BjuSPL10c gene, overexpression vector, construction method and application of BjuSPL10c, which regulates stem enlargement in stem mustard.

Abstract

The application belongs to the technical field of plant molecular breeding, and particularly relates to a BjuSPL10c gene for regulating stem swelling of tumorous stem, an overexpression vector, a construction method and application thereof. The BjuSPL10c gene for regulating stem swelling of tumorous stem has a nucleotide sequence as shown in SEQ ID NO. 1 and an amino acid sequence as shown in SEQ ID NO. 2. The application firstly clones a full-length sequence of the BjuSPL10c gene of tumorous stem, and obtains a protein sequence coded by the BjuSPL10c gene. By constructing an overexpression vector, a tumorous stem plant overexpressing the BjuSPL10c gene is obtained. Overexpression of the BjuSPL10c gene of tumorous stem can promote stem swelling of tumorous stem, which provides an effective gene for tumorous stem and other plant genetic engineering. The application also provides an important target for plant molecular genetic improvement of metamorphic stem development, and has important value and significance for cultivation and production of new varieties of tumorous stem.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to a BjuSPL10c gene for regulating stem tuber enlargement in *Strombus haematomarginatus*, its overexpression vector, construction method, and application. Background Technology

[0002] *Brassica juncea* var. *Tumida* Tsen et Lee is both a fresh vegetable and an important processed vegetable for pickling. The modified tuberous stem of *Brassica juncea*, as a crucial organ for both fresh consumption and processing, is a primary raw material and breeding trait for Fuling pickled mustard tuber. Therefore, the tuberous stem is a key target trait in *Brassica juncea* breeding. The tuberous stem of *Brassica juncea* is an above-ground modified stem, formed by 1-5 tubercular protrusions from the base of the petiole that expand laterally with the stem. Research on the related molecular regulatory mechanisms is scarce. Previous studies have found that the swelling of the tuberous stem in *Brassica juncea* is a process of increased cell number and volume. The cambium cells of *Brassica juncea* first divide tangentially, then radially, forming the long axis of the tuberous stem, and finally resulting in the swollen tuberous stem. Furthermore, intranuclear polyploidization also participates in the swelling of the tuberous stem. The external myelin cells are the source of the increased number of cells in the swollen tuberous stem, while intranuclear replication participates in the expansion of the internal myelin cells.

[0003] SPLs are plant-specific transcription factors containing a highly conserved 76-amino acid domain called the SBP, which acts on nuclear input and binds specifically to flanking regions containing the GTAC core motif and gene-specific structures. Studies have identified 17 SPL genes in Arabidopsis thaliana, whose proteins contain a highly conserved DNA-binding domain, SBP-DBD, and two zinc-finger-like structures of 79 amino acid residues each, participating in various stages of plant growth and development. Literature reports that 10 SPL members in Arabidopsis are downregulated by miR156. Based on their functions, these SPLs can be divided into three categories: 1. Regulating the transition from juvenile to adult vegetative growth and from vegetative to reproductive growth, including SPL2, SPL9, SPL10, SPL11, SPL13, and SPL15; 2. Promoting the transformation of floral meristems, including SPL3, SPL4, and SPL5; 3. Unknown functions, including SPL16. In addition, SPL1 and SPL12 are involved in regulating heat tolerance during the reproductive growth phase of plants, SPL8 is involved in GA-regulated plant development, SPL6 is involved in plant immune response, SPL7 is involved in regulating copper homeostasis within plants, and SPL14 is involved in regulating plant morphogenesis.

[0004] In summary, the problem with the existing technology is that although the SPL10 gene of Arabidopsis thaliana has been studied, there are no research reports on the function of the BjuSPL10c gene in Brussels sprouts, and its specific function remains unclear. Summary of the Invention

[0005] The purpose of this invention is to provide a BjuSPL10c gene for regulating stem tuber enlargement in *Strombus haematomarginatus*, an overexpression vector, a construction method, and its application. A gene that can directly regulate stem tuber development in *Strombus haematomarginatus* has been discovered.

[0006] The technical solution adopted in this invention is a BjuSPL10c gene for regulating stem tuber enlargement in mustard tubers, the nucleotide sequence of which is shown in SEQ ID NO.1 and the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] SEQ ID NO.1:

[0008] 5-’ATGGACTGCAACATGGTATCTCCGATGTGGGACTGGGAGCATTTGA TCATCCCCAACCTGTCAAAGACTGAAACTGACAAGAAACCGCCTTCTACTACTGATTGGGATATTGAGAAAGGTGAAGGGATTGAATCTATATTATTTCCCTCTTTCACTGGCTCCTCCTCCACCACCGGTTTCCTAAGCTCACAGTTGACCTCCACCAACTCATCATCTCCCAAACTCAAAAGTTCCCTTGGAGATTTTGACCAAGTCAAGGCATCCACAGCTCTCCAGGTGGTATCTGCTGAATCAGACCTTTGTTTAAAACTTGGGAAGCGGAGTTACTCTGATGAAGAGACTTGGGGTAGAAACAACAATAATGACATTTCAGCCGCTGTTTCTGTGAAGCTCTTGACTCCTCCATCTGTTGTTGCTCTGAAGAAATCCAAATCATCATGTGGTCAGAGCATGCAAGTACCTCGTTGTCAGATTGATGGCTGTGAACTGGATCTCTCATCTGCCAAGGATTATCATCGCAAGCATAGAGTCTGCGAACGCCATTCAAAGTGCCCTGTAGTTACTGTGGGTGGCTTTGAACGTCGATTCTGCCAACAGTGTAGCAGGTTGCATGCGGTCTCTGAATTTGATGAGAAGAAACGTAGCTGCCGTAAACGTCTTTCGCATCATAATGCGAGGCGCCGCAAGTCACAAGGAATGTTTCCTTTGAATCCAGAGAGGGTGTACAATGGAAGACAGCATACGAATATGTTGTGGAATGAGTTGTCCCTTAACACAGGATCTGAAGAAAAGTATGCATGGGGTACTACTACTTATGAGACAAAGCCTACGCAGATGGAAAGCGGCTTTACTCTGAACTTCCAGAGAGGTAGTGGCGCTGAGGAGCAGCAGGTGTTTGCTAGTAGCAACCTTTCTTTCTCTGCGTATCAACCCTCAGCAGGGAAATCCAACGTTCAACTTCCTAGCAAAGGTGTAGGAGAATACTCAGGAG GCCTCTATGAAACGCAAGATTTCTACAGTGCTCTCTCTCTTCTGTCAACGTCTTCGGATTCACATGGGACCAAACACCATCCCATGGTTGAATCACAGCCAATACATGGCACCTTCCCTACTCATTTTTTATGA-3'.

[0009] SEQ ID NO.2:

[0010] MDCNMVSPMWDWEHLIIPNLSKTETDKKPPSTTDWDIEKGEGIESILFPSFFTGSSSTTGFLSSQLTSTNSSSPKLKSSLGDFDQVKASTALQVVSAESDLCLKLGKRSYSDEETWGRNNNNNDISAAVSVKLLTPPSVVALKKSKSSCGQSMQVPRCQIDGCELDLSSAKDYHRKHRVCERHSKCPVVTVGGFE RRFCQQCSRLHAVSEFDEKKRSCRKRLSHHNARRRKSQGMFPLNPERVYNGRQHTNMLWNELSLNTGSEEKYAWGTTTYETKPTQMESGFTLNFQRGSGAEEQQVFASSNLSFSAYQPSAGKSNVQLPSKGVGEYSGGLYETQDFYSALSLLSTSSDSHGTKHHPMVESQPIHGTFPTHFL*, where * represents the termination signal.

[0011] An overexpression vector constructed using the BjuSPL10c gene of stem mustard.

[0012] An overexpression vector constructed using the BjuSPL10c gene from *Strombus haematomarginatus* is disclosed. The construction method includes the following steps: synthesizing a pair of primers, with primer sequences shown in SEQ ID NO.3 and SEQ ID NO.4; extracting total RNA from the stems of wild-type *Strombus haematomarginatus* and reverse transcribing it into cDNA; using the cDNA as a template, performing PCR amplification with the primers to obtain the *Strombus haematomarginatus* BjuSPL10c target gene; and sequentially digesting, ligating, and transforming the expression vector and the *Strombus haematomarginatus* BjuSPL10c target gene to obtain the overexpression vector.

[0013] Preferably, the tubercle is a tubercle in the middle stage of swelling.

[0014] Preferably, the expression vector is p1300-GFP.

[0015] Application of the BjuSPL10c gene or overexpression vector of BjuSPL10c in promoting stem enlargement in stem mustard.

[0016] The overexpression vector is used to prepare stem mustard plants overexpressing the BjuSPL10c gene. The preparation process includes the following steps: transforming the overexpression vector into Agrobacterium competent cells to obtain positive recombinant Agrobacterium; infecting the cotyledons of stem mustard seedlings with the positive recombinant Agrobacterium, transferring the cotyledons of the stem mustard seedlings to a co-culture medium, and culturing in the dark; sequentially transferring the cotyledons of the stem mustard seedlings to a callus induction medium to produce callus, an adventitious bud differentiation medium to produce adventitious buds, and a rooting medium to produce roots, thereby obtaining stem mustard plants overexpressing the BjuSPL10c gene.

[0017] Preferably, when the positive recombinant Agrobacterium tumefaciens infects the cotyledons of stem mustard seedlings, the OD of the bacterial solution of the positive recombinant Agrobacterium tumefaciens is... 600 It is 0.05.

[0018] Preferably, the callus induction culture medium is formulated as follows: 2 mg of 6-benzylaminopurine, 0.15 mg of α-naphthaleneacetic acid, 20 mg of hygromycin, 500 mg of termethin and 8 g of agar are added to each liter of MS medium.

[0019] The adventitious shoot differentiation medium formula is as follows: 2 mg of 6-benzylaminopurine, 0.15 mg of α-naphthaleneacetic acid, 20 mg of hygromycin, 500 mg of termethin and 8 g of agar are added to each liter of MS medium.

[0020] The rooting medium formula is as follows: add 0.1 mg of α-naphthaleneacetic acid, 20 mg of hygromycin, 500 mg of termethin and 8 g of agar to half a liter of MS medium.

[0021] The beneficial effects of this invention are that it is the first to clone the full-length sequence of the BjuSPL10c gene in *Mustela stenoptera*, obtain its encoded protein sequence, and construct an overexpression vector to obtain *Mustela stenoptera* plants overexpressing the BjuSPL10c gene. The results show that overexpression of the BjuSPL10c gene can promote the enlargement of the tuberous stems in *Mustela stenoptera*. This provides an effective gene for genetic engineering of *Mustela stenoptera* and other plants. This invention provides an important target for the molecular genetic improvement of plants with modified stem development, and has significant value and importance for the breeding and production of new *Mustela stenoptera* varieties. Attached Figure Description

[0022] Figure 1 This is an electrophoresis image of the target gene amplification and expression vector digestion of BjuSPL10c from stem mustard.

[0023] Figure 2 Electrophoresis diagram showing the gDNA detection results in stem mustard plants overexpressing the BjuSPL10c gene.

[0024] Figure 3 The figure shows the results of detecting the expression level of BjuSPL10c mRNA in stem mustard plants that overexpress the BjuSPL10c gene.

[0025] Figure 4 The results of comparing the tubercle weights of stem mustard plants overexpressing the BjuSPL10c gene with those of wild-type stem mustard plants are shown in Figure a. a) Tubercle weight statistics; b) Phenotypic diagram. Detailed Implementation

[0026] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0027] The inventive concept of this invention is as follows: This invention provides a BjuSPL10c gene for regulating stem tuber enlargement in *Strombus styrax*, the nucleotide sequence of which is shown in SEQ ID NO.1; the amino acid sequence of which is shown in SEQ ID NO.2.

[0028] The inventors discovered the BjuSPL10c gene and, by overexpressing it in *Musa coccinea*, promoted tuber enlargement, thus increasing yield. *Musa coccinea*, a biennial vegetative-season harvest crop, has tubers formed by the lateral expansion of the stem along the base of the petiole, consisting of 1-5 tubercular protuberances. This process involves multiple factors, including morphology, physiology, biochemistry, and genetics. Morphological studies revealed a close relationship between cell division and intranuclear replication and tuber enlargement. Specifically, the division of external medullary cells increases the number of cells in the enlarged tuber, while intranuclear replication promotes the enlargement of internal medullary cells. The tuber enlargement process in *Musa coccinea* is accompanied by continuous cell volume expansion. First, cambium cells undergo tangential division, increasing the number of cell layers. Then, radial division forms the long axis of the tuber. Subsequently, non-directional division occurs within the tuber, causing it to develop in various directions, ultimately forming the enlarged tuber. Previous studies have revealed intranuclear polyploidy during tuberculum enlargement, with a continuous increase in cell ploidy and the degree of intranuclear polyploidy. In mature stems, cell ploidy initially decreases and then increases from the outside in, with the lowest ploidy near the cambium and the highest in the inner pith. Further research has identified BjXTH1 (xyloglucanendotransglucosylase / hydrolase 1), BjXTH2 (xyloglucanendotransglucosylase / hydrolase 2), BjuBXL1-1 (β-D-xylosidase 1-1), and BjuBXL1-2 (β-D-xylosidase 1-2) as regulators of pith cell enlargement, while BjRBRs (Retinoblastoma-relateds family genes) regulate cell division and intranuclear replication.

[0029] Prior to the invention, the inventors had not anticipated that the SPL10c gene in stem mustard was associated with stem tuber enlargement. The inventors also promoted stem tuber enlargement and increased stem mustard yield by overexpressing the BjuSPL10c gene.

[0030] To achieve overexpression of the BjuSPL10c gene, this invention also provides an overexpression vector for the BjuSPL10c gene, the construction method of which includes the following steps:

[0031] A pair of primers were synthesized, and the primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0032] Total RNA was extracted from the stems of wild-type stem mustard and reverse transcribed into cDNA.

[0033] Using cDNA as a template, PCR amplification was performed with the primers to obtain the target gene BjuSPL10c from stem mustard.

[0034] The expression vector and the target gene BjuSPL10c from stem mustard were sequentially digested with enzymes, ligated, and transformed to obtain the overexpression vector.

[0035] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods and can be completed according to the described recombination technology, see Molecular Cloning, Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; MaXetal, Arobust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicotplants. Molecular Plant. 2015, 8(8):1274-1284.; the materials and reagents used can all be obtained commercially.

[0036] Example 1: Construction of the BjuSPL10c overexpression vector for stem mustard

[0037] Using the enlarged tuberous stems of wild-type *Strombyx mori* stored at -80℃ as material, total RNA was extracted from the enlarged tuberous stems of wild-type *Strombyx mori* using the RNAprep pure Plant Kit, following the instructions in the manufacturer's manual. 2 μL of total RNA was taken and reverse transcribed into first-strand cDNA using the All-in-One 5×RT MasterMix kit, purchased from abm (catalog number G592).

[0038] The reverse transcription program was: 37℃, 15 min; 60℃, 10 min; 95℃, 3 min.

[0039] Using first-strand cDNA synthesized by reverse transcription as a template, the coding sequence of BjuSPL10c was amplified using the high-fidelity enzyme MegaFi™ Fidelity2×PCRMasterMix. The PCR amplification system consisted of: 25 μL Primix, 2 μL of 10 μM BjuSPL10c-F forward primer, 2 μL of 10 μM BjuSPL10c-R reverse primer, 2 μL of cDNA template, and sterile ddH2O to a final volume of 50 μL. The forward primer sequence is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4.

[0040] SEQ ID NO. 3: BjuSPL10c-F, 5'-GAGAACACGGGGGACTCTAGAATGGACTGCAACATGGTATCTCCG-3'.

[0041] SEQ ID NO. 4: BjuSPL10c-R, 5'-GCCCTTGCTCACCATTCTAGATCATAAAAAATGAGTAGGGAAG-3'.

[0042] The obtained PCR amplification products were analyzed by agarose gel electrophoresis, such as... Figure 1 As shown, under ultraviolet light irradiation, a specific amplification band, namely the BjuSPL10c target gene, can be observed at a position of 1kb–2kb. The BjuSPL10c target gene was recovered using an agarose gel extraction kit D1200 purchased from Solarbio for later use.

[0043] Figure 1 The image shows the electrophoresis results of BjuSPL10c gene amplification and expression vector digestion in *Strombyx mori*. M: Marker; Lane 1: Electrophoresis results of BjuSPL10c target gene amplification; Lane 2: Electrophoresis results of p1300-GFP expression vector digestion products.

[0044] The p1300-GFP vector was linearized using Xba I restriction endonuclease (purchased from Thermo Scientific Fast Digest, catalog number 91252720). The amplified and recovered BjuSPL10c target gene was recombinantly ligated into the linearized p1300-GFP vector using 2×Pro Ligation-Free Cloning MasterMix (purchased from abm, catalog number E086-1). The ligation product was then transformed into *E. coli* DH5α. *E. coli* DH5α containing the ligation product was plated and cultured. Three positive clones were picked from an LB plate containing 50 mg / L kanamycin sulfate and analyzed by colony PCR. The amplified bacterial culture containing the target band was then sent to BGI Genomics for sequencing and screening to obtain the overexpression vector p1300-BjuSPL10c-GFP. The results showed that the full-length sequence of the stem mustard (BjuSPL10c) gene is shown in SEQ ID NO.1, containing a reading frame of 1125 bp including the stop codon. Based on SEQ ID NO.1, the amino acid sequence of stem mustard (BjuSPL10c) was obtained using Primer Premier software, as shown in SEQ ID NO.2, containing 375 amino acid residues, where * indicates a stop signal.

[0045] Example 2: Application of the BjuSPL10c gene overexpression vector in stem mustard.

[0046] The overexpression vector p1300-BjuSPL10c-GFP was transformed into Agrobacterium GV3101 competent cells. The Agrobacterium concentration OD was adjusted. 600The concentration of 0.05 mg / L was used to infect the cotyledons of *Streptococcus solani* seedlings for 10 minutes. The seedlings were then cultured in the dark for 2 days in a co-medium consisting of 2 mg 6-benzylaminopurine, 0.15 mg α-naphthylacetic acid, and 8 g agar per liter of MS medium. The co-cultured seedlings were then transferred to callus induction medium to induce callus formation. The callus induction medium consisted of 2 mg 6-benzylaminopurine, 0.15 mg α-naphthylacetic acid, 20 mg hygromycin, 500 mg termethin, and 8 g agar per liter of MS medium. The callus was then... The adventitious shoots were transferred to an adventitious shoot differentiation medium, which was formulated as follows: 2 mg of 6-benzylaminopurine, 0.15 mg of α-naphthylacetic acid, 20 mg of hygromycin, 500 mg of termethin, and 8 g of agar were added to each liter of MS medium. When the adventitious shoots reached 3 cm in length, they were transferred to a rooting medium, which was composed of 0.1 mg of α-naphthylacetic acid, 20 mg of hygromycin, 500 mg of termethin, and 8 g of agar added to each half liter of MS medium. The rooted lines were then transferred to nutrient soil and cultured in a light-controlled culture chamber to obtain stem mustard plants overexpressing the BjuSPL10c gene.

[0047] gDNA was extracted from stem mustard plants overexpressing the BjuSPL10c gene and detected by PCR. The results are as follows: Figure 2 As shown, the detection primers are p1300-GFP-F and p1300-GFP-R, and the primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6. Figure 2 In the middle, M: Marker; lanes 1 to 3: 3 wild-type stem-nourished mustard plants; lanes 4 to 13: 10 T0 generation stem-nourished mustard plants overexpressing the BjuSPL10c gene.

[0048] SEQ ID NO.5: p1300-GFP-F, 5'-GAATTCGCATGCCTGCAGGTC-3';

[0049] SEQ ID NO.6: p1300-GFP-R, 5'-GTGACAGATAGCTGGGCAATG-3'.

[0050] T0 generation *Strombyx mori* plants overexpressing the BjuSPL10c gene and capable of amplifying GFP nucleotide fragments, obtained from preliminary screening, were self-crossed for three generations. RNA was extracted from these plants, and the expression level of BjuSPL10c mRNA in the three self-crossed *Strombyx mori* plants was detected using qRT-PCR. Transgenic *Strombyx mori* plants with significantly increased BjuSPL10c mRNA expression were screened. The results of the BjuSPL10c mRNA expression level detection are as follows: Figure 3 As shown, the selected BjuSPL10c transgenic plants were transplanted into the experimental field to observe the stem phenotype. Figure 3 The image shows the results of BjuSPL10cmRNA expression level detection in BjuSPL10c stem mustard lines after 3 generations of self-pollination. WT is the wild-type control, and OE4, OE8, OE9, OE10, OE11, OE12 and OE13 are BjuSPL10c stem mustard lines after 3 generations of self-pollination.

[0051] OE4, OE8, OE9, OE10, OE11, OE12, and OE13 were cultured, and the weight of the nodule stem was observed and measured on days 30, 50, 70, 90, 100, and 130 of growth. OE12 showed the highest expression level of BjuSPL10c mRNA and the most significant phenotype. Only the phenotypic statistics of OE12 are presented here; the phenotypic results can be found in [link to phenotypic statistics]. Figure 4 Compared with the wild type, the stem weight and volume of BjuSPL10c-overexpressing stem mustard plants were significantly increased. Figure 4 Phenotypic images of BjuSPL10c overexpressing stem mustard lines and wild-type plants showing enlarged stem nodules. WT is the wild-type control, and OE12 is BjuSPL10c overexpressing line 12. Figure 4 a represents the statistical analysis of stem weight in BjuSPL10c-overexpressing plants; Figure 4 b represents the observation of the tuberculum swelling phenotype in plants overexpressing BjuSPL10c.

[0052] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0053] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A stem tumor of Brassica napus modulating tumor enlargement BjuSPL10c application characterized in that, stem mustard BjuSPL10c The nucleotide sequence of the gene is shown as SEQ ID NO. 1; the amino acid sequence is shown as SEQ ID NO. 2; The application refers to Brassica napobrassica BjuSPL10c gene or Brassica napobrassica BjuSPL10c application of overexpression vector of the gene in promoting tumorous stem enlargement of Brassica napobrassica 2. Use according to claim 1, characterized in that, The construction method of the overexpression vector comprises the following steps: A pair of primers are synthesized, and the primer sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4; Total RNA of the tumor stem of the wild type B. juncea is extracted and reversely transcribed into cDNA; PCR amplification was performed using the cDNA as template with the primers to obtain Brassica napus BjuSPL10c Gene of interest; Expression vector and stem canola BjuSPL10c The objective gene was sequentially subjected to enzyme digestion, ligation and transformation to obtain the overexpression vector.

3. Use according to claim 2, characterized in that, The tumor stem is a tumor stem in the middle of expansion.

4. Use according to claim 2, characterized in that, The expression vector is p1300-GFP.

5. The use according to claim 1, characterized in that, The overexpression vector is used to prepare a plant of Brassica rapa overexpressing BjuSPL10c the Brassica rapa plant overexpressing the gene to achieve the swollen stem of the Brassica rapa plant, and the swollen stem of the Brassica rapa plant is used to prepare a swollen stem of the Brassica rapa BjuSPL10c The preparation process of the Brassica rapa plant overexpressing the gene comprises the following steps: The overexpression vector is used to transform the competent cells of Agrobacterium, and positive recombinant Agrobacterium is obtained; The positive recombinant Agrobacterium is used to dip the cotyledon of the seedling of B. juncea, and the cotyledon of the seedling of B. juncea is transferred to co-culture medium and dark cultured; The cotyledon of the seedling of the stem canola was transferred to the callus induction medium, the adventitious bud differentiation medium and the rooting medium in sequence to generate callus, adventitious bud and root, respectively, and the stem canola plant overexpressing the gene was obtained. BjuSPL10c gene.

6. Use according to claim 5, characterized in that, When the cotyledon of the seedling of stem mustard is infiltrated by the positive recombinant Agrobacterium, the OD of the bacterial liquid of the positive recombinant Agrobacterium is 0.

05. 600 0.

05.

7. The use of the overexpression vector according to claim 5, characterized in that, The formula of the callus induction medium is: 2 mg of 6-benzylaminopurine, 0.15 mg of α-naphthaleneacetic acid, 20 mg of hygromycin, 500 mg of phosphinothricin and 8 g of agar are added into each liter of MS medium; The formula of the adventitious bud differentiation medium is: 2 mg of 6-benzylaminopurine, 0.15 mg of α-naphthaleneacetic acid, 20 mg of hygromycin, 500 mg of phosphinothricin and 8 g of agar are added into each liter of MS medium; The formula of the rooting medium is: 0.1 mg of α-naphthaleneacetic acid, 20 mg of hygromycin, 500 mg of phosphinothricin and 8 g of agar are added into each liter of half MS medium.

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