Brassica napus BnaA03.XTH4 gene and application thereof

Abstract

The invention belongs to the technical field of plant genetic engineering, and discloses a brassica napus BnaA03. XTH4 gene and application thereof, the nucleotide sequence of the BnaA03. XTH4 gene is as shown in SEQ ID NO.1, the coding sequence is as shown in SEQ ID NO.2, and the amino acid sequence is as shown in SEQ ID NO.3. The invention further discloses a preparation method of the BnaA03. XTH4 gene. The BnaA03.XHT4 gene has the remarkable effects that the BnaA03.XHT4 gene has a negative regulation effect on the plant height, so that the plant height of the rape is reduced, the lodging resistance and close planting resistance of the rape are enhanced, and a new germplasm is provided for genetic improvement of the plant type of the rape.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and specifically relates to a Brassica napus BnaA03.XTH4 gene and its application. Background Art

[0002] Brassica napus ( Brassica napus L . , AACC, 2n = 38), as the third largest oil crop in the world, precise regulation of plant height is a key breeding goal for achieving high yield, lodging resistance and mechanized production. Although previous studies have identified genes related to gibberellin and auxin pathways such as BnaA06.RGA (Zhao et al. 2017; Wang et al. 2020), BnaC05.IAA7 (Zhao et al. 2019; Zheng et al. 2019; Ping et al. 2022) on multiple chromosomes such as A02, A03, and C05 through QTL mapping, and obtained semi-dwarf and high-yield phenotypes by editing genes such as BnaMAX1 using CRISPR / Cas9 technology (Zheng et al. 2020; Fan et al. 2021). However, the existing regulatory network still has significant limitations: the cloned genes are mostly concentrated in the hormone metabolism pathway, and the genetic loci and molecular mechanisms of genes related to dynamic modification of the cell wall are not yet clear (Tan et al. 2024; Bhujbal et al. 2025).

[0003] The extensibility of the plant cell wall is the core mechanical basis for determining the elongation of stem cells. Among them, xyloglucan endotransglucosylase / hydrolase (XTH) mediates the relaxation process by reconstructing the cell wall polysaccharide network, and its function has been proven to be related to hypocotyl elongation in model plants such as Arabidopsis thaliana (Kushwah et al. 2020; Ishida and Yokoyama 2022). In Brassica napus, the cloning and functional research of the XTH family genes have lagged behind for a long time: although the cDNA sequences of genes such as BnXTH1 have been cloned, there is still a lack of experimental evidence for their biological functions in plant height regulation (Guan Rongzhan et al. 2014). It is worth noting that transcriptome analysis found that the expression of genes related to lignin monomer synthesis and pectin degradation changed in rapeseed dwarf mutants (Luo Jing et al. 2022). However, there is no report in the prior art on regulating rapeseed plant height using XTH gene editing or overexpression technology, and this gap provides an important breakthrough for developing new plant type improvement strategies.

[0004] The specific citations of the above-mentioned references are as follows: Bhujbal SK, Rai AN, Joshi-Saha A (2025) Dwarfs standing tall:breeding towards the 'Yellow revolution' through insights into plant heightregulation. Plant Mol Biol 115:34。

[0005] Fan S, Zhang L, Tang M, Cai Y, Liu J, Liu H, Liu J, Terzaghi W, WangH, Hua W, Zheng M (2021) CRISPR / Cas9-targeted mutagenesis of the BnaA03.BP gene confers semi-dwarf and compact architecture to rapeseed ( Brassica napus L.). Plant Biotechnol J 19:2383-2385。

[0006] Gu J, Guan Z, Jiao Y, Liu K, Hong D (2024) The story of a decade:Genomics, functional genomics, and molecular breeding in Brassica napus .Plant Commun 5:100884。

[0007] Ishida K, Yokoyama R (2022) Reconsidering the function of thexyloglucan endotransglucosylase / hydrolase family. J Plant Res 135:145-156 Kushwah S, Banasiak A, Nishikubo N, Derba-Maceluch M, Majda M, EndoS, Kumar V, Gomez L, Gorzsas A, McQueen-Mason S, Braam J, Sundberg B,Mellerowicz EJ (2020) Arabidopsis XTH4 andXTH9 contribute to wood cellexpansion and secondary wall formation. Plant Physiol 182:1946-1965。

[0008] Ping X, Ye Q, Yan M, Zeng J, Yan X, Li H, Li J, Liu L (2022)Integrated genetic mapping and transcriptome analysis reveal the BnaA03.IAA7protein regulates plant architecture and gibberellin signaling in Brassica napus L. Theor Appl Genet 135:3497-3510。

[0009] Tan Z, Han X, Dai C, Lu S, He H, Yao X, Chen P, Yang C, Zhao L, YangQY, Zou J, Wen J, Hong D, Liu C, Ge X, Fan C, Yi B, Zhang C, Ma C, Liu K,Shen J, Tu J, Yang G, Fu T, Guo L, Zhao H (2024) Functional genomics of Brassica napus : Progresses, challenges, and perspectives. J Integr Plant Biol66:484-509。

[0010] Wang X, Zheng M, Liu H, Zhang L, Chen F, Zhang W, Fan S, Peng M, HuM, Wang H, Zhang J, Hua W (2020) Fine-mapping and transcriptome analysis of acandidate gene controlling plant height in Brassica napus L. BiotechnolBiofuels 13:42。

[0011] Zhao B, Li H, Li J, Wang B, Dai C, Wang J, Liu K (2017) Brassica napus DS-3 , encoding a DELLA protein, negatively regulates stem elongationthrough gibberellin signaling pathway. Theor Appl Genet 130:727-741。

[0012] Zhao B, Wang B, Li Z, Guo T, Zhao J, Guan Z, Liu K (2019)Identification and characterization of a new dwarf locus DS-4 encoding anAux / IAA7 protein in Brassica napus . Theor Appl Genet 132:1435-1449。

[0013] Zheng M, Hu M, Yang H, Tang M, Zhang L, Liu H, Li X, Liu J, Sun X,Fan S, Zhang J, Terzaghi W, Pu H, Hua W (2019) Three BnaIAA7 homologs areinvolved in auxin / brassinosteroid-mediated plant morphogenesis in rapeseed( Brassica napus L.). Plant Cell Rep 38:883-897。

[0014] Zheng M, Zhang L, Tang M, Liu J, Liu H, Yang H, Fan S, Terzaghi W,Wang H, Hua W (2020) Knockout of two BnaMAX1 homologs by CRISPR / Cas9-targetedmutagenesis improves plant architecture and increases yield in rapeseed( Brassica napusL.). Plant Biotechnol J 18:644-654。

[0015] Guan RZ, Chu LY (2014) Cloning and expression analysis of BnXTH1 gene in Brassica napus L. Molecular Plant Breeding.

[0016] Luo J, Li C, Zhang RM, Zhao DG, Gao ZH, Wang F, Yang YY, Wang ZZ, Wang M (2022) Transcriptome sequencing analysis of the dwarf trait of Brassica napus DW871. Chinese Journal of Oil Crop Sciences 44:14-24. Summary of the Invention

[0017] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a Brassica napus BnaA03.XTH4 gene and its application, so as to overcome the disadvantages and deficiencies existing in the prior art.

[0018] To achieve the above object, the technical scheme adopted by the present invention is as follows: In the first aspect, the present invention provides a Brassica napus BnaA03.XTH4 gene, which is characterized in that: the nucleotide sequence of the BnaA03.XTH4 gene is as shown in SEQ ID NO.1, the coding sequence is as shown in SEQ ID NO.2, and the amino acid sequence is as shown in SEQ ID NO.3.

[0019] Furthermore, the nucleotide sequence of the BnaA03.XTH4 gene can also be: The nucleotide sequence obtained by substituting, deleting and / or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO.1 and expressing the same functional protein.

[0020] Furthermore, the amino acid sequence of the BnaA03.XTH4 gene can also be: The protein obtained by substituting, deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO.3 and derived from SEQ ID NO.3 and maintaining the function of the amino acid sequence shown in SEQ ID NO.3.

[0021] Furthermore, the ligation primer pair of the BnaA03.XTH4 gene includes: Forward ligation primer BnaA03.XTH4-BglII-F: CAAGCTGACTCTAGCAGATCTATGGCTGTTTCTTCAGCTCCATG; Reverse ligation primer BnaA03.XTH4-XbaI-R: TCCTTTGCCCATGGCTCTAGACACGTCTCTGTCCCTTTTACATTC。

[0022] In a second aspect, the present invention provides an application of the Brassica napus BnaA03.XTH4 gene, or the protein encoded thereby, or a biological material containing the gene, in rapeseed breeding.

[0023] Furthermore, the application in rapeseed breeding is an application in breeding dwarf rapeseed.

[0024] Furthermore, the nucleotide sequence of the Brassica napus BnaA03.XTH4 gene is as shown in SEQ ID NO.1, the coding sequence is as shown in SEQ ID NO.2, and the amino acid sequence is as shown in SEQ ID NO.3.

[0025] In a third aspect, the present invention provides an application of the Brassica napus BnaA03.XTH4 gene, or the protein encoded thereby, or a biological material containing the gene, in genetic improvement of plant plant type.

[0026] Furthermore, the nucleotide sequence of the Brassica napus BnaA03.XTH4 gene is as shown in SEQ ID NO.1, the coding sequence is as shown in SEQ ID NO.2, and the amino acid sequence is as shown in SEQ ID NO.3.

[0027] Furthermore, the plant is Brassica napus, Brassica oleracea or Arabidopsis thaliana.

[0028] The remarkable effect of the present invention is: The present invention first cloned the BnaA03.XTH4 gene from Brassica napus and constructed an overexpression vector of the BnaA03.XTH4 gene. After transforming Arabidopsis thaliana with this vector, the plant height of the transgenic lines was significantly lower than that of the wild type WT, proving that the BnaA03.XHT4 gene has a negative regulatory effect on plant height and has the value for breeding dwarf rapeseed. The method for overexpressing the Brassica napus BnaA03.XTH4 gene of the present invention can be applied to genetic engineering breeding of plants and can be applied to production practice for cultivating new germplasms of dwarf rapeseed. Description of the Drawings

[0029] Figure 1 is the electrophoresis diagram of the amplification of the BnaA03.XTH4 gene; Figure 2 is the schematic diagram of the construction of the pCAMBIA1300 - BnaA03.XTH4 overexpression recombinant vector and the detection diagram of the recombinant vector bacterial liquid PCR; Figure 3 is the identification diagram of the overexpressed transgenic Arabidopsis thaliana; Figure 4 This is the plant height phenotype diagram of transgenic Arabidopsis thaliana. Specific implementation manners

[0030] The following further elaborates on the specific implementation manners and working principles of the present invention in conjunction with the attached drawings.

[0031] The objective of the present invention is to extract genes that have a negative regulatory effect on plant height and obtain new germplasms with significantly reduced plant height. The present invention clones the BnaA03.XTH4 gene from Brassica napus, constructs an overexpression transformation vector of the BnaA03.XTH4 gene, and after transforming Arabidopsis thaliana with this vector, the plant height of the plant lines is significantly lower than that of the wild type WT, proving that the BnaA03.XHT4 gene has a negative regulatory effect on plant height.

[0032] Therefore, the present invention provides a Brassica napus BnaA03.XTH4 gene, which is characterized in that: the nucleotide sequence of the BnaA03.XTH4 gene is as shown in SEQ ID NO.1, the coding sequence is as shown in SEQ ID NO.2, and the amino acid sequence is as shown in SEQ ID NO.3.

[0033] Furthermore, the nucleotide sequence of the BnaA03.XTH4 gene can also be: The nucleotide sequence obtained by substituting, deleting, and / or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO.1 and expressing the same functional protein.

[0034] Furthermore, the amino acid sequence of the BnaA03.XTH4 gene can also be: The protein obtained by substituting, deleting, and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO.3 and derived from SEQ ID NO.3 and maintaining the function of the amino acid sequence shown in SEQ ID NO.3.

[0035] The present invention also provides the application of the above-mentioned Brassica napus BnaA03.XTH4 gene, or the protein encoded by it, or the biological material containing this gene in rapeseed breeding.

[0036] Furthermore, the application in rapeseed breeding is the application in breeding dwarf rapeseed.

[0037] Furthermore, the nucleotide sequence of the Brassica napus BnaA03.XTH4 gene is as shown in SEQ ID NO.1, the coding sequence is as shown in SEQ ID NO.2, and the amino acid sequence is as shown in SEQ ID NO.3.

[0038] The present invention also provides an application of the Brassica napus BnaA03.XTH4 gene, or the protein encoded thereby, or a biological material containing the gene in the genetic improvement of plant architecture.

[0039] Furthermore, the nucleotide sequence of the Brassica napus BnaA03.XTH4 gene is as shown in SEQ ID NO.1, the coding sequence is as shown in SEQ ID NO.2, and the amino acid sequence is as shown in SEQ ID NO.3.

[0040] Furthermore, the plant is Brassica napus, Brassica oleracea or Arabidopsis thaliana.

[0041] To further illustrate the present invention, the present invention will be described in detail below with reference to examples, but they should not be construed as limiting the protection scope of the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0042] Example 1: Amplification to obtain the full-length genomic sequence of the BnaA03.XTH4 gene: Fresh leaves of the Brassica napus inbred line DZ were taken, and genomic DNA was extracted using the Tiangen Plant Genomic DNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd., Beijing, product number DP302). The reference sequence of the BnaA03.XTH4 gene was downloaded from the BnIR website, and gene amplification primers, BnaA03.XTH4-F (SEQ ID NO.4: ATGGCTGTTTCTTCAGCTCCATG), BnaA03.XTH4-R (SEQ ID NO.5: CACGTCTCTGTCCCTTTTACATTC), were designed. Using the above-extracted DNA as a template, the full-length genomic sequence of BnaA03.XTH4 was amplified.

[0043] The PCR reaction was carried out using KOD-PLUS DNA polymerase (TOYOBO, OSAKA JAPAN, product number KOD-401). Refer to the following system (50 μL): Add 1 μL of the above-extracted DNA template, 0.5 μL of each of the BnaA03.XTH4-F / R primer pair, 1 μL of KOD-PLUS DNA polymerase, 5 μL of 10×KOD buffer, 1 μL of dNTP mixture, and sterile ultrapure water to make up to 50 μL in a sterile PCR tube. The reaction procedure was pre-denaturation at 94 °C for 2 min, followed by 37 cycles of denaturation at 98 °C for 10 s, annealing temperature (55 °C) for 30 s, extension at 68 °C (2 min), and finally extension at 72 °C for 5 min.

[0044] Take 4 μL of the reaction product and detect the target band by 1% agarose gel electrophoresis. If the target band is correct (as Figure 1 shown), it indicates that the primers can be used and the target gene can be correctly cloned. Figure 1 In BnaA03.XTH4 , N is the negative control without DNA template; the middle lane is the

[0045] gene amplification band, with a size of 1751 bp; M is the DNA Marker. Based on the BnaA03.XTH4 gene amplification primers obtained in Example 1, as well as the homologous arm sequences of the BglII and Xba I restriction enzyme sites of the pCAMBIA1300 backbone vector, a pair of ligation primers was designed for the construction of the recombinant vector, as specifically shown in Figure 2 Figures 2A and 2B. The pair of ligation primers includes: Forward ligation primer BnaA03.XTH4-BglII-F: SEQ ID NO.6: CAAGCTGACTCTAGCAGATCTATGGCTGTTTCTTCAGCTCCATG; Reverse ligation primer BnaA03.XTH4-XbaI-R: SEQ ID NO.7: TCCTTTGCCCATGGCTCTAGACACGTCTCTGTCCCTTTTACATTC.

[0046] Using this pair of ligation primers, DNA was used as the template again, and the target gene was amplified again with reference to the above reaction process and procedures.

[0047] Take the PCR product and detect the target band by 1% agarose gel electrophoresis. Subsequently, cut out the target band and recover the target product with Omega's gel extraction kit (product number D2500-01). The specific steps are as follows: Mix the gel block containing the target band with an equal volume of XP2 binding buffer, dissolve it at 60 °C and transfer it to a HiBind® DNA purification column. Centrifuge at 12,000 rpm to remove the waste liquid, wash it twice with SPW buffer, and dry it in a laminar flow hood. Finally, elute the DNA with 40 μL of sterile water, and detect the concentration and purity of the purified product by a NanoDrop One spectrophotometer for subsequent homologous recombination ligation reaction.

[0048] Meanwhile, using Bgl II and XbaThe pCAMBIA1300 backbone vector was linearized with restriction endonuclease. The reaction system contained 5 μL of 10× buffer, 3 μg of vector plasmid, 1 μL of restriction endonuclease, and sterile water was added to make up to 50 μL. Incubate at 37 °C for 3 h. The digestion efficiency was verified by 1% agarose gel electrophoresis. If it was not completely linearized, the digestion time was extended. After completion, the linearized vector was purified by the gel recovery method described in Example 1. The purified linearized vector was mixed with the target gene fragment at a volume ratio of 3:4. Then 2 μL of 5× CEII buffer of ClonExpress II One Step Cloning Kit (Vazyme, product number: C112) and 1 μL of ExnaseII were added, and ligated at 37 °C for 30 min.

[0049] Take 10 μL of the ligation product and transform it into Escherichia coli DH5α competent cells. The specific process includes: ice bath binding for 30 min, heat shock at 42 °C for 1 min, ice bath recovery for 2 min. Then 500 μL of antibiotic-free LB medium was added and cultured with shaking at 37 °C for 1 hour. Take 100 μL of the bacterial solution and spread it on LB solid medium containing 50 μg / mL kanamycin, and culture at 37 °C for 12 - 16 hours. When screening for positive clones, the primer pair (SEQ ID NO.8: 35S, ACTATCCTTCGCAAGACCCTTCCTC; SEQ ID NO.9: eGFP-R, TTGTGCCCATTAACATCACCATCTA) was used for colony PCR primary screening. Five clones with clear amplification bands were selected for sequencing verification. As Figure 2 shown in C, on the one hand, the specific gene sequence was determined, and on the other hand, the correctness of the recombinant vector sequence was confirmed.

[0050] In this example, Figure 2 among them, Figure 2 A is the overexpression backbone vector pCAMBIA1300; Figure 2 B is the digested and ligated recombinant vector pCAMBIA1300 - BnaA03.XTH4; Figure 2 C is a schematic diagram of the bacterial liquid PCR identification of recombinant vector monoclonal. N is the negative control, 1 - 6 are the amplification bands of the picked monoclonal with the detection primers (35S / eGRP-R). Among them, the ones with the target band (1900 bp) are positive clones, and the ones without bands are negative clones. M is the DNA Marker.

[0051] Through sequencing analysis, the full length of the BnaA03.XTH4 nucleic acid gene sequence is 1754 bp, as shown in SEQ ID NO.1; it includes 3 introns, the coding sequence is 888 bp, as shown in SEQ ID NO.2; the polypeptide amino acid sequence is 295, as shown in SEQ ID NO.3.

[0052] For the correctly connected positive clones, shake the bacteria overnight, and extract the recombinant plasmid using the TIANGEN Plasmid Mini Kit (product number: DP103). The specific steps for the improved large-scale plasmid extraction include: Centrifuge at 12,000 rpm for 1 min to collect 10 mL of bacterial solution (OD 600 = 0.8), resuspend the cells in 500 μL of Solution P1, sequentially add 500 μL of Solution P2 for lysis and 750 μL of Solution P3 for neutralization according to the steps. After centrifuging to remove the precipitate, the supernatant is combined with plasmid DNA through the adsorption column CP3 (pre-equilibrated treatment), double-washed with 600 μL of washing buffer PW in sequence, dried in a laminar flow hood, and finally eluted with 100 μL of sterile water. Detect the plasmid concentration (A260 / A280 = 1.8 - 2.0) and purity using NanoDrop One, and reserve the plasmid.

[0053] Example 3: Arabidopsis thaliana genetic transformation: Transform the above-extracted recombinant purified plasmid into GV3101 Agrobacterium competent cells by heat shock method: Take 1 μg of plasmid DNA and mix it with 100 μL of competent cells melted on ice. After quick-freezing in liquid nitrogen for 5 min, perform heat shock at 37 °C for 5 min, add 800 μL of antibiotic-free LB medium, and recover at 28 °C for 2 h; after centrifugation, take 300 μL of the bacterial solution and spread it on LB solid medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin, and culture at 28 °C for 48 h. After picking monoclonal colonies for amplification, perform PCR verification using vector-specific primers (SEQ ID NO.8: 35S, ACTATCCTTCGCAAGACCCTTCCTC; SEQ ID NO.9: eGFP-R, TTGTGCCCATTAACATCACCATCTA). Expand the positive bacterial solution at 28 °C and 200 rpm until OD 600 = 0.6, mix it with 50% glycerol at a volume ratio of 1:1, and store it at -80 °C for long-term use for Arabidopsis thaliana infection.

[0054] The present invention adopts an improved Agrobacterium-mediated floral dip method, which specifically includes: after surface sterilizing wild-type Arabidopsis thaliana seeds three times with 75% ethanol, drying them with sterile filter paper and evenly sowing them on 1 / 2 MS solid medium, culturing them in the dark at 4 °C for 3 days to synchronize the seeds, and then transferring them to an artificial climate chamber (photoperiod: 16 hours of light / 8 hours of darkness, temperature 22 ± 1 °C, relative humidity 60%) for 7 days; transplanting the seedlings into a substrate of vermiculite-perlite-nutrient soil (3:1:1), pruning the main stem when the bolting reaches 3 cm to induce lateral branch differentiation, screening the vigorously growing inflorescences after continuous culturing for 25 days, removing the opened flower organs and mature pods 24 hours before transformation, and performing substrate saturation irrigation to optimize the infection efficiency. Synchronously prepare the Agrobacterium infection solution: Take 100 μL of the frozen bacterial solution stored at -80 °C and inoculate it into LB liquid medium containing 25 μg / mL rifampicin and 50 μg / mL kanamycin, shake and culture at 28 °C and 200 rpm until OD 600 = 1.2 - 1.6, centrifuge to collect the bacterial cells and resuspend them in a 5% sucrose solution (containing 0.02% SilwetL-77) to an OD 600 = 0.8; Use a pipette to aspirate 200 μL of the bacterial suspension to infiltrate the inflorescence tissue, enhance the transformation efficiency by repeatedly pipetting the bacterial solution, immediately cover it with a black light-shielding bag to maintain a high-humidity dark environment for 20 hours after infection, restore the standard culture conditions after removing the cover, and repeat the secondary transformation every 7 days to improve the transformation success rate.

[0055] Example 4, Identification of overexpressing Arabidopsis thaliana: After harvesting the T1 generation seeds, use 1 / 2 MS solid medium containing 20 μg / mL hygromycin for germination screening, transplant the resistant plants into nutrient soil, collect leaf tissues from individual plants at the bolting stage, and perform single-plant DNA verification. The specific process includes: extracting genomic DNA by the CTAB method, performing PCR amplification using primer pairs (SEQ ID NO.8: 35S, ACTATCCTTCGCAAGACCCTTCCTC; SEQ ID NO.9: eGFP-R, TTGTGCCCATTAACATCACCATCTA), screening positive single plants and harvesting T2 generation seeds, as Figure 3 shown in A; Repeat the resistance screening and single-plant DNA verification for the T2 generation, and retain the lines with the insertion copy number conforming to the Mendelian single-gene dominant inheritance law (the ratio of resistant seedlings to non-resistant seedlings ≈ 3:1); Further expand and propagate the T3 generation seeds, screen for homozygous lines using hygromycin-resistant medium, collect mixed samples from multiple plants at the bolting stage, extract total RNA based on the TRIzol method and reverse transcribe it into cDNA. Use the RT-qPCR system (containing 10 μL of 2×FastFire qPCR PreMix, 0.6 μL each of primer SEQ ID NO.10, qBNXTH-F: ATGGCTGTTTCTTCAGCTCCATGG; primer primer SEQ ID NO.11, qBnXTH4-R: CCCTGTCCCAGTGTATTTGTCGAG), 1 μL of cDNA template, 0.4 μL of ROX reference dye, and RNase-Free water to make up to 20 μL) for expression analysis. Using the Actin7 gene (primers SEQ ID NO.12: athACT7-F GGAACTGGAATGGTGAAGGCTGG; SEQ ID NO.13: athACT7-R GCTCCTCAGGGGCAACACG) as an internal reference, calculate the relative expression of the target gene by the 2-ΔΔCT method, and screen for 3 homozygous lines with an expression level ≥ 2 times that of the wild type for phenotypic analysis. As a result, 3 successfully overexpressed transgenic lines were obtained, named OE1 / 2 / 3, as Figure 3 shown in

[0056] In Figure 3 , Figure 3 A is the genomic level PCR identification diagram. Among them, WT refers to wild-type Arabidopsis thaliana, M is DNA Marker, and the other lanes are the amplified bands of T1 generation transgenic Arabidopsis thaliana (1 - 10) using the detection primer (35S / eGRP-R). Those with the target band (1900 bp) are positive plants, and those without the band are negative. Figure 3 B is the related expression of transgenic Arabidopsis thaliana BnaA03.XTH4. The values in the figure are the mean ± standard deviation ( n = 3). Use the student t test to reveal statistically significant differences, ****P < 0.0001.

[0057] Example 5. Observation of the plant height trait of Arabidopsis thaliana: Since other plant XTH homologous gene families are involved in cell wall regulation, and the cell wall is related to plant height regulation, but it is still unknown whether the BnaA03.XTH4 gene can be involved in plant height regulation. Therefore, in this invention, the created BnaA03.XTH4 transgenic genetic material was used to observe plant height elongation.

[0058] Arabidopsis thaliana seeds (including wild-type Arabidopsis thaliana and BnaA03.XTH4 overexpression lines) were disinfected in 75% (v / v) ethanol for 30 seconds. After drying on a sterile workbench, these seeds were evenly sown on 1 / 2 MS medium containing 1% (w / w) sucrose. The seeds were placed at 4 °C for two days and then cultured under long-day conditions (16 hours of light, light intensity 100 μmol·m⁻²·s⁻¹, 8 hours of darkness, temperature 22 °C) for 7 days. Subsequently, the seedlings were transplanted into the soil and continued to be cultured in the greenhouse with the relative humidity set at 65%. When the seedlings matured, that is, about 6 weeks after planting, the PH (vertical distance from the base of the stem to the top of the inflorescence) was measured.

[0059] The investigation results showed that the plant height of the BnaA03.XHT4 gene overexpression lines was significantly lower than that of the wild-type WT, as Figure 4 shown in Figure 4 in Figure 4 A is a representative photo of the plants grown under long-day conditions at 22 °C for 42 days, and the scale bar is 10 cm; Figure 4 B is the statistical result of the plant height measurement at the mature stage. The values in the figure are the mean ± standard deviation ( n ≥ 20). Using the Student t test revealed a statistically significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001. These results indicate that the BnaA03.XHT4 gene has a negative regulatory effect on plant height, and this gene has the value for breeding dwarf rapeseed.

[0060] In summary, the present invention first cloned the BnaA03.XTH4 gene from Brassica napus, constructed a rapeseed transformation vector overexpressing the BnaA03.XTH4 gene. After the vector was transformed into Arabidopsis thaliana, the plant height of the lines was significantly lower than that of the wild-type WT, demonstrating that the BnaA03.XHT4 gene has a negative regulatory effect on plant height and has the value for breeding dwarf rapeseed. The method for overexpressing the BnaA03.XTH4 gene of Brassica napus in the present invention can be applied to the genetic engineering breeding of plants and can be applied to production practice for cultivating new germplasms of dwarf rapeseed.

[0061] The sequence listings SEQ ID NO.1 and SEQ ID NO.2 are respectively the nucleotide sequence and coding sequence of the BnaA03.XTH4 gene in the present invention, with sequence lengths of 1754 bp and 888 bp respectively; SEQ ID NO.3 is the amino acid sequence encoded by the BnaA03.XTH4 gene in the present invention, with a sequence length of 295 aa.

[0062] SEQ ID NO.1: SEQ ID NO.2: ATGGCTGTTTCTTCAGCTCCATGGGCTCTCGTAGCTCTGTTTCTGATGGCCTCTTCTACTGTAATGGCAATTCCTCCACGGAAGGCCATTGATGTGCCATTCGGCCGAAACTACGTTCCAACTTGGGCTTTTGACCACCAGAAGCAACTCAATGGCGGTTCCGAACTCCAACTCATCCTCGACAAATACACTGGGACAGGGTTTCAATCCAAAGGGTCATATTTGTTCGGACATTTCAGTATGCACATAAAGCTGCCAGCTGGTGATACCGCTGGGGTCGTCACTGCATTTTATCTGTCGTCGACTAACAACGAGCATGACGAGATAGATTTCGAGTTTCTCGGGAACAGGACAGGCCAGCCAGCAATATTGCAGACGAATGTGTTCACAGGAGGAAAGGGAAACAGAGAGCAACGCATCTATCTCTGGTTCGACCCTTCAAAGGCTTATCATACTTACTCCATCCTCTGGAACCTCTACCAAATTGTATTCTTTGTTGACAACATACCAATCCGTGTGTTCAAGAACGCTAAGGATCTAGGAGTACGTTTCCCATTCAACCAACCGATGAAGCTATACTCGAGCCTTTGGAACGCTGACGATTGGGCGACGAGAGGAGGGCTAGAGAAAACCAATTGGGCTAATGCACCCTTCATAGCTTCCTACAGAGGATTCCACATCGACGGCTGCCAAGCTTCTGTGGAGGCCAAGTACTGTGCTACCCAAGGCCGCATGTGGTGGGATCAGAATGAGTTCCGTGACCTTGATGCCGAACAATATCGTCGTCTCAAATGGGTCCGCATGAAATGGACCATCTACAACTACTGTACCGACCGTACTAGGTTCCCAGTTATGCCAGCCGAATGTAAAAGGGACAGAGACGTGTGA SEQ ID NO.3: MAVSSAPWALVALFLMASSTVMAIPPRKAIDVPFGRNYVPTWAFDHQKQLNGGSELQLILDKYTGTGFQSKGSYLFGHFSMHIKLPAGDTAGVVTAFYLSSTNNEHDEIDFEFLGNRTGQPAILQTNVFTGGKGNREQRIYLWFDPSKAYHTYSILWNLYQIVFFVDNIPIRVFKNAKDLGVRFPFNQPMKLYSSLWNADDWATRGGLEKTNWANAPFIASYRGFHIDGCQASVEAKYCATQGRMWWDQNEFRDLDAEQYRRLKWVRMKWTIYNYCTDRTRFPVMPAECKRDRDV* SEQ ID NO.4: ATGGCTGTTTCTTCAGCTCCATG SEQ ID NO.5: CACGTCTCTGTCCCTTTTACATTC SEQ ID NO.6: CAAGCTGACTCTAGCAGATCTATGGCTGTTTCTTCAGCTCCATG SEQ ID NO.7: TCCTTTGCCCATGGCTCTAGACACGTCTCTGTCCCTTTTACATTC SEQ ID NO.8: ACTATCCTTCGCAAGACCCTTCCTC SEQ ID NO.9: TTGTGCCCATTAACATCACCATCTA SEQ ID NO.10: ATGGCTGTTTCTTCAGCTCCATGG SEQ ID NO.11: CCCTGTCCCAGTGTATTTGTCGAG SEQ ID NO.12: GGAACTGGAATGGTGAAGGCTGG SEQ ID NO.13: GCTCCTCAGGGGCAACACG The technical solution provided by the present invention is introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A Brassica napus BnaA03.XTH4 gene, characterized in that: The nucleotide sequence of the BnaA03.XTH4 gene is shown in SEQ ID NO.1, the coding sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.

3.

2. The Brassica napus BnaA03.XTH4 gene according to claim 1, wherein: The nucleotide sequence of the BnaA03.XTH4 gene may also be: A nucleotide sequence obtained by substituting, deleting, and / or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO.1 and expressing the same functional protein.

3. The Brassica napus BnaA03.XTH4 gene according to claim 1, characterized in that: The amino acid sequence of the BnaA03.XTH4 gene may also be: A protein obtained by substituting, deleting, and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO.3 and derived from SEQ ID NO.3 while maintaining the function of the amino acid sequence shown in SEQ ID NO.

3.

4. The Brassica napus BnaA03.XTH4 gene according to claim 1, characterized in that: The ligation primer pair of the BnaA03.XTH4 gene includes: Forward ligation primer BnaA03.XTH4-BglII-F: CAAGCTGACTCTAGCAGATCTATGGCTGTTTCTTCAGCTCCATG; Reverse ligation primer BnaA03.XTH4-XbaI-R: TCCTTTGCCCATGGCTCTAGACACGTCTCTGTCCCTTTTACATTC.

5. Use of a Brassica napus BnaA03.XTH4 gene, or a protein encoded thereby, or a biological material containing the gene in rapeseed breeding.

6. The application according to claim 5, wherein The use in rapeseed breeding is the use in breeding dwarf rapeseed.

7. The application according to claim 5, characterized in that, The nucleotide sequence of the Brassica napus BnaA03.XTH4 gene is shown in SEQ ID NO.1, the coding sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.

3.

8. Use of a Brassica napus BnaA03.XTH4 gene, or a protein encoded thereby, or a biological material containing the gene in genetic improvement of plant plant type.

9. The application according to claim 8, wherein The nucleotide sequence of the Brassica napus BnaA03.XTH4 gene is shown in SEQ ID NO.1, the coding sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.

3.

10. The application according to claim 8, wherein, The plant is Brassica napus, Brassica oleracea or Arabidopsis thaliana.

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