Brassica napus bn tir1 gene and application thereof

Abstract

The application discloses a Brassica napus BnTIR1 gene and application thereof. The Brassica napus BnTIR1 gene comprises a DNA fragment of the BnTIR1 gene, the nucleotide sequence of the DNA fragment is shown as SEQ ID NO:1, the sequence length is 1788 bp, and the corresponding amino acid sequence is shown as SEQ ID NO:2. Function analysis of genetic transformation of Arabidopsis thaliana shows that the BnTIR1 is involved in regulation of seed length, which has very important significance for genetic breeding improvement of appearance quality of plant seeds.

Description

TECHNICAL FIELD

[0001] The present application relates to a Brassica napus BnTIR1 gene and its Brassica napus gene BnTIR1 in the application of improving the appearance quality of plant seeds. The present application adopts the method of RT-PCR to separate the DNA fragment containing Brassica napus BnTIR1, and the overexpression of BnTIR1 can affect the length of Arabidopsis thaliana seeds, which confirms the function of the gene and its application approach, belonging to the field of genetic improvement technology. BACKGROUND

[0002] Rape (Brassica napus L.) is the main oilseed rape species in China, with high yield, strong stress resistance, and wide adaptability. Given the economic value of Brassica napus, which is mainly used for oil and protein, seed quality traits are important traits for basic research and breeding improvement, including oil content, protein content, oleic acid content, linoleic acid content, etc. In addition, the appearance quality of seeds (such as seed coat color, seed morphology, etc.) also has an important influence on the oil content and protein content of rapeseed. Therefore, mining key genes that regulate seed quality (including appearance quality) and improving seed quality traits are important goals of Brassica napus genetic breeding. Currently, there are more reports on genetic mapping of seed size and thousand seed weight in Brassica napus (Wang H, Yan M, Xiong M, et al. Genetic dissection of thousand-seed weight and fine mapping of cqSW.A03-2 via linkage and association analysis in rapeseed (Brassica napus L.). Theor Appl Genet, 2020, 133: 1321-1335; Zhang X, Huang Q, Wang P, et al. A 24,482-bp deletion is associated with increased seed weight in Brassica napus L. Theor Appl Genet, 2021, 134: 2653-2669). However, there are fewer reports on the regulation of seed morphology (such as seed length) in Brassica napus, and a large number of appearance quality regulatory genes remain to be mined. The auxin receptor protein TIR1 is involved in auxin signal perception and Aux / IAA protein degradation, and Aux / IAA protein can inhibit the expression of auxin-related genes by interacting with ARF, thereby regulating plant growth and development, and response to biotic and abiotic stress (Parry G, Calderon-Villalobos LI, Prigge M, et al. Complex regulation of the TIR1 / AFB family of auxin receptors. Proc Natl Acad Sci USA, 2009, 106(52): 22540-22545). There is no report on obtaining Brassica napus BnTIR1 regulating seed length by transgenic technology. SUMMARY

[0003] Invention purposes: the first purpose of the present application is to provide a Brassica napus BnTIR1 gene, the second purpose of the present application is to provide the application of the Brassica napus BnTIR1 gene in improving the appearance quality of plant seeds, the third purpose of the present application is to provide a method for improving the length of plant seeds, and the fourth purpose of the present application is to provide a transgenic plant obtained by the improving method.

[0004] Technical scheme: the Brassica napus BnTIR1 gene provided by the present application comprises a DNA fragment of the BnTIR1 gene, the nucleotide sequence of the DNA fragment is shown as SEQ ID NO:1, and the length of the nucleotide sequence is 1788bp.

[0005] Further, the amino acid sequence of the DNA fragment is shown as SEQ ID NO:2.

[0006] The expression cassette, the recombinant vector, the recombinant microorganism or the transgenic cell line of the Brassica napus BnTIR1 gene provided by the present application.

[0007] The Brassica napus BnTIR1 gene provided by the present application is applied to genetic breeding of improving the appearance quality of plant seeds.

[0008] The present application also includes the application of the expression cassette, the recombinant vector, the recombinant microorganism or the transgenic cell line of the Brassica napus BnTIR1 gene in genetic breeding of improving the appearance quality of plant seeds.

[0009] Further, the appearance quality of the plant seeds includes the length of the plant seeds, and the plant seeds are Arabidopsis thaliana seeds.

[0010] The present application also includes a transgenic plant cultivation method for improving the length of plant seeds, comprising the following steps:

[0011] (1) the plasmid of the DNA fragment of the Brassica napus BnTIR1 gene in claim 1 or 2 is transformed into Agrobacterium by using the electric shock method, so as to obtain Agrobacterium with the transformed plasmid;

[0012] (2) the Agrobacterium with the transformed plasmid is transformed into target plants by using a transgenic method, so as to obtain transgenic plants.

[0013] Further, the target plants are wild-type Arabidopsis thaliana, and the length phenotype of the seeds of the transgenic plants is obviously different from that of the target plants.

[0014] Further, the DNA fragment of the BnTIR1 gene of Brassica napus is obtained by using RT-PCR with primer F and primer R as primers, wherein the nucleotide sequence of the primer F is shown as SEQ ID NO: 7, and the nucleotide sequence of the primer R is shown as SEQ ID NO: 8.

[0015] Further, the expression vector carrying the DNA fragment containing BnTIR1 of the present application can be introduced into plant cells by using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation and other conventional biological technology methods (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] Further, the DNA fragment containing BnTIR1 is recovered by using a DNA recovery kit, and the fragment is connected into the pCAMBIA1300-FLAG backbone vector by using the method of enzyme digestion and ligation, so as to construct the overexpression vector of the DNA fragment, which is named as pCAMBIA1300-BnTIR1-FLAG.

[0017] Further, the pCAMBIA1300-BnTIR1-FLAG vector is introduced into Agrobacterium tumefaciens by using the electroporation method, and the Agrobacterium tumefaciens strain is GV3101. The pCAMBIA1300-BnTIR1-FLAG is transformed into the Arabidopsis thaliana receptor material Col-0 by the method of genetic transformation mediated by Agrobacterium infection, and the transgenic Arabidopsis thaliana with significantly increased expression amount of BnTIR1 protein relative to the wild type is successfully obtained. It is found by observation that the seeds of the transgenic Arabidopsis thaliana overexpressing BnTIR1 are significantly longer than those of the wild type Arabidopsis thaliana, which indicates that BnTIR1 can regulate the length of plant seeds.

[0018] The present application also includes the transgenic plants obtained by the breeding method

[0019] Beneficial effects: Compared with the prior art, the present application has the following obvious advantages:

[0020] (1) The present application finds a candidate gene capable of regulating the length of plant seeds by analyzing the morphology of the seeds of the transgenic Arabidopsis thaliana overexpressing Brassica napus BnTIR1, and provides an important theoretical basis for the genetic breeding of crop appearance quality (such as seed morphology).

[0021] (2) The application takes Brassica napus and model plant Arabidopsis thaliana as research materials, and finds that BnTIR1 is obviously differentially expressed in the seed development process of Brassica napus through comparative analysis of RNA-seq of different development periods of Brassica napus in the early stage, which indicates that the gene may be involved in regulating the development of seeds. Therefore, the DNA fragment of BnTIR1 is isolated from Brassica napus, and the function of BnTIR1 in the seed development process of Arabidopsis thaliana is identified, which will have very important significance for the genetic breeding improvement of the appearance quality of plant seeds. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 2 is a diagram of the expression of BnTIR1 in various tissues of Brassica napus;

[0023] Figure 2 Figure 4 is a diagram of the overexpression of BnTIR1 in Arabidopsis thaliana;

[0024] Figure 3 Figure 5 is a phenotype diagram of BnTIR1 overexpression Arabidopsis thaliana seeds, in which Col-0 is the wild type of Arabidopsis thaliana control; 35S: BnTIR1 is the transgenic Arabidopsis thaliana of BnTIR1 overexpression. A: Seed phenotype; B: Seed length (*, p<0.05). DETAILED DESCRIPTION

[0025] The technical solutions of the application will be further described below in combination with the drawings.

[0026] The following examples define the application and describe the method for cloning the DNA fragment sequence containing BnTIR1 and verifying the function of BnTIR1. According to the following description and these examples, those skilled in the art can determine the basic characteristics of the application, and can make various changes and modifications to the application without departing from the spirit and scope of the application, so that the application can be adapted to different uses and conditions.

[0027] Example 1: qRT-PCR analysis of the expression of BnTIR1 in various tissues and organs of Brassica napus

[0028] Different tissue samples of Brassica napus at different periods were taken: mature leaf (Leaf), cotyledon (Cotyledon), hypocotyl (Hypocotyl), root (Root), stem apical meristem (SAM), stem (Stem), 3 mm length of bud (Bud-3mm), 6 mm length of bud (Bud-6mm), endosperm (Endosperm), silique 14 days after pollination (Silique-14DAP), seed 21 days after pollination (Seed-21DAP), seed 28 days after pollination (Seed-28DAP), seed 35 days after pollination (Seed-35DAP), seed 42 days after pollination (Seed-42DAP), seed 50 days after pollination (Seed-50DAP), and were quickly frozen in liquid nitrogen and moved to a -70°C refrigerator for storage until RNA extraction. Total RNA extraction was performed using the RNA Isolater Total RNA Extraction Reagent kit of Vazyme Company. The expression level of BnTIR1 was detected by qPCR using the poly(A) tailing method, a reverse transcription kit (HiScript II Q RT SuperMix for qPCR (+gDNA wiper)), and an expression detection kit SYBR Green (AceQ qPCR SYBR Green Master Mix). The upstream primer was SEQ ID NO: 3: 5'-AGGCTGAAGAGGATGGTGGTGA-3', and the downstream primer was SEQ ID NO: 4: 5'-CATCACTCTCACGCAAATCAAGC-3'. BnActin was used as an internal reference gene, the internal reference upstream primer was SEQ ID NO: 5: 5'-TCTTCCTCACGTATCCTCCG-3', and the internal reference downstream primer was SEQ ID NO: 6: 5'-AGCCGTCTCCAGCTCTTGC-3'. qPCR analysis was performed using an ABI7500 fluorescence quantitative PCR instrument. The reaction program was as follows: 95°C for 5 min, 95°C for 10 s, 60°C for 30 s, collection of fluorescence signal, a total of 40 cycles; 60°C to 95°C, collection of fluorescence signal every 1°C for 1 s. Three technical repeats were set for each sample, and after the reaction, the software (7500 Software v2.0.1) of the ABI7500 was used for analysis and drawing, and the relative expression of BnTIR1 in each tissue and organ of Brassica napus was calculated, as shown in FIG. 1. Figure 1

[0029] Figure 1 The expression of BnTIR1 in each tissue of Brassica napus is shown in FIG. 1. Figure 1 ​It can be seen that the BnTIR1 of Brassica napus is mainly highly expressed in shoot apical meristem (SAM) and seed, and the expression level of the gene increases with the development of seed, i.e. the expression level in seed 28-50 days after pollination is higher than that in seed 14-21 days after pollination. This indicates that the BnTIR1 of Brassica napus may play a role in the process of seed development.

[0030] Example 2: Molecular cloning of Brassica napus BnTIR1 DNA fragment

[0031] The Brassica napus variety "Darmor-bzh" was taken at the three-leaf one-core stage, quickly frozen in liquid nitrogen, and stored in a -70°C refrigerator for total RNA extraction. The total RNA was extracted using the RNA Isolater Total RNA Extraction Reagent kit of Vazyme Company. The synthesis of Brassica napus cDNA was performed according to the instructions of HiScriptII Q RT SuperMix for qPCR (+gDNA wiper) of Nanjing Novogene Bioinformatics Technology Co., Ltd. to synthesize the first strand. The above cDNA first strand was used as the amplification template, and the primers F: 5'-CCTCACAATCTAGTAATCTCCGTTAA-3' (SEQ ID NO: 7) and R: 5'-TGAAGCAGCAGCCCATAA-3' (SEQ ID NO: 8) were used as primers. RT-PCR was used for DNA amplification, and the amplification conditions were as follows: 95°C for 5 min, 95°C for 30 s, 59°C for 30 s, 72°C for 1 min, a total of 35 cycles; 72°C for 10 min. After PCR, electrophoresis analysis was performed, and the DNA recovery kit of Kangweishijie Biotechnology Co., Ltd. was used to recover the target amplification fragment. The amplification fragment was connected to the pEASY-Blunt T vector of Beijing Quan Shi Jin Biotechnology Co., Ltd., and the E. coli competent cells were transformed. White colonies were picked for colony PCR identification of positive clones, and the positive clones were sent to Yangzhou Qikexin Biotechnology Co., Ltd. for sequencing. The plasmid verified by sequencing without error was named BnTIR1-T. The nucleotide sequence length of the sequenced Brassica napus BnTIR1 DNA fragment was 1788 bp, and the nucleotide sequence was as shown in SEQ ID NO: 1:

[0032]

[0033]

[0034] The amino acid sequence of the DNA fragment is as shown in SEQ ID NO: 2:

[0035]

[0036] Example 3: Construction of BnTIR1 overexpression vector

[0037] In order to better analyze the function of BnTIR1, it is overexpressed in Arabidopsis thaliana, and the function of the gene is studied by observing the phenotype of the transgenic material. The overexpression vector is constructed as follows: using the BnTIR1 DNA sequence cloning vector plasmid BnTIR1-T verified by sequencing as the template, primers p1300-BnTIR1-F: 5'-CGGCGCGCCGGTACCATGTATAAGCGAGTGGCCT-3'(SEQ ID NO: 9) (sequence-specific primer plus Kpn I site linker) and p1300-BnTIR1-R: 5'-CTGCAGCCCGGGGGATCCTAACCCGTTAGTAGTGATG-3'(SEQ ID NO: 10) (sequence-specific primer plus BamH I site linker) are used for DNA amplification by PCR, and the amplification conditions are as follows: 94°C for 3 min, 94°C for 15 s, 60°C for 15 s, 72°C for 1 min, for a total of 35 cycles; 72°C for 10 min. After PCR, electrophoresis analysis is performed, and the DNA recovery kit of Kangweishijie Biotechnology Co., Ltd. is used to recover the target amplification fragment. The amplification fragment is ligated into the pEASY-Blunt T vector of Beijing Quanshi Gold Biotechnology Co., Ltd., and the E. coli competent cells are transformed, white colonies are picked for colony PCR to identify positive clones, and the positive clones are sent to Yangzhou Qinko Biotechnology Co., Ltd. for sequencing. The cloning vector plasmid containing the BnTIR1 DNA sequence is digested with Kpn I and BamH I, and the target DNA fragment is recovered using the DNA recovery kit. The fragment is ligated with the corresponding enzyme-digested pCAMBIA1300-FLAG backbone vector to construct the BnTIR1 overexpression vector, named pCAMBIA1300-BnTIR1-FLAG.

[0038] Example 4: Arabidopsis thaliana genetic transformation of pCAMBIA1300-BnTIR1-FLAG overexpression vector

[0039] The pCAMBIA1300-BnTIR1-FLAG plasmid is introduced into the competent cells of Agrobacterium tumefaciens GV3101 strain (purchased from Shanghai Weidi Biotechnology Co., Ltd.) by electroporation. Single colonies are picked and inoculated in 25 mL YEB medium (containing 50 mg / L rifampicin) and cultured overnight, 5 mL bacterial solution is transferred to 100 mL YEB medium (containing 50 mg / L rifampicin) and cultured to OD 600= 0.7-0.8, the bacteria liquid is placed on ice for 10 min, 5000 rpm 4°C centrifugal 10 min to collect bacteria, add 100 mL sterile double distilled water to wash twice. Add 4 mL 10% glycerol to suspend bacteria, transfer to 50 mL centrifuge tube. 4°C 5500 rpm centrifugal 10 min to collect bacteria, add 500 μL 10% glycerol to resuspend bacteria, transfer to 1.5 mL centrifuge tube, and obtain the resuspension.

[0040] Take 50 μL resuspension, add 5 μL pCAMBIA1300-BnTIR1-FLAG recombinant plasmid, mix with a gun head, and then transfer to a 0.1 cm electroporation cup. Electroporation parameters: 200Ω, 1.7KV, 2.5F, add 500 μL LB culture solution immediately after electroporation. After 37°C 220 rpm culture for 1 h, take 100 μL bacteria liquid to coat LB medium containing kanamycin resistance to screen transformants, and culture at 28°C for 16 h to obtain Agrobacterium containing pCAMBIA1300-BnTIR1-FLAG recombinant plasmid.

[0041] The genetic transformation of Arabidopsis (dip flower method) is as follows:

[0042] (1) Select Arabidopsis wild type Col-0 with good growth vigor, about one month old, and about 5 cm bolting for infection;

[0043] (2) Two days before preparing for infection, Agrobacterium containing the plasmid to be transformed is expanded in a 15 mL shaking tube overnight, and then 1 mL Agrobacterium liquid containing pCAMBIA1300-BnTIR1-FLAG recombinant plasmid is added to a 200 mL conical flask for overnight expansion;

[0044] (3) Centrifuge the bacteria liquid at 5000 rpm for 5 min, resuspend the bacteria liquid to OD 600 ≈0.8 with infection buffer (2.5 g / L MS + 50 g / L Sucrose + 0.5 g / L MES + 200 μ / L Silwet L-77, pH 5.8), remove the Arabidopsis that has flowered and siliques, and immerse the Arabidopsis inflorescences in the resuspension for 30 s-1 min;

[0045] (4) After infection, cover the Arabidopsis plants with a preservative bag and place them in the dark, remove the preservative bag after 24 h, spray sterile water to wash off Silwet L-77 on the surface of the plants, and finally place them in a light incubator for normal growth;

[0046] (5) After 7-10 days, perform secondary infection, and the infection process is the same as steps (2)-(4). After the siliques are fully mature, collect the seeds.

[0047] Example 5: Screening and identification of positive transgenic Arabidopsis thaliana plants

[0048] The positive screening procedure of the transgenic Arabidopsis thaliana is as follows:

[0049] (1) After soaking the Arabidopsis thaliana seeds received in Example 4 in 75% alcohol for 1 min, soak them in 4% NaClO for 10 min, and finally rinse them with sterile water for 4-5 times;

[0050] (2) Place the sterilized Arabidopsis thaliana seeds in step (1) on 1 / 2MS + 50 mg / L Hyg fixed medium, and culture them in a light incubator, with the culture conditions being 16 h light / 8 h dark, 22°C, and 70% humidity;

[0051] (3) After about 10 days of culture, transplant the plants with obvious growth of hypocotyls and roots into soil;

[0052] (4) After about 14 days of growth of the seedlings, extract DNA from the leaves, perform positive identification by PCR, and collect seeds from the positive plants;

[0053] (5) Repeat the above steps (1)-(4) until a homozygous transgenic line is screened (i.e., all Arabidopsis thaliana seeds can normally germinate and grow on the screening medium containing Hyg).

[0054] In step (4), the Arabidopsis thaliana genomic DNA is extracted by a rapid plant DNA extraction method, with the specific steps being as follows:

[0055] (1) Take two young leaves (about 0.2 g), cut them into pieces, and place them in a 2 mL centrifuge tube, add 250 μL of DNA buffer (500 mM Tris-HCl, 300 mM NaCl, 300 mM Sucrose, pH = 7.5) and two steel balls (diameter 6.7 mm), and use a sample machine to crush the leaf samples at 50 Hz for 180 s;

[0056] (2) Incubate the crushed leaf samples at 95°C for 10 min;

[0057] (3) Take out the incubated leaf samples, cool them to room temperature, and centrifuge them at 12000 rpm for 5 min;

[0058] (4) Transfer 50 μL of the supernatant to a new 1.5 mL centrifuge tube, dilute it 5 times, and reserve it for use.

[0059] Take 1 μL DNA as a template, and use primer 35S-F: 5'-CTTCGCAAGACCCTTCCTC-3'(SEQ ID NO: 11) and primer 35S-R: 5'-ACTCTTCTCCATCCATTTCC-3'(SEQ ID NO: 12) to perform PCR amplification, and the amplification conditions are: 94℃ 5 min; 94℃ 30 s, 58℃ 30 s, 72℃ 2 min, for 35 cycles; 72℃ 10 min. The transgenic Arabidopsis DNA is used as a template, and a specific target fragment can be amplified, which proves that the target vector pCAMBIA1300-BnTIR1-FLAG has been integrated into the Arabidopsis genome.

[0060] Example 6: Overexpression of BnTIR1 changes the morphology of Arabidopsis seeds

[0061] The present application adopts the method of immunoblotting to detect the protein expression level of BnTIR1 in transgenic Arabidopsis, and the specific steps are as follows:

[0062] (1) Take two young leaves (seedlings grow for about 14 days, about 0.1 g), cut and put into 2 mL centrifuge tube, then add two steel balls (diameter 6.7 mm), and use a sample machine to break the leaf sample at 50 Hz for 180 s;

[0063] (2) Add 100 μL of plant Western and IP cell lysis solution of Shanghai Biyun Tian Biotechnology Co., Ltd., and incubate at 4℃ for 1 h;

[0064] (3) Centrifuge the sample obtained in the above step (3) at 12000 rpm for 5 min;

[0065] (4) Transfer 40 μL of supernatant to a new 1.5 mL centrifuge tube, add 10 μL of 5×SDS Loading buffer of Jiangsu Kangwei Shijide Biological Technology Co., Ltd., and heat at 100℃ for 5 min to obtain the sample for Western blotting detection.

[0066] The specific steps of Western blotting are as follows:

[0067] (1) Prepare polyacrylamide gel according to the One-Step PAGE Gel Fast Preparation Kit (10%) kit of Nanjing Novozyme Biological Technology Co., Ltd.;

[0068] (2) After the gel is solidified, load 10-20 μl of sample per well, and electrophorese: concentrate gel at 20 mA for 15 min, and separate gel at 40 mA for 1.5 h;

[0069] (3) After electrophoresis, take out the gel, rinse with water for 10 min, and change the solution once;

[0070] (4) Transfer the membrane by wet transfer method, 300 mA, 1.5 h;

[0071] (5) After transferring the membrane, take out the PVDF membrane and rinse with TBST for 5 min;

[0072] (6) Add 10 mL of blocking solution One Step Western Blocking Buffer (purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.), incubate at room temperature for 1-2 h;

[0073] (7) Rinse the PVDF membrane after incubation in step (6) with TBST for 3 times, 10 min each time;

[0074] (8) Add 10 mL of Jiangsu Kangwei Century Biotechnology Co., Ltd. primary antibody Anti Flag-Tag Mouse Monoclonal Antibody (diluted 1000 times with antibody diluent), incubate at room temperature for 1-2 h or at 4°C overnight;

[0075] (9) Rinse the PVDF membrane after incubation in step (8) with TBST for 3 times, 10 min each time.

[0076] (10) Add 10 mL of Jiangsu Kangwei Century Biotechnology Co., Ltd. secondary antibody Goat Anti-Mouse IgG, HRP Conjugated (diluted 10000 times with antibody diluent), incubate at room temperature or at 37°C for 1 h.

[0077] (11) Rinse the PVDF membrane after incubation in step (10) with TBST for 3 times, 5 min each time.

[0078] (12) Color development with BeyoECL Plus (ultra-sensitive ECL chemiluminescence kit) from Shanghai Biyun Tian Biotechnology Co., Ltd.

[0079] (13) Mix equal volumes of appropriate amount of BeyoECL Plus A and B to prepare working solution;

[0080] (14) Dry the TBST on the PVDF membrane, according to the size of the PVDF membrane, add 1 mL of BeyoECL Plus working solution to the PVDF membrane, incubate at room temperature for 3-5 min in the dark; 2

[0081] (15) Transfer the PVDF membrane after incubation in step (14) to a new clean plastic film, avoiding the formation of bubbles;

[0082] ​(16) Exposure to X-ray film or development using a developer.

[0083] The results are as follows Figure 2 As shown, Figure 2 The results showed that transgenic plants with significantly higher BnTIR1 protein expression levels compared with wild-type Arabidopsis were successfully obtained, that is, BnTIR1 protein accumulation was obvious in BnTIR1 overexpressing plants.

[0084] Phenotypic analysis of seeds of transgenic Arabidopsis thaliana overexpressing BnTIR1 was performed. Figure 3 As shown. Figure 3 The researchers found that, compared to wild-type Arabidopsis Col-0, transgenic Arabidopsis plants overexpressing BnTIR1 exhibited significant seed morphology changes, with seeds becoming significantly longer. The length of seeds in transgenic Arabidopsis plants overexpressing BnTIR1 increased by approximately 10% compared to the control.

[0085] The present invention isolates genes from Brassica napus related to plant seed morphological development, enabling targeted identification of candidate genes regulating plant seed development and providing theoretical guidance for the study of genes associated with crop seed appearance quality. The genes associated with seed length development isolated by the present invention originate from the plant itself and have minimal environmental impact. Functional studies of the isolated genes can provide a basis for breeding and improvement of crops such as Brassica napus, and are of great significance for modifying the appearance quality traits of Brassica napus seeds.

[0086] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. Overexpression of Brassica napus BnTIR1 Application of a gene for increasing plant seed length in genetic breeding, the Brassica napus BnTIR1 The nucleotide sequence of the gene is shown in SEQ ID NO: 1, Brassica napus BnTIR1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2, and the plant is Arabidopsis thaliana or rapeseed.

2. A method for cultivating transgenic plants to increase the length of plant seeds, characterized in that: comprising the following steps: (1) Using electric shock method to BnTIR1 The plasmid of the gene is transformed into Agrobacterium to obtain Agrobacterium with the transformation plasmid; the Brassica napus BnTIR1 The nucleotide sequence of the gene is shown in SEQ ID NO: 1, Brassica napus BnTIR1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2; (2) using the transgenic method to transform the target plant with the agrobacterium carrying the transformation plasmid, to obtain a transgenic plant; the plant is Arabidopsis thaliana or Brassica napus.

3. The breeding method according to claim 2, characterized by, The target plant is wild-type Arabidopsis thaliana, and the seed length phenotype of the transgenic plant is obviously different from that of the target plant.

4. The breeding method according to claim 2, characterized by, Brassica napus BnTIR1 The gene was obtained by DNA amplification using RT-PCR with primer F and primer R as primers, the nucleotide sequence of the primer F is shown as SEQ ID NO: 7, and the nucleotide sequence of the primer R is shown as SEQ ID NO: 8.

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