Use of bnaa09.mapk7 in promoting stem growth of brassica napus

Abstract

This invention belongs to the field of agricultural bio-genetic engineering technology, and specifically discloses... BnaC09.gluA2 BnaC09.gluA2 BnaC09.gluA2 BnaC09.gluA2 BnaC09.gluA2 BnaC09.gluA2 BnaC09.gluA2 BnaC09.gluA2 BnaC09.gluA2 BnaC09.gluA2 BnaC09.gluA2 BnaC09 Application in promoting rapeseed stem growth. Specifically: ​ The application of genes in promoting rapeseed stem growth, the aforementioned ​ The nucleotide sequence of the gene is shown in SEQ ID NO.1. This invention discloses... ​ Its application in promoting rapeseed stem growth provides a new option for improving rapeseed lodging resistance. This application involves... ​ Genes are integrated into rapeseed through expression vectors, effectively promoting the growth and development of rapeseed stems, which has significant application value for rapeseed breeding.

Description

Technical Field

[0001] This invention belongs to the field of agricultural bio-genetic engineering technology, specifically involving BnaA09.MAPK7 Application in promoting rapeseed stem growth. Background Technology

[0002] Brassica napus ( Brassica napus As the world's third largest oilseed crop, rapeseed accounts for over 13% of global oil production, holding a vital position in agricultural production. With increasing planting density, widespread mechanized harvesting, and frequent extreme weather events, lodging has become a core bottleneck restricting the yield and quality improvement of rapeseed. Lodging damages the vascular tissue of the rapeseed plant, hindering the absorption and transport of water and nutrients. Secondary branching fails to form siliques normally, and the plant becomes more susceptible to pests and diseases, severely impacting plant growth, development, and ultimately, yield and quality.

[0003] In traditional variety breeding, reducing plant height or increasing stem diameter can improve lodging resistance to some extent, but this method is often accompanied by a decrease in biomass and source-sink imbalance. As a tetraploid crop, rapeseed has only been domesticated for a few hundred years, and has limitations such as a narrow genetic background and a single genetic basis, making it difficult for traditional breeding methods to achieve precise improvement of lodging resistance traits.

[0004] The mechanical strength of plant stems is primarily determined by the degree of development of secondary xylem and the level of cell wall lignification. Lignin, as a core component of the secondary cell wall, is synthesized via the phenylpropane pathway and forms a rigid network through the oxidative polymerization of monomers such as coniferyl alcohol and sinapyl alcohol, providing the stem with resistance to compression and bending. Studies have confirmed that inhibited lignin metabolism leads to thinner and softer stems and a significantly increased risk of lodging, while increasing lignin content helps to thicken the secondary cell wall and enhance stem rigidity. However, lignin metabolism is regulated by multiple signaling networks, including mechanical stimulation, reactive oxygen species (ROS), and plant hormones. The key regulatory nodes in lignin metabolism in Brassica napus have not yet been systematically elucidated, severely hindering the molecular design breeding process for highly lodging-resistant and stable-yielding rapeseed varieties.

[0005] The mitogen-activated protein kinase (MAPK) cascade is a core pathway for transmitting environmental-developmental signals in plants, consisting of three levels of components: MAPKKK-MAPKK-MAPK. Downstream MAPKs can be divided into AD groups based on their amino acid sequence, kinase domains, and TXY motifs, with relatively limited research on C-group MAPKs. Existing studies have shown that Arabidopsis MAPK1 / 2 / 7 can regulate plant growth in an ABA-dependent manner through protein kinase activity, and that MAPKKK62-MAPKK3-MAPK7 / 14 mediates ABA signaling in rice, participating in seed dormancy. However, the biological function of C-group MAPK7 in dicotyledonous oilseed crops has not been systematically reported. Summary of the Invention

[0006] This invention aims to provide BnaA09.MAPK7 Its application in promoting rapeseed stem growth provides a new option for improving rapeseed lodging resistance. This application involves... BnaA09.MAPK7 Genes are integrated into rapeseed through expression vectors, effectively promoting the growth and development of rapeseed stems, which has significant application value for rapeseed breeding.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] BnaA09.MAPK7 The application of genes in promoting rapeseed stem growth, the aforementioned BnaA09.MAPK7 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0009] Preferably, the BnaA09.MAPK7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0010] Preferably, the promotion of rapeseed stem growth is achieved by regulating... BnaA09.MAPK7 Gene expression levels promote rapeseed stem growth.

[0011] The present invention also provides an overexpression vector that promotes rapeseed stem growth, the overexpression vector comprising the... BnaA09.MAPK7 Gene.

[0012] The present invention also provides a strain that promotes the growth of rapeseed stems, the strain comprising the overexpression vector described above.

[0013] The present invention also provides a method for utilizing the above-mentioned BnaA09.MAPK7 The method for promoting rapeseed stem growth using genes includes the following steps:

[0014] S1, Cloned Rapeseed BnaA09.MAPK7 Gene;

[0015] S2, Construction BnaA09.MAPK7 Overexpression vector;

[0016] S3. The overexpression vector obtained in S2 was transformed into Agrobacterium tumefaciens, and transgenic plants were obtained after identification.

[0017] Preferably, in S1, the clone BnaA09.MAPK7 The genes are specifically:

[0018] Total RNA was extracted from rapeseed and reverse transcribed into cDNA. This cDNA was then used as a template for PCR amplification using primers ORF7-F and ORF7-R.

[0019] Preferably, the nucleotide sequence of primer ORF7-F is shown in SEQ ID NO.3, and the nucleotide sequence of primer ORF7-R is shown in SEQ ID NO.4.

[0020] This invention also provides a method for screening rapeseed germplasm with excellent stem characteristics, including detecting the aforementioned characteristics in rapeseed samples. BnaA09.MAPK7 Gene expression level or the stated BnaA09.MAPK7 The activity of gene-encoded proteins.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects:

[0022] This invention discloses BnaA09.MAPK7 Application in promoting rapeseed stem growth, overexpression BnaA09.MAPK7 The stem traits of transgenic rapeseed plants (OE) were significantly improved. Compared with wild type (WT), the average stem diameter increased significantly by 42.4%, fresh weight and dry weight increased by 58.8% and 86.5% respectively, and the breaking strength and compressive strength increased by 57.9% and 63.8% respectively. Conversely, RNAi lines that inhibited the expression of this gene had thinner stems, decreased biomass and significantly reduced mechanical strength, which clearly confirmed that this gene can positively regulate the radial growth and mechanical strength of rapeseed stems.

[0023] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0024] Figure 1 for BnaA09.MAPK7 Figure showing the results of amino acid sequence identity comparison analysis between the gene and reference genes BnaA07G0021500ZS, BnaA09G0115700ZS, and BnaC09G0119100ZS.

[0025] Figure 2 for BnaA09.MAPK7 Schematic diagrams of overexpression and RNAi-inhibited expression vectors, where, Figure 2 A in the text is BnaA09.MAPK7 Schematic diagram of overexpression vector. Figure 2 B in the diagram represents the RNAi repressive expression vector.

[0026] Figure 3 for BnaA09.MAPK 7. Phenotypic diagrams of stems from overexpression, suppressed expression, and wild-type plants, among which... Figure 3 In the diagram, A represents the phenotypic diagram of fresh plant stems during the flowering period. Figure 3 The B-diameter statistics chart in the image. Figure 3 C in the graph represents the fresh weight statistics. Figure 3 D in the graph represents the dry weight statistics. Figure 3 E in the figure represents the statistical chart of breaking strength. Figure 3 F in the figure represents the compressive strength statistics.

[0027] Figure 4 for BnaA09.MAPK7 Figures showing the results of metabolite identification in stems of overexpressed, suppressed, and wild-type plants;

[0028] Figure 5 This is a graph showing the overall detection status of metabolites, where... Figure 5 In the figure, A represents the positive ion chromatogram of the QC sample mass spectrometry detection and its correlation analysis. Figure 5 In the diagram, B represents the negative ion flow chromatogram detected by QC sample mass spectrometry and its correlation analysis. Figure 5 C in the figure represents the principal component analysis of the positive ion group in the total sample. Figure 5 D in the figure represents the principal component analysis diagram of negative ion groups in the total sample;

[0029] Figure 6 for BnaA09.MAPK7 The results of screening and identification of differentially expressed metabolites in stems by overexpression and inhibition are shown in the figure. Figure 6 In the figure, A represents the total metabolite volcano diagram of overexpressed relative wild-type stems. Figure 6 In the diagram, B represents the suppression of total metabolite expression relative to wild-type stems. Figure 6 In the diagram, C represents the total metabolite volcano plot of the stem, where overexpression is relatively suppressed. Figure 6 D in the diagram represents the Venn diagram of differential metabolites among the groups. Figure 6 E in the figure represents the K-means clustering analysis of total differential metabolites;

[0030] Figure 7 for BnaA09.MAPK7 The results of the enrichment analysis of differentially expressed and inhibited metabolites are shown in the figure. Figure 7 In the diagram, A represents a heatmap of clusters of differentially expressed metabolites in the stems of the overexpressing wild type. Figure 7 In the diagram, B represents the heatmap of clustering metabolites that suppress expression relative to wild-type stems. Figure 7In the diagram, C represents the KEGG pathway enrichment analysis of overexpressed differential metabolites in the stem relative to the wild type. Figure 7 The diagram in Figure D represents the KEGG pathway enrichment analysis that inhibits the expression of differentially expressed metabolites in the stem relative to the wild type. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0032] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0033] Source of experimental materials:

[0034] The inbred line ZY821, a black-seeded rapeseed variety, originated from the Chongqing Rapeseed Engineering Technology Research Center.

[0035] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0036] Example 1

[0037] I. Rapeseed BnaA09.MAPK7 Gene cloning and bioinformatics analysis.

[0038] Roots, stems, and leaves of one-month-old healthy seedlings of the inbred line ZY821 (black-seeded Brassica napus) were used to prepare a mixed sample, and total RNA was extracted using the Tiangen RNAprep Pure Plant Kit. The mixed sample was flash-frozen in liquid nitrogen and ground into powder using a plant tissue homogenizer. 450 µL of lysis buffer was added, vortexed, and incubated in a 56°C water bath for 5 min. Then, 200 µL of chloroform was added for emulsification for 5 min, followed by centrifugation at 12000 rpm for 2 min at room temperature. The supernatant was transferred to a filter column, and the remaining steps were performed according to the kit instructions.

[0039] RNA was analyzed for concentration and quality using 1.2% agarose gel electrophoresis and NanoDrop 2000c. After passing the quality test, cDNA was prepared by reverse transcription according to the Takara PrimeScript RT reagent Kit manual for gene cloning. Using sequence information from the Brassica napus BnIR database (BnaA07G0021500ZS, BnaA09G0115700ZS, BnaC09G0119100ZS) and the Arabidopsis thaliana TAIR database (AT2G18170), specific primers were designed using ZY821 cDNA as a template, and the full-gold EasyPfu DNA Polymerase was used to clone the C-group of Brassica napus. MAPK7 Gene.

[0040] The cloning primers are ORF7-F and ORF7-R. The nucleotide sequence of primer ORF7-F is shown in SEQ ID NO.3, and the nucleotide sequence of primer ORF7-R is shown in SEQ ID NO.4.

[0041] SEQ ID NO. 3: 5'-caccATGGCGATGTTAGTTGAGCC-3'.

[0042] SEQ ID NO. 4: 5'-GGGGTTTTGCAGTTTCAGCTCCAG-3'.

[0043] An open reading frame with a sequence length of 1104 bp was obtained, as shown in SEQ ID NO.1; encoding Brassica napus C-group mitogen-activated protein kinase 7, with a sequence length of 368 amino acids, as shown in SEQ ID NO.2.

[0044] SEQ ID NO.1:

[0045]

[0046] SEQ ID NO.2:

[0047] MAMLVEPPNGIKQQGKHYYSMWQTLFEIDTKYVPIKPIGRGAYSVVRSSINRETNEKVAIKKIHNVFENRVDALRTLRELKLLRHVRHDNVIALKDVMLPTNKSSFKDVYLVYELVDTDLHQIIKSSQSLSDDHCKYFLFQLLRGLKYLHSANILHRDLKPGNLLVNANCDLKICDFGLARTSQ GNEQFMTEYVVTRWYRAPELLLCCDNYGTSIDVWSVGCIFAEILGRKPIFPGTECLNQLKLIINVVGSQQESDIRFIDNPKARRFIKSLPYSRGTHLSNLYPQANPLAIDLLQRMLVFNPTKRISVTDALSHPYMAGLFDPGSNPPAHVPISLDIDENMEERMIREMMWDEMLYYHPGAETANP.

[0048] The amino acid sequence encoded by the cloned gene was compared with that of the three reference sequences in the aforementioned BnIR database using ESPript 3.0. Figure 1 It can be seen that the amino acid sequence identity of the cloned gene with BnaA07G0021500ZS, BnaA09G0115700ZS, and BnaC09G0119100ZS is 95.4%, 98.9%, and 98.4%, respectively. It is named... BnaA09.MAPK7 .

[0049] Example 2

[0050] BnaA09.MAPK7 Construction of overexpression and suppression expression vectors and investigation of plant phenotypic traits.

[0051] In order to investigate BnaA09.MAPK7 The biological function of the gene was determined by constructing an overexpression vector using Gateway technology, following the procedures outlined in the Thermo Fisher pENTR / D-TOPOCloning Kit and LR Clonase II Enzyme Mix Kit. Simultaneously, recombination technology was used to construct the gene expression vector, following the procedures outlined in the Novizan ClonExpress II One Step Cloning Kit. BnaA09.MAPK7 RNAi repressive expression vector targeting the 376-578bp open reading frame region, among which... BnaA09.MAPK7The open reading frame (376-578 bp) sequence is shown in SEQ ID NO. 5. Schematic diagrams of overexpression and RNAi-inhibited expression vectors are shown below. Figure 2 As shown.

[0052] Depend on Figure 2 It can be seen that the overexpression vector is driven by the 35S promoter. BnaA09.MAPK7 The open reading frame was followed by a fluorescent tag eYFP, named pEarleyGate101-BnaA09.MAPK7-eYFP; the RNAi repressive expression vector with segments A and B being complementary and linked by rapeseed introns was named pFGC5941M-BnaA09.MAPK7.

[0053] SEQ ID NO.5:

[0054] TCCTCTCAGTCTCTTTCTGATGATCACTGCAAATACTTCTTGTTCCAGTTGCTAAGAGGACTGAAGTATCTTCACTCTGCAAACATCCTTCACCGAGATTTGAAGCCAGGGAACCTCTTAGTCAACGCTAACTGCGATCTAAAGATATGCGACTTCGGGTTAGCTAGAACGAGCCAGGGAAATGAACAGTTCATGACCGAGTA.

[0055] The cloning primers for segment A of the RNAi repressor expression vector are RNAi7A-F (SEQ ID NO.6) and RNAi7A-R (SEQ ID NO.7), and the cloning primers for segment B of the complementary sequence are RNAi7B-F (SEQ ID NO.8) and RNAi7B-R (SEQ ID NO.9).

[0056] The detection primers for the overexpression recombinant vector are 35S-F (SEQ ID NO.10) and OCS-R (SEQ ID NO.11), the detection primers for the B segment of the RNAi-inhibited expression recombinant vector are 35S-F (SEQ ID NO.10) and Intron-R (SEQ ID NO.13), and the detection primers for the A segment are Intron-F (SEQ ID NO.12) and OCS-R (SEQ ID NO.11).

[0057] The expression vector construction and vector detection primers are as follows:

[0058] RNAi7A-F (SEQ ID NO.6):

[0059] 5'-TGCTTAGGTGGATCCATTAAATCCTCTCAGTC-3'.

[0060] RNAi7A-R (SEQ ID NO.7):

[0061] 5'-AATTAACTCTCTAGAACCACATACTCGGTCATG-3'.

[0062] RNAi7B-F (SEQ ID NO.8):

[0063] 5'-TTACAATTACCATGGACCACATACTCGGTCAT-3'.

[0064] RNAi7B-R (SEQ ID NO.9):

[0065] 5'-TGTAAACCTGACCGTCATTAAATCCTCTCAGTCT-3'.

[0066] 35S-F (SEQ ID NO.10):

[0067] 5'-ACTATCCTTCGCAAGACCCTTCC-3'.

[0068] OCS-R (SEQ ID NO.11):

[0069] 5'-GCGGTAAGGATCTGAGCTACACA-3'.

[0070] Intron-F (SEQ ID NO.12):

[0071] 5'-ATTTCGTATCCTCATTCGGTCT-3'.

[0072] Intron-R (SEQ ID NO.13):

[0073] 5'-GCATGCATTTGAAAAATTAGGG-3'.

[0074] The sequencing-verified overexpression and RNAi repressor expression vectors were transformed into Agrobacterium tumefaciens GV3101, and then transformed into wild-type recipient material Westar (WT) using hypocotyl tissue culture technology. After screening for herbicide Basta resistance, DNA PCR, and RT-PCR detection, seeds of T0 generation plants that were positive and showed significant differences in expression levels were collected for propagation.

[0075] Following the aforementioned screening and testing, the positive / negative ratios of T1, T2, and T3 generation plants were statistically analyzed, and homozygous positive plants conforming to Mendel's laws of inheritance were selected for further experiments. Phenotypic traits of the stably expressing T3 generation lines (OE-23, OE-52, Ri-71, Ri-75) were investigated. Ten healthy plants from each of the wild-type WT, OE overexpression, and RNAi suppressed expression lines, exhibiting consistent growth and flowering, were selected. The diameter of the thickest part of the stem was measured using calipers; the fresh weight of the stems was weighed using an electronic balance, and the dry weight was measured after drying at 80℃ to constant weight. The YYD-1 stem strength tester was used to measure the stem's breaking strength and compressive strength: A 15cm section of fresh stem from the lower part was placed on a clamp support frame with a 10cm spacing. The U-shaped bending probe was slowly pressed down from 8cm above the support frame to the center of the stem internode, and the peak breaking strength of the stem was measured. The clamp support frame spacing was adjusted to 5cm, and a cylindrical 1cm... 2 The compressive strength probe was slowly and evenly pressed down until it crushed the stem epidermis. The peak compressive strength of the upper 15cm section of the fresh stem was read. Three different center points of each stem were measured, and the average value was recorded as the compressive strength of that plant. Results are as follows: Figure 3 As shown.

[0076] Depend on Figure 3 It can be seen that, compared with the stem diameter of the control WT plant (1.34±0.17cm), the stem diameter of OE-23 and OE-52 increased to 1.94±0.32cm and 1.77±0.14cm, respectively. P <0.01), Ri-71 and Ri-75 decreased to 0.89±0.12 and 0.86±0.13 cm, respectively. P <0.01). The fresh weight of the stems of OE-23 and OE-52 was significantly increased by 63.4% and 54.1% respectively compared with WT (42.1±7.3 g), while that of the RNAi lines Ri-71 and Ri-75 was significantly decreased by 62.2% and 55.1% respectively. After drying, the dry weight of the stems of OE-23 and OE-52 was 2.1 and 1.7 times that of WT (4.2±1.0 g), respectively, while that of both RNAi lines decreased to 0.5 times that of WT. In terms of mechanical properties, the breaking strength and compressive strength of the stems of OE-23 and OE-52 were 93.6-146.7 N and 204.5-303.2 N·cm, respectively. -2 It is significantly higher than WT (54.5-88.3 N, 140.8-187.2 N·cm). -2 The N-weight of Ri-71 and Ri-75 plants was significantly reduced to 35.2-54.1 N and 72.4-138.3 N·cm, respectively. -2 The above results show that BnaA09.MAPK7 Positive gene regulation of radial stem growth in Brassica napus can significantly enhance the plant's resistance to lodging.

[0077] Example 3

[0078] BnaA09.MAPK7 Evaluation and analysis of stem metabolites from overexpressing and suppressing expression plants.

[0079] To further explore BnaA09.MAPK7 The metabolic mechanisms regulating stem radial growth were investigated. UPLC-MS / MS was used to identify primary and secondary metabolites in the stems of WT, OE, and RNAi lines using non-targeted metabolomics. Three biological replicates were set up for each sample, and five plants were used to prepare a pooled sample from each replicate. Metabolite detection and analysis were performed as follows: Figure 4 As shown, the overall detection status of metabolites is analyzed as follows: Figure 5 As shown.

[0080] Depend on Figure 4 It was found that a total of 2,370 metabolites were obtained (1,797 in positive ion mode and 933 in negative ion mode), mainly including 749 amino acids and their derivatives (27.44%), 387 organic acids (14.18%), 265 benzene and its derivatives (9.71%), 127 alkaloids (4.65%), and 111 lipids (4.07%).

[0081] Depend on Figure 5 It was found that preparing quality control (QC) samples by mixing extracts from nine samples showed good repeatability of metabolite extraction and detection according to total ion chromatogram overlap analysis. The Pearson correlation coefficient was 0.9998-0.9999, indicating strong detection stability and high data quality. Principal component analysis (PCA) results of the overall detection showed that QC, overexpression (OE), suppressed expression (RNAi), and wild-type (WT) all showed a trend of significant clustering within groups and significant separation between groups. The first principal component (PC1) and the second principal component (PC2) explained more than 60% of the features of the original dataset, indicating that the metabolite distribution was highly similar among biological replicates within groups and that there were significant differences in metabolite composition among groups.

[0082] Example 4

[0083] BnaA09.MAPK7 Screening and identification of differential metabolites in the stems of overexpressing and inhibiting plants.

[0084] Using variable importance projection (VIP) > 1, Fold Change ≥ 2, or Fold Change ≤ 0.5 as thresholds, differentially expressed metabolites (DAMs) were compared and screened between the OE and WT groups (OE_vs_WT), RNAi and WT groups (RNAi_vs_WT), and OE and RNAi groups (OE_vs_RNAi). To investigate the relative content trends of metabolites in different groups, the relative contents of all DAMs in the three comparison groups were processed using Unit Variance Scaling (UV), and clustered using the K-Means clustering algorithm. The results are shown below. Figure 6 As shown.

[0085] Depend on Figure 6 The results showed that the OE_vs_WT group yielded 478 DAMs, of which 230 were upregulated and 248 were downregulated; the RNAi_vs_WT group yielded 928 DAMs, of which 366 were upregulated and 562 were downregulated; and the OE_vs_RNAi group yielded 841 DAMs, of which 492 were upregulated and 349 were downregulated. K-Means clustering divided the 1264 DAMs into 10 clusters. Compared with WT, cluster 4 contained 129 DAMs that were significantly upregulated in the OE stem and significantly downregulated in the RNAi stem; conversely, cluster 10 contained 106 DAMs that were significantly downregulated in OE and significantly upregulated in RNAi. The DAMs with the largest proportion in both clusters were amino acids and their derivatives, at 31.8% and 32.1%, respectively. In the fourth cluster, the proportions of organic acids, flavonoids, alcohols / amines, lignans and coumarins, and phenolic acid-related metabolites were 14.7%, 3.9%, 3.1%, 2.3%, and 1.6%, respectively.

[0086] Cluster 4 was enriched with several DAMs involved in lignin metabolism pathways, such as dimethylfraxetin, coumarin, 2,4-diacetylphloroglucinol, and 4-hydroxy-5-(4-hydroxy-3-methoxyphenyl)pentanoic acid, which act as precursors for lignin synthesis. These DAMs were upregulated in OE stems and downregulated in RNAi stems. Furthermore, some amino acids, His-Thr-Lys, Leu-Gln-Gln, and Glu-Tyr-Asp-Lys, were also identified as exhibiting similar metabolic trends. This indicates that BnaA09.MAPK7 is involved in regulating cell wall lignification and cell wall structural protein synthesis in Brassica napus stem tissues, contributing to secondary cell wall thickening and stem thickening.

[0087] To further explore BnaA09.MAPK7 The regulatory pathways were analyzed in 478 DAMs from the OE_vs_WT group and 928 DAMs from the RNAi_vs_WT group, and the results are as follows: Figure 7 As shown. By Figure 7 It was found that both groups identified 77 amino acids and their derivatives, 38 organic acids, 26 benzenes and their derivatives, 21 lipids, 13 flavonoids, 12 alkaloids, 12 glycerophospholipids, 11 terpenes, 10 nucleotides and their derivatives, 10 alcohols / amines, 9 heterocyclic compounds, 8 phenolic acids, 4 lignans and coumarins, 3 fatty acyls, 1 quinone, 1 glycerol, and 36 other classes, totaling 292 DAMs in 17 categories. Using a threshold of Log2Fold Change(OE_vs_WT) / Log2Fold Change(RNAi_vs_WT) < 0 between the two comparison groups, a total of 59 DAMs were screened, including amino acids and their derivatives (32.2%), lipids (18.6%), benzene and its derivatives (11.9%), nucleotides and their derivatives (5.1%), alkaloids (5.1%), glycerophospholipids (3.4%), alcohols / amines (3.4%), organic acids (3.4%), lignans and coumarins (3.4%), fatty acyls (1.7%), heterocyclic compounds (1.7%), flavonoids (1.7%), and terpenes (1.7%). The amino acid and its derivative tetrapeptides Arg-Tyr-Leu-Lys and tripeptide Phe-Pro-Lys can mimic systemin-induced JA and lignin pathways; the coumarin Rutarin promotes lignification by activating phenylalanine lyase PAL / peroxidase POD through antioxidant activity; and the lactone compound 1-peroxyferuric acid can mediate the reactive oxygen species (ROS) signaling pathway to promote lignin cross-linking. These metabolites were significantly upregulated in the stems of OE plants and significantly downregulated in RNAi (compared to WT). KEGG enrichment analysis showed that DAMs in the OE_vs_WT group were significantly enriched in metabolic pathways such as unsaturated fatty acid biosynthesis, alanine, aspartate and glutamate metabolism, biotin metabolism, and indole alkaloid biosynthesis; while in the RNAi_vs_WT group, DAMs were significantly enriched in pathways such as arachidonic acid metabolism, as well as indole alkaloid biosynthesis and biotin metabolism. These results indicate that... BnaA09.MAPK7It may positively regulate lignification and stem strength in rapeseed through processes such as carbon skeleton supply and lipid signaling cascade, which has a positive effect on the plant's lodging resistance.

[0088] This invention constructs BnaA09.MAPK7 Overexpression (OE) and suppression (RNAi) vectors were used to obtain stable OE and RNAi transgenic rapeseed lines. Phenotypic results showed that OE plants had a significantly increased average stem diameter of 42.4%, increased fresh weight and dry weight of 58.8% and 86.5%, respectively, and increased breaking strength and compressive strength of 57.9% and 63.8%, respectively. Conversely, RNAi plants had thinner stems, decreased biomass, and significantly reduced mechanical strength. Non-targeted metabolomics analysis revealed that lignin monomer synthesis intermediates such as phenylalanine and its derivatives, rutarin, and peroxycinnamic acid derivatives such as 1-peroxyferolic acid were significantly upregulated in OE lines, while RNAi lines showed the opposite trend, indicating that... BnaA09.MAPK7 This invention participates in the lignin pathway, promoting lignin cross-linking and thus thickening the secondary stem wall, positively regulating the radial growth of rapeseed stems. This invention provides new gene resources and molecular design strategies for the creation of lodging-resistant rapeseed varieties and for breeding high-yield and stable-yield varieties.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. BnaA09.MAPK7 The application of genes in promoting rapeseed stem growth is characterized by, The BnaA09.MAPK7 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The BnaA09.MAPK7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The promotion of rapeseed stem growth is achieved by regulating... BnaA09.MAPK7 Gene expression levels promote rapeseed stem growth.

4. An overexpression vector that promotes rapeseed stem growth, characterized in that, The overexpression vector comprises the one described in claim 1. BnaA09.MAPK7 Gene.

5. A bacterial strain that promotes the growth of rapeseed stems, characterized in that, The strain includes the overexpression vector as described in claim 4.

6. A method utilizing the method described in claim 1 BnaA09.MAPK7 The method for promoting rapeseed stem growth using genes is characterized by, Includes the following steps: S1, Cloned Rapeseed BnaA09.MAPK7 Gene; S2, Construction BnaA09.MAPK7 Overexpression vector; S3. The overexpression vector obtained in S2 was transformed into Agrobacterium tumefaciens, and transgenic plants were obtained after identification.

7. The method according to claim 6, characterized in that, In S1, the clone BnaA09.MAPK7 The genes are specifically: Total RNA was extracted from rapeseed and reverse transcribed into cDNA. This cDNA was then used as a template for PCR amplification using primers ORF7-F and ORF7-R.

8. The method according to claim 7, characterized in that, The nucleotide sequence of primer ORF7-F is shown in SEQ ID NO.3, and the nucleotide sequence of primer ORF7-R is shown in SEQ ID NO.

4.

9. A method for screening rapeseed germplasm with excellent stem characteristics, characterized in that, Including the detection of rapeseed samples as described in claim 1 BnaA09.MAPK7 Gene expression level or as described in claim 2 BnaA09.MAPK7 The activity of gene-encoded proteins.

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