Bjuubp15 gene and its encoded protein from brassica juncea and application

Abstract

This invention discloses a stem-nodular mustard BjuUbp15 This invention relates to genes and their encoded proteins and their applications, belonging to the field of plant genetic engineering technology. This invention cloned *Strombyx mori* using homologous cloning. BjuUbp15 Genes. Using Agrobacterium-mediated transformation, ... BjuUbp15 Genetic transformation of stem mustard with a gene overexpression vector yielded overexpressing plants. Experimental results showed that... BjuUbp15 The tuberous stems of the gene-overexpressing plants were significantly larger than those of the wild type. Therefore, the tuberous stem mustard of this invention... BjuUbp15 The gene has a positive regulatory effect on the size of the tuberous stem of *Mustela stenoptera*. Therefore, the present invention... BjuUbp15 Genes have great potential for increasing the yield of stem mustard.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a stem-nodulous mustard. BjuUbp15 Genes and their encoded proteins and their applications. Background Technology

[0002] Stem mustard ( Brassica juncea var. tumida Tsen et Lee (Brassica oleracea var. tectorum) is a crop belonging to the Brassicaceae family and the Brassicaceae genus. It is a variety of mustard. The tuberous stem of Tsen et Lee is an important organ for fresh consumption and processing, and is the main raw material and breeding trait for making Fuling pickled mustard tuber. Therefore, the tuberous stem is a major target trait for breeding Tsen et Lee. The tuberous stem of Tsen et Lee is an above-ground modified stem, formed by 1-5 tuberous protrusions formed from the base of the petiole as the stem expands laterally. Research on the related molecular regulatory mechanisms is scarce. Previous studies have found that the swelling of the tuberous stem of Tsen et Lee is a process of increasing cell number and volume. The cambium cells of Tsen et Lee first divide tangentially, then divide radially to form the long axis of the tuberous stem, and finally form the swollen tuberous stem. In addition, intranuclear polyploidization also participates in the swelling of the tuberous stem of Tsen et Lee. 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 volume of the internal myelin cells.

[0003] UBP15 It is a ubiquitin-specific protease with deubiquitination activity. It can prune ubiquitin chains by hydrolyzing isopeptide bonds between ubiquitin molecules and remove covalently bound ubiquitin from proteins; this activity is crucial for its function in vivo. Researchers found that the number of external integument cells in the Arabidopsis mutant upp15-1 ovules was significantly reduced compared to wild-type ovules, resulting in flattened and narrow rosette leaves, earlier flowering, and reduced fertility. Conversely, in lines overexpressing UBP15, the plants exhibited the opposite phenotype. Currently, although... UBP15 The gene has been reported in Arabidopsis thaliana, rapeseed, maize, and rice, but not in stem-nodular mustard. BjuUbp15 Research reports on gene function have been published, but the specific functions remain unclear. Summary of the Invention

[0004] To address the aforementioned shortcomings of the existing technology, the objective of this invention is to provide a stem-nodular mustard. BjuUbp15 Genes and their encoded proteins and their applications to promote the enlargement of stem tubers in stem mustard, thereby increasing its yield.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A stem-nodular mustard is provided. BjuUbp15 A gene has any of the following nucleotide sequences: (A1) The nucleotide sequence shown in SEQ ID NO.1; (A2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases of the nucleotide sequence shown in SEQ ID NO.1; (A3) A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in SEQ ID NO.1.

[0006] This invention provides the above-mentioned stem nodule mustard. BjuUbp15 The gene encodes a protein having any of the following amino acid sequences: (B1) The amino acid sequence as shown in SEQ ID NO.2; (B2) An amino acid sequence of a protein having the same function as the amino acid sequence shown in SEQ ID NO.2, obtained by substitution, insertion or deletion of one or more amino acids; (B3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO.2; (B4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins defined in (B1)-(B3).

[0007] This invention provides a method for overexpressing the above-mentioned stem mustard. BjuUbp15 Application of genes in promoting the enlargement of tuberous stems in stem mustard.

[0008] This invention provides a method for promoting the enlargement of the tuberous stems of *Strombyx mori*, by increasing the concentration of *Strombyx mori* in... BjuUbp15 Gene expression levels are used to increase the size and weight of the tuberous stems of stem mustard.

[0009] This invention provides the application of the above-mentioned encoded protein in the preparation of a formulation that promotes the enlargement of tuberous stems of *Strombyx mori*.

[0010] The present invention also provides an agent for promoting the enlargement of the tuberous stem of *Strombyx mori*, comprising the above-described encoded protein.

[0011] The present invention has the following beneficial effects: The present invention clones stem mustard using the homologous cloning method. BjuUbp15 Genes. Using Agrobacterium-mediated transformation, ... BjuUbp15 Genetic transformation of stem mustard with a gene overexpression vector yielded overexpressing plants. Experimental results showed that... BjuUbp15 The tuberous stems of the gene-overexpressing plants were significantly larger than those of the wild type. Therefore, the tuberous stem mustard of this invention... BjuUbp15 The gene has a positive regulatory effect on the size of the tuberous stem of *Mustela stenoptera*. Therefore, the present invention... BjuUbp15 Genes have great potential for increasing the yield of stem mustard. Attached Figure Description

[0012] Picture 1 for BjuUBP15 Phylogenetic tree analysis results of genes and their electrophoretic detection graph; Picture 2 The vector map of the overexpression vector pBin35Sred3; Picture 3 For the overexpression vector pBin35SRed3N-H- BjuUBP15 Enzyme digestion verification electrophoresis image; Picture 4 The image shows the identification results of transgenic positive plants of stem mustard; Picture 5 stem mustard BjuUBP15 Phenotypic identification diagram of gene overexpression plants; Picture 6 stem mustard BjuUBP15 Histograms of tubercle weight and tubercle length-to-width ratio in gene-overexpressing plants. Detailed Implementation

[0013] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0014] Example 1: Stem nodule mustard BjuUBP15 Cloning of genes (1) Extraction of total RNA: Collect 80-120 mg of fresh stem mustard leaves, quickly place them in a mortar pre-cooled with liquid nitrogen, add an appropriate amount of liquid nitrogen and grind them thoroughly into powder, immediately transfer to a 1.5 mL enzyme-free centrifuge tube, add 450 μL of cell lysis buffer RL (pre-added with 1% β-mercaptoethanol), and vortex to mix; transfer the above-mentioned homogenate to a filter column CS (CS is placed in a collection tube), centrifuge at 12000 rpm for 5 min, carefully aspirate the supernatant from the collection tube into an RNase-free centrifuge tube; add 250 μL of anhydrous ethanol, gently mix, transfer the solution to an adsorption column CR3, centrifuge at 12000 rpm for 50 s, and discard the filtrate; add 350 µL of protein removal buffer RWl to CR3, centrifuge at 12000 rpm for 45 s, and discard the filtrate; add 80 µL of DNase I working solution to the center of the CR3 adsorption membrane, let stand at room temperature for 15 min, and add 350 µL of DNase I working solution. Centrifuge RW1 at 12000 rpm for 45 s and discard the filtrate. Add 500 µL of wash buffer RW (for the first use, add 48 mL of anhydrous ethanol to 12 mL of RW) to the adsorption column CR3. Let stand at room temperature for 2 min, centrifuge at 12000 rpm for 45 s and discard the waste liquid. Repeat this step once. Centrifuge at 12000 rpm for 2 min, open the tube cap, discard the waste liquid, and let CR3 stand at room temperature for 4-6 min to dry the residual wash buffer. Place CR3 on a new RNase-free centrifuge tube, and add 30-100 µL of RNase-free double-distilled water to the center of the adsorption membrane. Let stand at room temperature for 2-5 min, centrifuge at 12000 rpm for 2 min to obtain the RNA solution.

[0015] RNA concentration and integrity testing: Concentration was measured by taking 2 μL of diluted RNA solution and placing it on a Nanodrop 2000 micro spectrophotometer to ensure the peak value was correct and the OD value was between 1.8 and 2.1. Integrity testing was performed by soaking the equipment and electrophoresis tank in hydrogen peroxide for 20 min, then preparing a 1.0% (w / v) agarose gel on an enzyme-free, clean work surface. Total RNA was extracted and electrophoresed immediately. When the electrophoresis result showed at least two clear bands, and the brightness of the upper 28S band was approximately twice that of the middle 18S band, it indicated that the extracted *Strombax cuneata* total RNA had good integrity and could be used for subsequent experiments. Finally, it was stored at -80℃ for later use.

[0016] (2) First-strand cDNA synthesis: The concentration of RNA extracted in step (1) was uniformly adjusted to 100 ng / μl, and a 14.5 μL reaction system was prepared: total RNA: 4 μL, Oligo (dT) 15Primer: 1 μL (50 μmol / L), pd(N)6: 0.5 μL (50 μmol / L), RNase-free double-distilled water: 9 μL. Reaction program: 70℃ for 10 min, 4℃ for 2 min), yielding product A. Next, prepare a 20 μL reverse transcription reaction system: Product A: 14.5 μL, 5×M-MLV buffer: 4 μL, M-MLV: 0.25 μL, dNTP (10 mM): 1 μL, recombinant RNase inhibitor: 0.25 μL. Reaction program: 42℃ for 60 min, 70℃ for 10 min, 4℃ for 2 min), yielding first-strand cDNA, which is stored at -80℃ for later use.

[0017] (3) Stem mustard BjuUBP15 Gene cloning: Searching for Arabidopsis thaliana on the TAIR website (https: / / www.arabidopsis.org) UBP15 Gene( AT1G17110 Based on its full-length CDS sequence, a BLAST (with the parameter Nucleotide databases: Braju tum V 2.0 cds) was performed in the Brassica genus database (http: / / brassicadb.cn) to obtain the homologous genes of *Brassica juncea*. BjuVB03G06640 Primers were designed using Oligo 7 software: forward primer F1: 5'-ATGCTTGAACCAAGGGGAGC-3' (SEQ ID NO.3), and reverse primer R1: 5'-CTACCAGTTTTGTTCATAACTGT-3' (SEQ ID NO.4). Using the reverse-transcribed first-strand cDNA as a template, PCR amplification was performed using the above-mentioned forward and reverse primer combinations. BjuUBP15 Gene. The PCR amplification system (50 μL) was: 2×Fast Pfu 26 μL Master Mix enzyme, 2 μL each of F1 and R1, 2 μL template cDNA, and 18 μL double-distilled water. The PCR amplification program was: 95℃ for 90 s; 94℃ for 20 s, 58℃ for 20 s, 72℃ for 90 s, 34 cycles; 72℃ for 5 min.

[0018] After the PCR reaction, the target band was detected by 1.5% (w / v) agarose gel electrophoresis. The target band was then excised and recovered using a gel extraction / DNA purification kit (DC301). Specific steps were described in the kit's instruction manual. The recovered gel product was then combined with the cloning vector. pEASY ®After ligation with Blunt Simple, the cells were transformed into *E. coli* DH-5α competent cells. Single-clone plaques were picked for detection, and correct plaques were sent to the company for sequencing. BjuUBP15 The coding sequence (CDS) of the gene was verified, and based on the sequence assembly results, stem mustard was obtained. BjuUBP15 The CDS sequence of the gene (as shown in SEQ ID NO.1) was obtained simultaneously. BjuUBP15 Gene plasmid.

[0019] stem mustard BjuUBP15 Phylogenetic trees were constructed in Geneious 9.0.2 software using the gene and its homologous genes from Arabidopsis thaliana, Chinese cabbage, Brassica napus, black mustard, Brassica napus-type rapeseed, and Ethiopian mustard. The results showed that... BjuUBP15 With black mustard BniB015334-PA And Ethiopian mustard Bca_GomB03g11270 , Bca_GomB03g11520 The genes are on the same branch, and all four genes are located in the B subgenome of the Yu triangle (see...). Picture 1 A), ultimately, the stem mustard was successfully cloned. BjuUBP15 Genes (see) Picture 1 B).

[0020] Example 2: BjuUBP15 Construction of overexpression vectors (1) The pBin35Sred3N-H expression vector was developed using the overexpression vector pBin35SRed3 (a gift from Professor Qian Wei of the Chongqing Rapeseed Engineering Technology Center; vector map can be found in...). Picture 2 Based on the above, a hygromycin expression cassette was added. The specific modification method is as follows: Bacterial culture containing the pBin35SRed3 plasmid was cultured overnight in LB liquid medium (Kana, 50 µg / mL), and the plasmid was extracted. Subsequent operations are as follows: 1. Preparation of novel multiple cloning sites (MCS): Primer MCS-F: 5'-GGAATTCGGATCCGCTCTAGACCTTAATTAATCGCGACCGCTCGAGGCGG-3' (SEQ ID NO.5) and MCS-R: 5'-CCGCCTCGAGCGGTCGCGATTAATTAAGGTCTAGAGCGGATCCGAATTCC-3' (SEQ ID NO.6) were mixed in double-distilled water to form a 0.5 μmol / L mixture. The mixture was reacted at 90 °C for 30 s and then cooled to room temperature to complete the annealing.

[0021] 2. Enzyme digestion: The pBin35SRed3 plasmid was digested with the new multiple cloning site MCS using... EcoR I and Xho I. Double enzyme digestion, reaction system (50 μL): QuickCut™ EcoRI and QuickCut™ Xho Ⅰ 1.5 μL each, 5 μL of 10X QuickCut Green Buffer, 21 μL of MCS / pBin35SRed3, and 21 μL of double-distilled water, mix well and place in a 37℃ water bath for 2 h.

[0022] 3. Ligation and Transformation: Take 1 μL of pBin35SRed3 digested and purified product, 4 μL of MCS digested and purified product, and mix with 5 μL of Solution I. React at 16℃ for 2 h. Take 5 μL of the ligation product and transform DH5α by heat shock. Spread the transformed competent cells evenly on LB solid medium (Kana, 50 µg / mL) and incubate overnight.

[0023] 4. Screening of positive clones: A small number of bacteria from the colony were picked up with a sterile toothpick and PCR was performed using QW586F: 5'-CGCACAATCCCACTATCCTT-3' (SEQ ID NO.7) and MCS-R. Multiple positive clones were selected for sequencing. The correctly sequenced vector was named: pBin35SRed3N The bacterial culture containing pBin35SRed3N and pCAMBIA1300 (provided by our laboratory) plasmids was cultured overnight in LB liquid medium (Kana, 50 µg / mL), and the plasmids were extracted. Subsequent procedures were as follows: ① Hygromycin expression cassette cloning: 2 μL of pCAMBIA1300 plasmid, 2 μL of Hyp-F: 5'-CACCATGTTGGGCCCGGCGCGCCTAATTCGGGGGATCTGGATTT-3' (SEQ ID NO.8), 2 μL of Hyp-R: 5'-GTTGTCACATACATCGGCGCGCCATGGTGGAGCACGACACTCTCG-3' (SEQ ID NO.9), 25 μL of 2×Fast Pfu Master Mix, and 19 μL of double-distilled water were mixed thoroughly. 30 PCR cycles were performed: 94℃ for 20 s, 58℃ for 20 s, and 72℃ for 2 min. The mixture was then purified using a PCR product purification kit.

[0024] ② Enzyme digestion: Take 21 μL of pBin35SRed3N, add 22.5 μL of double-distilled water and 1 μL of... Asc I. 5 μL of 10×QuickCut Green Buffer was reacted at 37 °C for 2 h and then purified using a PCR product purification kit.

[0025] ③ One-step cloning and transformation: Mix 5 μL of pBinGly35SRed3N digestion product, 1 μL of hygromycin expression cassette, 4 μL of 5×CE II buffer, 2 μL of Exnase II, and 8 μL of double-distilled water thoroughly on ice. Take 5 μL of the ligation product and transform DH5α by heat shock. Spread the transformed competent cells evenly on LB solid medium (Kana, 50 µg / mL) and incubate overnight.

[0026] ④ Screening of positive clones: A small number of bacteria from the colony were picked up with a sterile toothpick and PCR was performed using p35s-F: 5'-CCAACCACGTCTTCAAAGCA-3' (SEQ ID NO.10) and p35s-R: 5'-CAGTGAACAAAAGGCAAGCTA-3' (SEQ ID NO.11). Multiple positive clones were selected for sequencing. The correctly sequenced vector was named: pBin35SRed3N-H.

[0027] (2) BjuUBP15 The overexpression vector was constructed using the pBin35Sred3N-H expression vector. EcoR I and Pac I restriction site. PCR amplification was performed at... BjuUBP15 Primers were designed at both ends of the coding region of the gene, and corresponding restriction enzyme sites and protective bases were added to the 5' end of the primers. Using the target gene plasmid as a template, the gene was introduced into the genome at F2:5′-CGGAATTCATGCTTGAACCAAGGGGAGC-3′ (SEQ ID NO.12). EcoR I. Enzyme cleavage site and protective base (CG) and R2:5′-CCTTAATTAACTACCAGTTTTGTTCATAAC-3′ (SEQ ID NO.13), introduced Pac Using the I restriction site and the protective base C(C) as primers, and employing 2×Fast... Pfu Master Mix enzyme was used for PCR amplification. The reaction volume (25 μL) was: 2×Fast Pfu Master Mix enzyme 13μL, F2 and R2 1μL each BjuUBP15 1 μL of gene plasmid and 9 μL of double-distilled water were used. The PCR amplification program was as follows: 94℃ for 90 s; 94℃ for 20 s, 58℃ for 20 s, 72℃ for 90 s, 34 cycles; 72℃ for 5 min.

[0028] After the PCR reaction, the PCR products were subjected to 1.5% (w / v) agarose gel electrophoresis. The PCR products were recovered using a gel recovery / DNA purification kit (DC301). The products and the plasmid of the expression vector were then subjected to double enzyme digestion reactions. The enzyme digestion reaction system (25 μL) consisted of: 10.5 μL of the gel-recovered vector or gene product and 52.5 μL of Fastcut Buffer. EcoR I 0.75μL, Pac 0.75 μL of Solution I and 10.5 μL of double-distilled water were mixed and placed in a 37°C water bath for 2 hours. After enzyme digestion, the target gene and expression vector were recovered using a DC301 to obtain the digested products. The digested target gene and expression vector were ligated using T4-DNA ligase. The ligation system (10 μL) consisted of: 5 μL Solution I (containing T4-DNA ligase), 4 μL target gene, and 1 μL expression vector. The mixture was thoroughly mixed using a small centrifuge and incubated in a 16°C metal bath for 2 hours. After ligation, the cells were transformed into competent *E. coli* DH-5α cells. The culture plates were incubated overnight in an inverted position at 37°C. Colonies were detected using primers F3 (5′-AAAAGACAAAAGTGGGGTAG-3′, SEQ ID NO.14) and R3 (5′-ACACTTTTCAATGCCAACCT-3′, SEQ ID NO.15). Positive colonies were shaken and sent to the company for sequencing verification. After successful verification, the cells were shaken again and plasmids were extracted to obtain the overexpression vector pBin35SRed3N-H-. BjuUBP15 ,use EcoR I and Pac I was subjected to double enzyme digestion for verification, followed by agarose gel electrophoresis. The result showed two bands (see...). Picture 3 The band above 5000bp represents the vector pBin35SRed3N-H, and the target gene is located at 2600bp. BjuUBP15 (bands), proving that the overexpression vector pBin35SRed3N-H- BjuUBP15 Successfully built.

[0029] (3) Agrobacterium transformation: Take 2 μL of pBin35SRed3N-H- prepared in step (1) BjuUBP15The recombinant plasmid was added to 100 μL of Agrobacterium competent cells GV3101 that had been thawed on ice beforehand, and mixed thoroughly. The mixture was then incubated on ice for 5 min, frozen in liquid nitrogen for 5 min, and quickly placed in a 37°C water bath for 5 min, followed by an ice bath for 5 min. 800 μL of antibiotic-free YEB liquid medium was added, and the mixture was incubated at 28°C and 250 rpm for 2 h with shaking. The bacterial culture was collected by centrifugation at 6000 rpm for 1 min, and approximately 100 μL of the supernatant was gently resuspended and evenly spread onto YEB solid medium (containing 60 μg / mL Kan and 25 μg / mL Rif antibiotics). The culture was incubated upside down at 28°C for 48 h. Single colonies were detected using primers F3 and R3. Positive colonies were preserved by shaking (700 μL bacterial culture + 300 μL 60% glycerol) to obtain the plasmid containing pBin35SRed3N-H-. BjuUBP15 Agrobacterium tumefaciens culture containing recombinant plasmids was used for genetic transformation of stem mustard.

[0030] Example 3: Overexpression vector pBin35SRed3N-H- BjuUBP15 Genetically transformed stem mustard (1) Pre-culture: Select 20 plump seeds of Stem Mustard 1w06 and place them in a 200mL sterile tissue culture bottle. Disinfect the seeds by soaking them in 75% alcohol for 45s, then rinse them with sterile deionized water for 60s, and then soak them in 10% NaClO solution for 15min, shaking them constantly. Continue rinsing them with sterile deionized water for 60s, repeating the rinsing process 5 times to complete the seed disinfection. Sow the disinfected seeds onto MS solid medium and culture them in a culture room for 7 days. Cut the hypocotyls of the germinated seedlings into small segments of about 1cm and culture them on a pre-culture medium (MS solid medium + 3mg / L 6-benzylaminopurine (6-BA) + 0.2mg / L α-naphthaleneacetic acid (NAA)) for 2 days.

[0031] (2) Activation and dark culture: Dip a small amount of pBin35SRed3N-H- into the inoculation loop. BjuUBP15 Agrobacterium bifidum culture containing recombinant plasmids was streaked onto YEB solid medium containing 25 µg / mL rifampin (Rif) and 60 µg / mL kanamycin (Kan) and incubated at 28 °C for 2 days. After colony PCR detection, positive colonies were picked and cultured. 4 mL of the culture was activated in 50 mL of YEB liquid medium (containing 25 mg / L Rif and 60 mg / L Kan) and amplified to OD0.05. 600The concentration should be 0.5-0.6. Transfer 50 mL of bacterial culture to a 50 mL sterile centrifuge tube, centrifuge at 4000 rpm for 10 min at 4℃, discard the supernatant, resuspend the bacterial cells in 25 mL of pre-cooled MS liquid medium, and infect the pre-cultured hypocotyls with the resuspended medium for 10 min, gently shaking continuously during the infection process. Finally, blot the bacterial culture on the hypocotyls dry with sterile filter paper, spread them evenly on the pre-medium (MS solid medium + 3 mg / L 6-BA + 0.2 mg / L NAA), and incubate in the dark for 2 days.

[0032] (3) Differentiation and rooting culture: After dark culture, the co-cultured hypocotyls were transferred to MS selection medium (MS solid medium + 3 mg / L 6-BA + 0.2 mg / L NAA + 25 mg / L hygromycin B (Hyg) + 400 mg / L carbenicillin (Cb)) and cultured at 25℃ under 16 h light and 8 h dark conditions. The selection medium was changed every 7 days until the hypocotyls differentiated into adventitious shoots. After multiple subcultures, the surviving adventitious shoots were cut off and inserted into rooting medium (MS solid medium + 0.2 mg / L NAA + 25 mg / L Hyg + 400 mg / L Cb) to induce rooting. When the roots grew to 3-5 cm, the tissue culture bottle was opened for hardening off for 2-3 days. After removing the culture medium from the roots, the seedlings were transplanted into the substrate, thus obtaining stem mustard with a single mutation site. BjuUBP15 Genetically modified strains.

[0033] Example 3: Screening and phenotypic identification of stem mustard overexpression plants The DNA extraction method described in Example 1 was used to extract the DNA created in Example 2. BjuUBP15 DNA from the genetically modified strain was used as a template, and F3 and R3 were used as primers, with 2× Taq PCR amplification was performed using PCR Master Mix enzyme. Agarose gel electrophoresis results showed that 13 positive plants were selected from 14 transgenic lines, and the bands were consistent with the positive control (see...). Picture 4 A, where M is the DNA molecular weight standard, and 5-18 represents the obtained... BjuUBP15 The gene-transgenic lines were used, with P representing the positive control overexpression vector pBin35SRed3N-H-BjuUBP15 and N representing the negative control. Two positive plants were randomly selected for quantitative RT-qPCR analysis, ultimately yielding two *Mustela stenoptera* overexpression plants (see [link to relevant documentation]). Picture 4 B, where * and ** indicate that the difference is significant at the 0.05 and 0.01 levels, respectively.

[0034] The obtained stem-nodular mustard overexpression plants were simultaneously planted with wild-type mustard tuber in an experimental plot with identical environmental conditions. Phenotypic observation and statistical analysis revealed that the weight and size of the nodular stems in the positive stem-nodular mustard overexpression plants were significantly larger than those in the wild type, and they grew normally (see...). Picture 5-6 ; where * and ** indicate that the difference is significant at the 0.05 and 0.01 levels, respectively.

[0035] In this invention BjuUBP15 In the present invention BjuUBP15 The amino acid sequence of the encoded protein of the gene is shown below: MLEPRGADIPLLFLVLVVFPVVAYILLGKWSDISKKRGRANFLAQMAAEEALRAEFEVNVNRGVRFEAVATENRALRSKTKAVPAVRDDFVAGVSGTVAEQRSESMAATCGVPVSNEIHVCARCFSPAKTRCSRCKSVRYCSGQCQIIHWRLAHKDECIPVETCSSSSERASFENESVLYDQDTDSAMYSNNTKQKAKGKASKSSVEFESLGVSTPQVNTQGRKSVLKPVSSKFNGGSFSGEAASAGGDIKKGHTRHKSRSNIGAAETNSRRQSVDNSCMQMNGQTFVSGMQESCESNLGARSSFGCPNAQFPANGTRTATLRKSGEQSSPETSKKGQVAAVSKTLRSKDTCIAEESNGISSTVGLMKMMGLRNSTKHDDRHKNLRMLFPYEEFVKFFQCEVFYLSPRGLVNCGNSCYANAVLQSLTCTKPLVAYLLRRSHSRSCSGKDWCLMCELEQHVMMLRESGGPLSASRILSHMRSINCQIGDGSQEDAHEFLRLLVASMQSICLERLGGETKVDPRLQETTLVQHMFGGRLHSKVKCLRCGHESERYENIMDLTLEIYGWVESLQDALTQFTRPEDLDGDNMYRCSRCAGYVRARKELSIHEAPNILTIVLKRFQEGRYGKINKCISFPEMLDMIPFMTRTGDVPPLYMLYAVIVHLDTLNASFSGHYISYVKDLRGNWFRIDDSEIHQVPMTQVMSEGAYMLFYMRSYPRPQRGEHNGKAQVRHSQARDEMKEQRKPVNRFKPRADHHKKVESSSEWSLFTSSDEASFTTESTRDSFSTVDYTDGCNVLDSSSPFSIFNNLHQRVEPSPHNTVACRMFSGTKSETRYFVEEETNHNNTVMMDSSPSPHDYYQQSMFVNYDTNPGFNCQDQTYSYEQNW (SEQ ID NO.2) The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of stem-nodular mustard BjuUbp15 Genes, characterized by, The stem-nodular mustard BjuUbp15 A gene has any of the following nucleotide sequences: (A1) The nucleotide sequence shown in SEQ ID NO.1; (A2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases of the nucleotide sequence shown in SEQ ID NO.1; (A3) A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in SEQ ID NO.

1.

2. The stem mustard as described in claim 1 BjuUbp15 The protein encoded by the gene is characterized by, The encoded protein has any of the following amino acid sequences: (B1) The amino acid sequence as shown in SEQ ID NO.2; (B2) An amino acid sequence of a protein having the same function as the amino acid sequence shown in SEQ ID NO.2, obtained by substitution, insertion or deletion of one or more amino acids; (B3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO.2; (B4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins defined in (B1)-(B3).

3. Overexpression of the stem mustard described in claim 1 BjuUbp15 Application of genes in promoting the enlargement of tuberous stems in stem mustard.

4. A method for promoting the enlargement of the tuberculate stems of *Strombyx mori*, characterized in that, By increasing the content of stem mustard BjuUbp15 Gene expression levels are used to increase the size and weight of the tuberous stems of stem mustard.

5. The use of the encoded protein of claim 2 in the preparation of a formulation that promotes the enlargement of tuberous stems of *Strombyx mori*.

6. A preparation for promoting the enlargement of tuberous stems in stem mustard, characterized in that, Includes the encoded protein as described in claim 2.

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