-flowering materials of Brassica napus, and have important application value for promoting the breeding improvement of growth period traits of Brassica napus. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1The phenotypes of wild-type plants J9712 and BnaTOE1-overexpressing Brassica napus under long-day conditions; 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 strains of BnaTOE1-overexpressing Brassica napus; LD is long-day conditions (22h light / 2h dark); 42DAG is 42 days of seedling age.

[0029] Figure 2 The result diagram of using BnaTOE1 as a specific molecular marker to distinguish early-flowering and late-flowering materials of Brassica napus. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0031] The following examples define the present invention, describe the method of cloning a DNA fragment sequence containing BnaTOE1, and verifying the function of BnaTOE1, and provide a molecular marker primer and application for rapid and efficient identification of early-flowering materials and late-flowering materials of Brassica napus. Based on the following description and these examples, those skilled in the art can determine the basic characteristics of the present invention, and can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention to make it suitable for different uses and conditions.

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

[0033] The seedlings of the Brassica napus variety "Darmor-bzh" at the three-leaf and one-core stage were taken, quickly frozen in liquid nitrogen, and stored in a -70°C refrigerator for total RNA extraction. Total RNA was extracted using the RNA Isolater Total RNA Extraction Reagent kit from Vazyme. The synthesis of Brassica napus cDNA was performed according to the instructions of Nanjing Novozyme Biotechnology Co., Ltd. HisScriptIII RT SuperMix for qPCR (+gDNA wiper) for first-strand synthesis. The first chain of cDNA was used as an amplification template, and the designed F primer: 5'-ACTCTTCGCCGTCACTTT-3' (SEQ ID NO.3) and R primer: 5'-ATTCAATCCTGGTGCCTTA-3' (SEQ ID NO.4) were used as primers. Phanta Max Super-Fidelity DNA Polymerase of Nanjing Novogene Biotechnology Co., Ltd. was used to perform DNA amplification by RT-PCR. The amplification system was: 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 was added to 50 μL. The amplification conditions were: 95°C for 5 min, 95°C for 30 s, 59°C for 30 s, 72°C for 1 min 30 s, for a total of 35 cycles; 72°C for 5 min. After PCR, electrophoresis analysis was performed, and the target amplified fragment was recovered using the DNA recovery kit of Kangwei Century Biotechnology Co., Ltd. The amplified fragment was connected to the pEASY-Blunt T vector of Beijing Quanshijin Biotechnology Co., Ltd., transformed into Escherichia coli competent cells DH5α, white colonies were picked for colony PCR identification of positive clones, and the positive clones were sent to Yangzhou Qingke Biotechnology Co., Ltd. for sequencing. The plasmid verified by sequencing was named BnTOE1-T, and the cloned DNA fragment containing the BnaTOE1 gene was 1254 bp in length, the nucleotide sequence was shown in SEQ ID NO.1, and its amino acid sequence was 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] In order 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 construction method is as follows: using the BnaTOE1 gene DNA sequence cloning vector plasmid BnaTOE1-T verified by sequencing as a template, using primers pMDC83-TOE1-F: 5'-GACTAGTCATGCTGCTGGATCTCAACC-3' (SEQ ID NO.5) (sequence-specific primer plus adapter Spe I site) and pMDC83-TOE1-R: 5'-GGCGCGCCAAAATTGTGGATAAAAAT-3' (SEQ ID NO.6) (sequence-specific primer plus adapter Asc I site), PCR was used for DNA amplification, and the amplification system was: 20μL 2×Taq Master Mix (Dye Plus) (Nanjing Novogene Biotechnology Co., Ltd.), 1μL F primer (10μM), 1μL R primer (10μM), 1μL template DNA, add ddH2O to 50μL, amplification conditions: 95℃5min, 95℃30s, 59℃30s, 72℃1min 30s, 35 cycles; 72℃5min. After the PCR, electrophoresis analysis was performed, and the target fragment was recovered using the DNA recovery kit of Wanhe Biotechnology Co., Ltd. The amplified fragment was connected to the pEASY-Blunt T vector of Beijing Quanshijin Biotechnology Co., Ltd., transformed into Escherichia coli competent cells DH5α, and white single colonies were picked for colony PCR identification of positive clones. The positive clones were sent to Yangzhou Qingke Biotechnology Co., Ltd. for sequencing. The cloning vector plasmid containing the BnaTOE1 DNA sequence was double-digested with Spe I and Asc I, and the target DNA fragment was recovered using a DNA recovery kit. This fragment was connected to the correspondingly digested pMDC83 backbone vector to construct an overexpression vector of BnaTOE1, named pMDC83-BnaTOE1.

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

[0043] A single colony of Agrobacterium tumefaciens GV3101 was selected and inoculated into 25 mL YEB medium (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) containing 50 mg / L rifampicin and cultured overnight. 5 mL of the bacterial solution was transferred to 100 mL YEB medium (containing 50 mg / L rifampicin) and cultured until OD 600 =0.7-0.8, place the bacterial solution on ice for 10 minutes, centrifuge at 5000rpm at 4℃ for 10 minutes to collect the bacteria, add 100mL of sterile double distilled water to wash twice. Add 4mL of 10% glycerol to suspend the bacteria, and transfer to a 50mL centrifuge tube. Centrifuge at 4℃5500rpm for 10 minutes to collect the bacteria, add 500μL of 10% glycerol to resuspend the bacteria, and transfer to a 1.5mL centrifuge tube. Store the competent cells of the above-mentioned Agrobacterium tumefaciens GV3101 strain in a -70℃ refrigerator for later use.

[0044] The pMDC83-BnaTOE1 plasmid was introduced into the competent cells of Agrobacterium tumefaciens GV3101 strain by electroporation. Take 50 μL of competent cells of Agrobacterium tumefaciens GV3101 strain, add 5 μL of pMDC83-BnaTOE1 recombinant plasmid, mix well with a pipette tip and transfer to a 0.1 cm electroporation cup. Electroporation parameters: 200Ω, 1.7KV, 2.5F, add 500 μL of LB culture medium immediately after electroporation. After culturing at 28℃ and 220rpm for 1 hour, take 100 μL of bacterial solution and apply it to LB medium containing kanamycin resistance to select transformants, and culture at 28℃ for 16 hours.

[0045] The genetic transformation method of Brassica napus is an improved method based on the transformation method of the State Key Laboratory of Crop Genetic Improvement of 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 methodusing hypocotyl as explants for Brassica napus. Mol Breed, 2020, 40: 96). The hypocotyls of sterile Brassica napus seedlings are used as explants, and the genetic transformation of exogenous fragments in Brassica napus is achieved by Agrobacterium-mediated method.

[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 medium (M3): MURASHIGE&SKOOG MEDIUM (Duchefa Biochemie) + 10 g / L glucose + 0.25 g / L xylose + 0.6 g / L MES + 2 mg / L Zeatin + 0.1 mg / L IAA + 300 mg / L Timentin + 25 mg / L Hygromycin B.

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

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

[0053] The specific steps are as follows:

[0054] (1) Sterilization:

[0055] a. First, soak the wild type Brassica napus J9712 seeds in 75% alcohol for 1 minute. Be careful not to soak for too long.

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

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

[0058] (2) Seeding:

[0059] a. Sow sterilized wild-type Brassica napus J9712 seeds on M0 medium using sterile tweezers, 30 seeds per dish;

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

[0061] (3) Shaking bacteria:

[0062] 5-6 days after sowing, inoculate Agrobacterium GV3101 into a sterile Erlenmeyer flask or centrifuge tube containing LB liquid culture medium and culture it in a shaking incubator at 28°C and 180-220 rpm.

[0063] (4) Explant preparation and infection:

[0064] a. Use sterile forceps and a scalpel to cut the seedlings that have grown for 6-7 days after sowing, and cut their hypocotyls into explant segments with a length of 0.8-1 cm. When cutting the seedlings, place their hypocotyls in M1 liquid culture medium for better cutting results. Cut quickly and accurately without dragging;

[0065] b. Measure the OD of Agrobacterium 600 Value (OD in LB medium 600 = 0.3 is better), centrifuge the pre-cultured bacterial solution at 6000rpm for 10min, discard the supernatant, and resuspend the bacteria in an equal volume of MS liquid medium containing 100μM acetosyringone AS, and then repeat once. Finally, take 2mL of bacterial solution and dilute it with 20mL of MS liquid medium containing 100μM AS;

[0066] c. Place the cut explants in the resuspended bacterial solution with adjusted concentration and immerse for 10 minutes. Be careful not to infect for too long, otherwise the explants will die. It is appropriate to infect 150-200 explants in every 20mL bacterial solution.

[0067] (5) Transfer the infected explants to M1 medium, preferably 20-25 explants per dish, and culture them at 24°C in dim light for 36-48 h;

[0068] (6) The explants were transferred from M1 to M2 medium and cultured in a light incubator (24°C, 16 h light / 8 h dark) for 3 weeks;

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

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

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

[0072] The genomic DNA of Brassica napus cultured in Example 3 was extracted using a method for rapid plant DNA extraction, and the specific steps are as follows:

[0073] (1) Take two young 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 (diameter 6.7 mm), and break the leaf samples with a sampler at 50 Hz for 180 s;

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

[0075] (3) The sample was taken out and cooled to room temperature, and centrifuged at 12000 rpm for 5 min;

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

[0077] 1 μL DNA was used as template, and PCR amplification was performed with primers 35S (5'-TCCCACTATCCTTCGCAAG-3') (SEQ ID NO.7) and GFP (5'-TCAGGGTAACGGGAGAAGC-3') (SEQ ID NO.8). The amplification system was: 10 μL 2×TaqMaster Mix (Dye Plus) (Nanjing Novozyme Biotechnology Co., Ltd.), 0.5 μL F primer (10 μM), 0.5 μL R primer (10 μM), 1 μL template DNA, add ddH2O to 20 μL, and the amplification conditions were: 95℃5min, 95℃30s, 59℃30s, 72℃1min 30s, 35 cycles in total; 72℃5min. Using transgenic Brassica napus DNA as a template, a specific target fragment can be amplified, proving that the target vector pMDC83-BnaTOE1 has been integrated into the Brassica napus genome.

[0078] Example 5 Overexpression of BnaTOE1 delays flowering time of Brassica napus

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

[0080] RNA was extracted using the RNA Isolater Total RNA Extraction Reagent kit from Vazyme, and the expression level of BnaTOE1 was detected by qPCR using the reverse transcription kit HisScriptIII RT SuperMix for qPCR (+gDNA wiper) and the expression detection kit 2×Q3 SYBR qPCR Master mix (Universal). 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 the internal reference gene, the upstream primer used was qBnaActin-F: 5'-TCTTCCTCACGCTATCCTCCG-3' (SEQ ID NO.11), the downstream primer was qBnaActin-R: 5'-AGCCGTCTCCAGCTCTTGC-3' (SEQ ID NO.12), and the qPCR analysis was performed using a StepOnePlus fluorescence quantitative PCR instrument. The amplification system was: 5μL 2×Q3 SYBR qPCR Master mix (Nanjing Novogene Biotechnology Co., Ltd.), 0.2μL F primer (10μM), 0.2μL R primer (10μM), 3μL cDNA, add ddH2O to 10μL, and the reaction program was: 95℃30s, 95℃10s, 60℃30s, collect fluorescence signals, a total of 40 cycles; 60℃to 95℃, collect fluorescence signals every 1℃ for 1s. Three technical replicates were set for each sample. After the reaction, the software provided by the StepOnePlus fluorescence quantitative PCR instrument (StepOne Software v2.3) was used for analysis and drawing to calculate the relative expression level of BnaTOE1 in the transgenic materials of Brassica napus.

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

[0082] After growing the wild-type plants J9712 and the overexpressing plants OE-BnaTOE1 under 16h light / 8h dark conditions for 14 days, they were transferred to 22h light / 2h dark conditions and grown for 28 days (seedling age 42 days). Their phenotypes were observed and the flowering time was counted. Figure 1The square pots used for rapeseed planting are 13cm×13cm in size, and each pot is divided into two areas for planting, namely Figure 1 The left side of A shows the wild-type control material J9712, and the right side shows the transgenic material. Three rapeseeds were planted in each area. The results showed that the flowering time of the transgenic rapeseed overexpressing BnaTOE1 was delayed by 5 days compared with the wild type ( Figure 1 Middle C).

[0083] Example 6 Development and application of BnaTOE1 specific molecular marker

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

[0085] 1. The CTAB method was used to extract genomic DNA of Brassica napus, and the steps were as follows:

[0086] (1) Take 0.5 g of young leaf tissue and place it in a 2 mL clean centrifuge tube, add liquid nitrogen and grind it into powder;

[0087] (2) Add 450 μL of CTAB extraction buffer preheated at 65°C and place in a 65°C water bath for 30 min. Gently mix every 5 min to allow the solution to fully contact and react with the tissue.

[0088] (3) After the reaction, the sample was removed and cooled to room temperature, and then an equal volume of chloroform / isoamyl alcohol (v / v = 24:1) was added, gently inverted and mixed for 15 min, and then centrifuged at 15°C and 10,000 rpm for 5 min;

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

[0090] (5) Transfer the supernatant to a new tube, add 0.7 times the volume of pre-cooled isopropanol, gently invert upside down to mix, so that the DNA precipitates into flocculent precipitates, and place in a -20°C refrigerator for half an hour;

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

[0092] (7) Add 1 mL of 75% anhydrous ethanol and wash twice, then 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 min to accelerate dissolution;

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

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

[0096] 1 μL DNA was taken as a template, and PCR amplification was performed on the BnaTOE1 sequence of the early-flowering material and the late-flowering material of the natural population of Brassica napus, respectively. The amplification primers were TOE1(C03)-F: 5'-CAACGTCTCAAAACCGTTAGTTC-3' (SEQ ID NO.13) and TOE1(C03)-R: 5'-CACACATTTCTTTTGTAAGCCTCC-3' (SEQ ID NO.14), and the PCR system and amplification conditions were the same as those shown in Example 1.

[0097] The PCR product was recovered and sequenced with TOE1(C03)-R (SEQ ID NO.14). The differences in the BnaTOE1 sequence between the early-flowering materials and the late-flowering materials of Brassica napus were determined by comparing the sequencing peaks, namely, there was a C / T polymorphism at the 17011360 base of the ChrC03 chromosome, and there was an insertion of a small fragment sequence AATTTCCTTGGTA (as shown in SEQ ID NO.17) at the 17011623 base.

[0098] Molecular marker primers were designed based on the above mutation sites, and the sequences were as follows: 11360-MF: 5'-TCTTTCTCCTGAGAATGCGGT-3' (SEQ ID NO. 15); 11623-MR: 5'-CACTGTCTGGAATTTACCAAGGA-3' (SEQ ID NO. 16). The above primers were used to perform PCR amplification on DNA of five early-flowering varieties of Brassica napus (Andor, Okara-B9, Dara, Risalpur, Kurram Agency) and five late-flowering varieties (Ceres, Jumbo, Cobra, CR3252, Zhongshuang9). The amplification system was: 10 μL 2×Taq Master Mix (Dye Plus) (Nanjing Novogene Biotechnology Co., Ltd.), 0.5 μL F primer (10 μM), 0.5 μL R primer (10 μM), 1 μL template DNA, add ddH2O to 20 μL, and the amplification conditions were: 94°C for 5 min; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, for a total of 35 cycles; 72°C for 5 min. Subsequently, agarose gel electrophoresis analysis revealed that the early-flowering materials all had obvious bands between 250 bp and 500 bp, while the late-flowering materials did not have this band ( Figure 2The above results indicate that the BnTOE1 specific molecular marker can be used to identify early-flowering and late-flowering materials of Brassica napus.

[0099] The present invention isolates the gene BnTOE1 that regulates the flowering time of Brassica napus, provides a method for influencing the flowering time of Brassica napus and a molecular marker primer for quickly and efficiently distinguishing early-flowering materials and late-flowering materials of Brassica napus. The specific molecular marker primer designed by the present invention can clearly distinguish early-flowering varieties and late-flowering varieties of Brassica napus, which is of great significance to the improvement of 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, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. Application of BnaTOE1 gene in regulating flowering time of Brassica napus.

2. The use of the BnaTOE1 gene according to claim 1 in regulating the flowering time of Brassica napus, characterized in that: The DNA nucleotide sequence of the BnaTOE1 gene is shown in SEQ ID NO.

1.

3. The use of the BnaTOE1 gene according to claim 2 in regulating the flowering time of Brassica napus, characterized in that: The amino acid sequence encoded by it is shown in SEQ ID NO.

2.

4. Use of the expression cassette, recombinant vector, recombinant microorganism or transgenic cell line of the BnaTOE1 gene according to any one of claims 1 to 3 in regulating the flowering time of Brassica napus or in genetic breeding of Brassica napus.

5. A transgenic Brassica napus, characterized in that: The BnaTOE1 gene described in any one of claims 1 to 3 is used to transform Brassica napus to obtain transgenic Brassica napus.

6. A method for cultivating transgenic Brassica napus, characterized in that: The following steps are involved: (1) Cloning the DNA fragment of the BnaTOE1 gene of Brassica napus; (2) using the electroporation method to transform the plasmid carrying the DNA fragment of the Brassica napus BnaTOE1 gene into Agrobacterium; (3) The Agrobacterium carrying the transformation plasmid is used to transform the target Brassica napus by a transgenic method to obtain transgenic Brassica napus.

7. The method for cultivating transgenic Brassica napus according to claim 6, characterized in that: 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, 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.

8. A molecular marker primer for identifying the flowering time of Brassica napus, characterized in that: The molecular marker primers are 11360-MF and 11623-MR. The sequence of primer 11360-MF is shown in SEQ ID NO.15, and the sequence of primer 11623-MR is shown in SEQ ID NO.

16.

9. A method for identifying the flowering time of Brassica napus, characterized in that: The following steps are involved: (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; (2) Using molecular marker primers 11360-MF and 11623-M, PCR amplification was used to molecularly identify early-flowering and late-flowering materials of Brassica napus.

10. The method for identifying the flowering time of Brassica napus according to claim 9, characterized in that: In step (1), the mutation site of BnaTOE1 is a C / T polymorphism at base 17011360 of chromosome ChrC03, and an insertion of a small sequence AATTTCCTTGGTA at base 17011623.

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