Application of Brassica napus BnaTRM5 gene in regulating seed size and flowering time length

Abstract

The application discloses application of Brassica napus BnaTRM5 genes in regulation of seed size and flowering time length, and belongs to the technical field of genetic engineering. The application is directed to Brassica napus BnaTRM5 genes, and a double-copy mutant Bnatrm5a of BnaTRM5A09 (nucleotide sequence shown in SEQ ID NO. 1) and BnaTRM5C08 (nucleotide sequence shown in SEQ ID NO. 2) is constructed by using CRISPR technology. Experimental results show that the seed size of the double-copy mutant Bnatrm5a plant is significantly increased, and the flowering time is significantly prolonged. The application successfully creates new Brassica napus germplasm, provides important experimental materials for subsequent research, and provides gene resources and breeding parents for further Brassica napus single-plant yield and flowering period adaptability, and is helpful to promote the development of the Brassica napus breeding industry.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the BnaTRM5 gene in the regulation of seed size and flowering time in Brassica napus. Background Technology

[0002] Brassica napus, a globally important oilseed crop, plays a crucial role in edible oil production and biofuels. Seed size and flowering period are two key traits affecting rapeseed yield and quality. Seed size directly relates to oil accumulation, while the length of the flowering period influences the crop's reproductive cycle and environmental adaptability. Traditional breeding and single-gene improvement methods often suffer from drawbacks in regulating these traits, such as long cycles, unstable effects, and sensitivity to external environments. Therefore, using precise gene editing technology to modify key regulatory genes has become an important research direction for improving the overall traits of rapeseed.

[0003] Existing research has shown that seed development is a complex biological process involving the coordinated processes of cell division, cell expansion, and regulation by multiple hormones. Meanwhile, the regulation of flowering time involves the interaction of multiple factors, including endogenous signals, external photoperiod, and temperature. These genes have received widespread attention and application in crop breeding. Nevertheless, the molecular mechanisms underlying these processes are not yet fully understood, and traditional breeding methods have limitations in improving seed yield and quality, particularly in genetic improvement and germplasm resource development, often hampered by phenotypic instability, complex trait interactions, and environmental factors.

[0004] In recent years, with the advancements in gene editing technology and molecular marker-assisted breeding, an increasing number of studies have focused on improving germplasm through precise regulation of specific genes or proteins. BnaTRM5, a methyltransferase protein found in Brassica napus, has been shown to potentially participate in mechanisms of cell expansion, metabolic regulation, and endogenous signal transduction during plant development. This dual effect may be attributed to the alterations in cell cycle regulation, hormone homeostasis, and signal transduction processes induced by the double mutant Bnatrm5a. This allows seeds more time and resources for nutrient accumulation during development, while the extended flowering period provides a wider window for crop reproduction, thereby further improving overall crop yield and adaptability.

[0005] In current technologies, the improvement of seed size and flowering time mainly relies on traditional hybridization breeding or transgenic techniques. However, these methods often suffer from problems such as poor variety stability, long breeding cycles, and a lack of breeding materials. Therefore, there is an urgent need to provide a more effective gene modification strategy to achieve efficient breeding for regulating seed size and flowering time. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the BnaTRM5 gene in the regulation of seed size and flowering time in rapeseed, in order to solve the problems existing in the prior art. This invention utilizes gene editing technology to target and knock out the BnaTRM5a gene. Through this technical means, not only can the seed size of rapeseed be increased and the flowering time be extended, but it can also provide new ideas and methods for germplasm innovation of other crops.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides an application of the Brassica napus methyltransferase BnaTRM5 protein in any of the following:

[0009] (1) Application in regulating the seed size of Brassica napus;

[0010] (2) Application in the breeding of rapeseed varieties with increased seed size;

[0011] (3) Application in regulating the flowering time of Brassica napus;

[0012] (4) Application in the cultivation of rapeseed varieties with extended flowering time;

[0013] The gene encoding the Brassica napus methyltransferase BnaTRM5 protein includes nucleotide sequences such as BnaTRM5A09 as shown in SEQ ID NO.1 and BnaTRM5C08 as shown in SEQ ID NO.2.

[0014] This invention also provides an application of the BnaTRM5 gene expressing methyltransferase from Brassica napus in any of the following:

[0015] (1) Application in regulating the seed size of Brassica napus;

[0016] (2) Application in the breeding of rapeseed varieties with increased seed size;

[0017] (3) Application in regulating the flowering time of Brassica napus;

[0018] (4) Application in the cultivation of rapeseed varieties with extended flowering time;

[0019] The BnaTRM5 gene consists of nucleotide sequences BnaTRM5A09 as shown in SEQ ID NO.1 and BnaTRM5C08 as shown in SEQ ID NO.2.

[0020] Preferably, the BnaTRM5 gene is knocked out in Brassica napus to increase the seed size and / or prolong the flowering time of the Brassica napus.

[0021] The present invention also provides the application of a knockout vector targeting the BnaTRM5 gene described above in any of the following:

[0022] (1) Application in increasing the seed size of Brassica napus;

[0023] (2) Application in the breeding of rapeseed varieties with increased seed size;

[0024] (3) Application in prolonging the flowering time of Brassica napus;

[0025] (4) Application in the cultivation of rapeseed varieties with extended flowering time.

[0026] The present invention also provides the use of engineered bacteria comprising the above-described knockout vector in any of the following:

[0027] (1) Application in increasing the seed size of Brassica napus;

[0028] (2) Application in the breeding of rapeseed varieties with increased seed size;

[0029] (3) Application in prolonging the flowering time of Brassica napus;

[0030] (4) Application in the cultivation of rapeseed varieties with extended flowering time.

[0031] The present invention also provides a method for increasing the seed size of Brassica napus, including the step of knocking out the BnaTRM5 gene in Brassica napus;

[0032] The BnaTRM5 gene consists of nucleotide sequences BnaTRM5A09 as shown in SEQ ID NO.1 and BnaTRM5C08 as shown in SEQ ID NO.2.

[0033] Preferably, knocking out the BnaTRM5 gene in Brassica napus includes the following steps: constructing knockout vectors with BnaTRM5A09 and BnaTRM5C08 as target sites, respectively;

[0034] The knockout vector was transferred into Brassica napus using Agrobacterium-mediated transformation to obtain single mutants that knocked out BnaTRM5A09 and BnaTRM5C08. These mutants were then hybridized to obtain double mutants, thus completing the knockout of the BnaTRM5 gene.

[0035] The present invention also provides a method for prolonging the flowering time of Brassica napus, including the step of knocking out the BnaTRM5 gene in Brassica napus;

[0036] The BnaTRM5 gene consists of nucleotide sequences BnaTRM5A09 as shown in SEQ ID NO.1 and BnaTRM5C08 as shown in SEQ ID NO.2.

[0037] The present invention also provides a method for cultivating Brassica napus with increased seed size, including the step of knocking out the BnaTRM5 gene in Brassica napus;

[0038] The BnaTRM5 gene consists of nucleotide sequences BnaTRM5A09 as shown in SEQ ID NO.1 and BnaTRM5C08 as shown in SEQ ID NO.2.

[0039] The present invention also provides a method for cultivating Brassica napus with extended flowering time, including the step of knocking out the BnaTRM5 gene in Brassica napus;

[0040] The BnaTRM5 gene consists of nucleotide sequences BnaTRM5A09 as shown in SEQ ID NO.1 and BnaTRM5C08 as shown in SEQ ID NO.2.

[0041] The present invention discloses the following technical effects:

[0042] This invention uses the Brassica napus 'Xiaoyun' as the transformation recipient. Through genetic transformation and hybridization, a double-copy mutant Bnatrm5a with BnaTRM5A09 and BnaTRM5C08 knockout sites was obtained. This mutant significantly increased the seed size and prolonged the flowering time of the rapeseed. It successfully created a new germplasm with increased seed size and prolonged flowering time, providing important experimental materials for subsequent research. This invention will help promote the development of genomic breeding in the rapeseed industry and provides an important theoretical foundation for future research. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a comparison diagram of the TRM5-encoding DNA sequences in Arabidopsis thaliana and Brassica napus.

[0045] Figure 2This is a schematic diagram of the pKSE401 carrier used in this invention;

[0046] Figure 3 Seed size phenotype of Bnatrm5a double mutant;

[0047] Figure 4 Seed size statistics for Bnatrm5a double mutants; **** indicates P < 0.0001;

[0048] Figure 5 Phenotypic observation of Bnatrm5a double mutant plants at different growth stages;

[0049] Figure 6 The chart shows the flowering period statistics for the Bnatrm5a double mutant; **** indicates P<0.0001. Detailed Implementation

[0050] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0051] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0052] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0053] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0054] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0055] The antibiotics used in this invention include kanamycin, gentamicin, and rifamycin.

[0056] The culture medium formula used in this invention is as follows:

[0057] M0 germination medium: 1.1g 1 / 2 MS, 15g sucrose, bring to a final volume of 500mL, adjust pH to 5.84-5.88, add 3.5g agar, and sterilize.

[0058] M1 medium: MS 2.2g, sucrose 15g, mannitol 9g, 2,4-D 0.5mL (stock solution 1mg / mL), KT 0.5mL (stock solution 0.3mg / mL), bring to a final volume of 500mL, adjust pH to 5.84-5.88, add 4g agar, and sterilize. Add antibiotic 0.25mL As (stock solution 200mM) while rapidly cooling.

[0059] M2 medium: MS 2.2g, sucrose 15g, mannitol 9g, 2,4-D 0.5mL (stock solution 1mg / mL), KT 0.5mL (stock solution 0.3mg / mL), bring to a final volume of 500mL, adjust pH to 5.84-5.88, add 4g agar, and sterilize. While rapidly cooling, add antibiotics: STS 75μL, TMT 0.5mL (stock solution 1mg / mL), Hyg 250μL (stock solution 50mg / mL).

[0060] M3 medium: MS 2.2g, glucose 5g, sucrose 0.125g, MES 0.3g, bring to a final volume of 500mL, adjust pH to 5.84-5.88, add 4g agar, and sterilize. While rapidly cooling, add antibiotics: ZT 0.5mL (stock solution 2mg / mL), IAA 0.1mL (stock solution 0.1mg / mL), TMT 0.5mL (stock solution 300mg / mL), Hyg 250μL (stock solution 50mg / mL), AgNO3 75μL (stock solution 20mg / mL).

[0061] M4 rooting medium: MS 2.2g, sucrose 5g, bring to a final volume of 500mL, adjust pH to 5.84-5.88, add 4g agar, sterilize, and add antibiotic TMT 0.5mL (stock solution 0.3g / mL) while cooling rapidly.

[0062] Example 1

[0063] The methyltransferase BnaTRM5 protein in rapeseed has two copies on chromosome A (BnaTRM5A09 and BnaTRM5A01) and two copies on chromosome C (BnaTRM5C01 and BnaTRM5C08). Using CRISPR / Cas9 gene editing technology, two copies of the rapeseed methyltransferase synthesis gene (BnaTRM5), BnaTRM5A09 and BnaTRM5C08, were edited. The two single mutants were then hybridized to obtain the double-copy mutant Bnatrm5a. Although the AtTRM5a gene also exists in Arabidopsis thaliana, its sequence differs from that of BnaTRM5A09 and BnaTRM5C08 in rapeseed. Figure 1 As shown, BnaTRM5A09 lacks 63 bases at positions 300-370, BnaTRM5C08 has 3 bases inserted at positions 242-244, and there are other differences of multiple bases at other positions.

[0064] Example 2

[0065] 1. Agrobacterium-mediated genetic transformation of Bnatrm5a double mutant materials in Brassica napus

[0066] 1.1 Construction of two CRISPR knockout vectors

[0067] BnaTRM5A09 and BnaTRM5C08 knockout vector: pKSE401 ( Figure 2 ).

[0068] Agrobacterium GV3101 strain was extracted from the -80℃ cryopreservation environment and transformed into the constructed vector to obtain Agrobacterium infection solution.

[0069] The CDS sequences of the BnaTRM5A09 and BnaTRM5C08 genes are as follows:

[0070] CDS sequence of the BnaTRM5A09 gene (SEQ ID NO.1):

[0071]

[0072] The CDS sequence of the BnaTRM5C08 gene (SEQ ID NO.2):

[0073]

[0074] 1.2 Tissue Culture of Genetic Materials

[0075] a) Preparation of sterile recipient material. Select plump and clean rapeseed seeds, soak them in 75% ethanol for 1 min, then sterilize them in 50% 84 disinfectant for 8 min, rinse them aseptically 4-5 times, blot off excess water with sterile filter paper, inoculate them on M0 germination medium, and incubate them in the dark at 25℃-28℃ for 5 days.

[0076] b) Donor strain culture. Remove the inoculum tube stored at -80℃ and place it on ice. Under aseptic conditions, scrape the frozen culture surface with an inoculation needle and quickly streak the Agrobacterium adhering to the needle onto an LB agar plate containing 50 g / L Kana + 50 mg / L Gen + 50 mg / L LRif. Incubate the inoculated agar plate in the dark at 28℃ for 36-48 hours. Use a sterile toothpick to pick a well-grown single colony from the agar plate and inoculate it into liquid LB medium (containing 50 mg / L Kana + 50 mg / L Gen + 50 mg / L LRif). Incubate overnight (16-18 hours) at 28℃ with shaking (200 rpm) to allow the Agrobacterium to reach the logarithmic growth phase. 600 The value reaches approximately 0.4. Alternatively, 5 μL of bacterial culture can be directly inoculated into 5 mL of LB (50 mg / L Kana + 50 mg / L Gen + 50 mg / L L Ref) liquid medium and cultured at 28°C and 180 rpm for approximately 14-16 hours to allow Agrobacterium to reach the logarithmic growth phase. 600 The value is around 0.4-1.0.

[0077] c) Explant preparation and infection. Transfer 2 mL of the cultured bacterial solution to a sterile centrifuge tube, centrifuge at 6000 rpm for 3 min, and discard the supernatant. Add 2 mL of DM (+AS, acetylsuccinyl ethylstilbestrol), centrifuge at 6000 rpm for 3 min, and discard the supernatant. Resuspend in 2 mL of DM (+AS) again and store at 4°C. Add 18 mL of DM (+AS) to a sterile Petri dish. Use sterile scissors to cut hypocotyls from seedlings into the dish, each explant being 0.8 cm-1.0 cm in length. Cut the explants vertically in one stroke, 150-200 explants per dish (2-3 seeding boxes).

[0078] Pour 2 mL of the prepared DM bacterial solution into the cut explant dish and incubate for 8-10 minutes, shaking every 2 minutes. Three minutes before the incubation is complete, pipette off the DM bacterial solution. Use sterile forceps to transfer the explants to sterile filter paper and let them rest briefly, then aspirate any excess bacterial solution. Transfer the explants to M1 medium and incubate them at 24°C in a light-protected incubator.

[0079] d) Co-culture of explants and Agrobacterium. After co-culturing explants and Agrobacterium for 36-48 hours, the explants were transferred to M2 medium and cultured for 36 hours.

[0080] e) Induction of callus culture. Transfer the explants from M1 to M2 medium in sequence and arrange them neatly. Place them in a light-controlled culture room and culture for about 18 days.

[0081] f) Induction of shoot culture. Select green explants from M2 and transfer them to M3 medium, arranging them neatly. Place them in a light-controlled culture room and subculture every 2-3 weeks until green shoots appear. Cut the shoot tissue differentiated from the callus tissue at the adhesion point with the callus and place it in a new medium until the shoot grows to 1-2 cm.

[0082] g) Rooting culture. Green shoots with intact growth points are transferred to M4 rooting medium to grow and develop roots.

[0083] 2. Identification of positive plants from transgenic materials

[0084] 2.1 Experimental Procedure

[0085] DNA was extracted from transgenic rapeseed using a rapid leaf DNA extraction method. PCR identification was performed using the Hi-TOM method with two sets of primers, and positive plants were selected for phenotypic verification.

[0086] The two sets of primers are: Group A: target 1 (A09-T1-FT1 and A09-T1-RT1) and target 2 (A09-T2-FT1 and BnaA09-T2-R); Group B: target 1 (BnaC08-T1-R and C08-T1-FT) and target 2 (BnaC08-T2-R and C08-T2-FT). The primers for the control transgenic lines are as follows: BnaTRM5A09: Group A primers; BnaTRM5C08: Group B primers.

[0087] Group A primers:

[0088] A09-T1-FT1: ggagtgagtacggtgtgcTTCCGAGTGCCGAAGTTCG (SEQ ID NO. 3);

[0089] A09-T1-RT1:gagttggatgctggatggGTCTCTCCAGGTTTGAACAG(SEQ ID NO.4);

[0090] A09-T2-FT1: ggagtgagtacggtgtgcCTGTTCAAACCTGGAGAGAC (SEQ ID NO. 5);

[0091] BnaA09-T2-R: gagttggatgctggatggACCGAACACTATCCGGATTC (SEQ ID NO. 6).

[0092] Group B:

[0093] BnaC08-T1-R: gagttggatgctggatggTAGTCCAGCTTGAATCTCGC (SEQ ID NO. 7);

[0094] C08-T1-FT: ggagtgagtacggtgtgcGACAGCAAAGAAGTCTGATC (SEQ ID NO. 8);

[0095] BnaC08-T2-R: gagttggatgctggatggCTTCAAGTACCGAACACTGTC (SEQ ID NO.9);

[0096] C08-T2-FT: ggagtgagtacggtgtgcCTGTTTAAACCTGGAGAGACCG (SEQ ID NO. 10).

[0097] PCR procedure:

[0098] Group A primer target 1: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 5 min.

[0099] Group A primer target 2: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 54℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 5 min.

[0100] Group B primer target 1: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 53℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 5 min.

[0101] Group B primer target 2: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 53℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 5 min.

[0102] PCR products were subjected to 1.5% agarose gel electrophoresis at 140V for 15 min. After detection and identification, positive single plants and a small number of negative single plants were retained as controls. The plants were then managed normally in the greenhouse, and subsequent experiments were conducted after the seeds were harvested.

[0103] 2.2 Results Statistics

[0104] A total of 359 T0 generation edited materials were identified. The identification results are shown in Table 1. Table 1 shows the identification results of some positive single plants and their offspring T1 materials (obtained through bagging and self-pollination).

[0105] Table 1. Statistics on the editing of transgenic positive plants

[0106]

[0107] 3. Investigation of agronomic traits in the edited materials

[0108] 3.1 Methods for seed size determination and flowering period recording

[0109] 1) Seed size determination. Measure the seed diameter using a ruler and vernier calipers. Take 20 rapeseed seeds, arrange them in a row, and measure the sum of their diameters. Take the average of their radii and determine their area. Repeat three times.

[0110] 2) Record the rapeseed flowering period. Record the opening time of the first flower and the withering time of the last flower.

[0111] 3.2 Seed size and flowering time records of transgenic lines

[0112] T1-C08-51-8 and T1-A09-96-21 (genotype completely identical to T1-A09-97 in Table 1) were crossed, and the progeny double mutant material was collected and denoted as C-1; C-1 was bagged and self-crossed, and the progeny double mutant material was collected and denoted as C-1-4.

[0113] 1) Determine seed size

[0114] The average radius of the wild type was 0.0783 cm, while the average radius of the Bnatrm5a double mutant (C-1) was 0.1117 cm, which was 1.43 times that of the wild type. Figure 3 The average seed area of ​​wild-type seeds is 0.0193 cm². 2 The average seed area of ​​the Bnatrm5a double mutant (C-1) was 0.0392 cm². 2 It is 2.03 times that of the wild type. Figure 4 ).

[0115] 2) Record of rapeseed flowering period

[0116] Phenotypic data were recorded by photographing different growth stages of the same batch of plants. Figure 5 The average flowering days for the wild type was 19.5 days, while the average flowering days for the Bnatrm5a double mutant (C-1-4) was 26.3 days, which was 1.35 times that of the wild type. Figure 6 ).

[0117] In summary, this invention constructed a double-copy mutant Bnatrm5a at the BnaTRM5A09 and BnaTRM5C08 sites. Through the investigation of its agronomic traits, it significantly improved seed size and prolonged flowering period. The successful creation of a new germplasm with increased seed size and prolonged flowering time provides a new method for improving crop yield and ecological adaptability through genetic engineering. It also provides important experimental materials for subsequent research, which will help promote the development of the rapeseed industry system and enhance its application potential in various fields.

[0118] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a Brassica napus methyltransferase BnaTRM5 protein in any of the following, characterized in that, The gene encoding the Brassica napus methyltransferase BnaTRM5 protein includes the nucleotide sequence shown in SEQ ID NO.

1. BnaTRM5A09 And as shown in SEQ ID NO.2 BnaTRM5C08 ; (1) The application of knocking out the gene encoding the Brassica napus methyltransferase BnaTRM5 in increasing the seed size of Brassica napus; (2) Application of knocking out the gene encoding the Brassica napus methyltransferase BnaTRM5 in the breeding of Brassica napus varieties with increased seed size; (3) The application of knocking out the gene encoding the Brassica napus methyltransferase BnaTRM5 in prolonging the flowering time of Brassica napus; (4) Application of knocking out the gene encoding the Brassica napus methyltransferase BnaTRM5 in the cultivation of Brassica napus varieties with extended flowering time.

2. A method for expressing methyltransferases from Brassica napus. BnaTRM5 The application of genes in any of the following, characterized in that, The BnaTRM5 The gene consists of a nucleotide sequence as shown in SEQ ID NO.

1. BnaTRM5A09 And as shown in SEQ ID NO.2 BnaTRM5C08 composition; (1) Knock out the above BnaTRM5 Application of genes in increasing seed size in Brassica napus; (2) Knock out the above BnaTRM5 Application of genes in breeding rapeseed varieties with increased seed size; (3) Knock out the above BnaTRM5 Application of genes in prolonging the flowering time of Brassica napus; (4) Knock out the above BnaTRM5 Application of genes in breeding rapeseed varieties with extended flowering time.

3. A targeting method as described in claim 2 BnaTRM5 The application of gene knockout vectors in any of the following: (1) Application in increasing the seed size of Brassica napus; (2) Application in the breeding of rapeseed varieties with increased seed size; (3) Application in prolonging the flowering time of Brassica napus; (4) Application in the cultivation of rapeseed varieties with extended flowering time.

4. The use of an engineered bacterium comprising the knockout vector of claim 3 in any of the following: (1) Application in increasing the seed size of Brassica napus; (2) Application in the breeding of rapeseed varieties with increased seed size; (3) Application in prolonging the flowering time of Brassica napus; (4) Application in the cultivation of rapeseed varieties with extended flowering time.

5. A method for increasing the seed size of Brassica napus, characterized in that, Including knockout in Brassica napus BnaTRM5 The steps of gene generation; The BnaTRM5 The gene consists of a nucleotide sequence as shown in SEQ ID NO.

1. BnaTRM5A09 And as shown in SEQ ID NO.2 BnaTRM5C08 composition.

6. The method as described in claim 5, characterized in that, Knockout in the Brassica napus BnaTRM5 The gene includes the following steps: respectively using the described BnaTRM5A09 and stated BnaTRM5C08 We constructed a knockout vector targeting the target site. The knockout vector was transferred into Brassica napus using Agrobacterium-mediated transformation to obtain the knockout vector. BnaTRM5A09 and knockout BnaTRM5C08 The single mutant was hybridized to obtain a double mutant, thus completing the knockout process. BnaTRM5 Gene.

7. A method for prolonging the flowering time of Brassica napus, characterized in that, Including knockout in Brassica napus BnaTRM5 The steps of gene generation; The BnaTRM5 The gene consists of a nucleotide sequence as shown in SEQ ID NO.

1. BnaTRM5A09 And as shown in SEQ ID NO.2 BnaTRM5C08 composition.

8. A method for cultivating Brassica napus with increased seed size, characterized in that, Including knockout in Brassica napus BnaTRM5 The steps of gene generation; The BnaTRM5 The gene consists of a nucleotide sequence as shown in SEQ ID NO.

1. BnaTRM5A09 And as shown in SEQ ID NO.2 BnaTRM5C08 composition.

9. A method for cultivating Brassica napus with an extended flowering time, characterized in that, Including knockout in Brassica napus BnaTRM5 The steps of gene generation; The BnaTRM5 The gene consists of a nucleotide sequence as shown in SEQ ID NO.

1. BnaTRM5A09 And as shown in SEQ ID NO.2 BnaTRM5C08 composition.

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