Huperzia serrata AbMYB01 gene and application thereof

Abstract

This application belongs to the field of plant functional gene development and provides information on Polygonum sambac. AbMYB01 Genes and their applications, the aforementioned Polygonum sambac AbMYB01 The nucleotide sequence of the gene is SEQ ID No. 1. Based on the drought-resistant transcriptome data of *Polygonum sambac*, this application screened for MYB transcription factors related to drought resistance in *Polygonum sambac*; combining bioinformatics analysis, qRT-PCR expression analysis, gene cloning, and transgenic techniques, the MYB transcription factors associated with drought resistance in *Polygonum sambac* were identified. AbMYB01 The drought-resistant function and regulatory mechanism of the gene provide a theoretical basis and excellent gene resources for the further development and utilization of Polygonum sambac and the genetic improvement of forest trees.

Description

Technical Field

[0001] This application belongs to the field of plant functional gene development, specifically, this application provides Polygonum sambac. AbMYB01 Genes and their applications. Background Technology

[0002] Polygonum sambac ( Atraphaxis bracteata A. Los) is a broad-leaved shrub belonging to the Polygonaceae family and the Polygonum genus. It has characteristics such as resistance to wind erosion, sand burial, cold resistance, and drought resistance. Its wild community has few species and a simple composition, dominated by xerophytic and hyperxerophytic shrubs and perennial herbs. It has obvious desert steppe vegetation characteristics. Studying its drought resistance is of great significance for enriching the tree species for sand control and afforestation in desert areas.

[0003] MYB transcription factors are one of the largest transcription factor families in plants, playing crucial roles in signal transduction, lignin synthesis, and abiotic stress regulation. They can regulate various types of downstream target genes, thus functioning in physiological and metabolic processes in response to abiotic stresses. Based on the number of MYB repeat sequences (R) in their DNA-binding domains, the plant MYB family is mainly divided into four subfamilies: 1R-MYB / MYB-related, 2R-MYB / R2R3-MYB, 3R-MYB / R1R2R3-MYB, and 4R-MYB. The MYB-related subfamily is the second largest group after R2R3-MYB, and its members contain only one MYB domain, which can be located at different positions on the polypeptide chain. As important telomere-binding proteins, they play a key role in maintaining chromosome structure, regulating gene transcription, responding to stress, and in flower and fruit development. Summary of the Invention

[0004] The technical solution in this application is one of the research results of the following projects: Gansu Provincial Science and Technology Plan Project: "Study on the Drought Resistance Function and Regulatory Mechanism of Transcription Factor AbMYB01 in the Desert Plant Polygonum sambac" (24JRRH007), "Study on the Regulatory Mechanism of Exogenous Abscisic Acid (ABA) on Polygonum sambac under Drought Stress" (25JRRH004); Wuwei Municipal Science and Technology Plan Project: "Analysis of Molecular Mechanism of Drought Resistance in Polygonum sambac and Mining of Key Genes from the Perspective of Genomics" (WW25A01RYFL002). The implementing unit of both projects is Wuwei Forestry Science Research Institute.

[0005] Based on previously analyzed drought-resistant transcriptome data of Polygonum sambac, this application screened out MYB transcription factors associated with drought resistance in Polygonum sambac, confirmed their functions, and elucidated their mechanisms of action.

[0006] On the one hand, this application provides Polygonum sambac. AbMYB01 Genes, the Polygonum sambac AbMYB01 The nucleotide sequence of the gene is SEQ ID No. 1.

[0007] On the other hand, this application provides the aforementioned Polygonum hydropiper. AbMYB01 Proteins encoded by genes.

[0008] Furthermore, the amino acid sequence of the protein is SEQ ID No. 2.

[0009] On the other hand, this application provides the aforementioned Polygonum hydropiper. AbMYB01 Application of genes or proteins in regulating plant drought resistance.

[0010] Furthermore, overexpression of *Polygonum sambac* in the plant AbMYB01 Genes are used to enhance the drought resistance of the plant.

[0011] Furthermore, the plant is Polygonum sambac or Arabidopsis thaliana.

[0012] On the other hand, this application provides a drought-inducible promoter for Polygonum sambac, the nucleotide sequence of which is SEQ ID No. 3.

[0013] On the other hand, this application provides the above-mentioned drought-inducible promoter of Polygonum sambac in regulating the above-mentioned Polygonum sambac AbMYB01 Applications in gene expression.

[0014] On the other hand, this application provides the aforementioned Polygonum hydropiper. AbMYB01 Application of genes, proteins, or drought-inducible promoters of Polygonum sambac in the breeding of drought-resistant Polygonum sambac plants.

[0015] Furthermore, in the application, the above-mentioned Polygonum hydropiper is selected with high expression. AbMYB01 Plants that express the above-mentioned Polygonum hydropiper gene or through genetic engineering. AbMYB01 Breeding plants with specific genes.

[0016] The beneficial effects of this application include: by combining bioinformatics analysis, qRT-PCR expression analysis, gene cloning, and transgenic techniques, the application has clarified the characteristics of Polygonum sambac. AbMYB01 The drought-resistant function and regulatory mechanism of the gene provide a theoretical basis and excellent gene resources for the further development and utilization of Polygonum sambac and the genetic improvement of forest trees. Attached Figure Description

[0017] Figure 1 For genes AbMYB01 Results of cloning and subcellular localization experiments.

[0018] Figure 2 Genetically modified AbMYB01 Positive seedling identification results.

[0019] Figure 3The phenotypic characteristics of different Arabidopsis thaliana lines under normal irrigation and drought conditions are shown. The first row, from left to right, shows the phenotypes of wild-type (WT) and transgenic Arabidopsis thaliana lines (OE1, OE2) under normal growth conditions; the second row, from left to right, shows the phenotypes of wild-type (DWT) and transgenic Arabidopsis thaliana lines (DOE1, DOE2) under drought stress.

[0020] Figure 4 The malondialdehyde content and antioxidant enzyme activity of different Arabidopsis thaliana strains were determined under normal irrigation and drought conditions.

[0021] Figure 5 The proline and soluble protein content of different Arabidopsis thaliana strains under normal irrigation and drought conditions.

[0022] Figure 6 The soluble sugar and starch content of different Arabidopsis thaliana strains under normal irrigation and drought conditions.

[0023] Figure 7 for AbMYB01 Promoter activity detection results.

[0024] Figure 8 These are the results of the self-activation test.

[0025] Figure 9 for AbMYB01 promoters and AbMYB01 Results of protein-protein interactions. Detailed Implementation

[0026] Example 1: Identification of the MYB-related gene family in Polygonum sambac and its expression pattern in response to drought stress. (1) Phylogenetic analysis of MYB-related gene family proteins in Polygonum sambac: The full-length transcriptome sequencing data of *Polygonum sambac* used in this study were all derived from previous research findings of the research group. The data were uploaded to the NCBI database (accession number: PRJNA 1 028 835), and redundant and incomplete sequences were removed before screening. The MYB-related Hidden Markov Model (SANT) (PF00249) was downloaded from the online website PFAM. The Simple HMM Search function in TBtools II software was then used to search and compare the *Polygonum sambac* genome protein data with the MYB-related Hidden Markov Model, selecting protein sequences with an E value ≤ e⁻¹⁰ as candidate *Polygonum sambac* MYB-related gene families. The conserved domains of MYB-related gene family proteins were analyzed using the CDD database on the NCBI online website. Further analysis was performed using the Visualize NCBI CDD Domain Pattern function in TBtools II software, eliminating sequences with incomplete conserved domains. Based on the analysis results, the members of the *Polygonum sambac* MYB-related gene family were determined. The MYB-related gene family sequences of Polygonum sambac were sorted and compared using the ClustalW function in MEGA7 software. The comparison results were used to construct a phylogenetic tree using the neighbor-joining method. The Bootstrap value was set to 1000, and other parameters were used at their default values. The phylogenetic tree was then beautified using the online website iTOL.

[0027] A total of 55 *Polygonum sambac* MYB-related gene families were obtained through screening. To further investigate the phylogenetic relationships among members of these gene families, multiple sequence alignment was performed on the 55 *Polygonum sambac* MYB-related proteins. According to the phylogenetic tree of the *Polygonum sambac* MYB-related gene families, the 55 proteins were divided into 5 subgroups with varying numbers of members. Subgroup V contained the most members (20); subgroups I and III contained the fewest (4 each); subgroup II had the second most members (18); and subgroup VI contained 9 members. Phylogenetic analysis revealed that gene family members with closer phylogenetic relationships exhibited higher similarity in gene structure (e.g., exon-intron composition), indicating potentially high functional similarity or homology.

[0028] (2) Physicochemical properties of members of the MYB-related gene family of Polygonum sambac: The Protparam function in the online website Expasy was used to analyze the number of amino acids, molecular weight, theoretical isoelectric point, instability index, aliphatic index, and grand average of hydrophobicity of the MYB-related gene family members of Polygonum sambac. The WoLFPSORT function in the online website GenScript was used to analyze the subcellular localization of the MYB-related gene family of Polygonum sambac.

[0029] As shown in Table 1, the amino acid number of members of the Polygonum sambac MYB-related gene family ranges from a minimum of 79 to a maximum of 995, with an average of 364; the molecular weight ranges from 9732.04 to 109938.85, with an average molecular weight of 40169.56; the isoelectric point ranges from 4.81 to 11.84, with an average isoelectric point of 8.18; the instability index ranges from a minimum of 32.66 to a maximum of 71.22, with an average of 53.16; the adipose coefficient ranges from 50.41 to 111.21, with an average of 65.16; the average hydrophobicity ranges from -1.247 to 0.290; and most members are subcellularly located in the cell nucleus.

[0030] Table 1. Physicochemical properties of members of the MYB-related gene family of Polygonum sambac.

[0031] (3) Conserved motif analysis of the MYB-related gene family of Polygonum sambac: Conserved motifs of the *Polygonum sambac* MYB-related gene family were analyzed using the online website MEME, and then visualized using the Visualize Motif Pattern function in TBtools II software. Conserved domains of the *Polygonum sambac* MYB-related gene family were analyzed using the BatchCD-Search function in the online website NCBI, and then visualized using the Visualize Domain Pattern function in TBtools II software. Finally, the Gene StructureView function in TBtools II software was used to comprehensively plot the conserved motifs, conserved domains, and phylogenetic tree of the *Polygonum sambac* MYB-related gene family.

[0032] The results showed that all proteins in the *Polygonum sambac* MYB-related gene family possessed conserved domains. Conserved motifs varied significantly across different subgroups, but showed high similarity within the same subgroup. The number of motifs varied among different genes, typically ranging from 1 to 8. Motif 2 had the highest frequency, accounting for 61.8%, suggesting it is a characteristic motif of the *Polygonum sambac* MYB-related gene family. Motifs 4, 5, and 9 had the lowest frequency, with only 8 motifs in total. Most members of the *Polygonum sambac* MYB-related gene family had conserved motifs at their N-terminus, such as Motif 1, 2, and 8, which are mostly located at the N-terminus, indicating that these motifs may perform the same function. Conserved motifs were almost entirely absent at the C-terminus. The shortest motif, Motif 8, contains only 15 amino acid residues, while the longest motifs are Motif 3, Motif 4, and Motif 10, each containing 50 amino acid residues. Visualization analysis of conserved motifs shows that Motif 4 has the highest conservation.

[0033] (4) Secondary and tertiary structures of Polygonum sambac MYB-related gene family proteins: The α-helix, extended strand, β-sheet, and random coil of the MYB-related gene family proteins from *Polygonum sambac* were predicted using the SOPMA SECONDARY STRUCTURE PREDICTION METHOD function in the online website Prabi. The tertiary structure of the MYB-related gene family proteins from *Polygonum sambac* was then predicted using the SWISS-MODEL function in the online website Expacy.

[0034] Secondary structure prediction of proteins from the *Polygonum sambac* MYB-related gene family (Table 2) showed that random coils accounted for the largest proportion of these proteins, averaging 68.12%; followed by α-helices, averaging 26.13%; while extended strands and β-sheets had the lowest average proportions, at 4.03% and 1.72%, respectively. This indicates that random coils are the main components of the secondary structure of *Polygonum sambac* MYB-related gene family proteins. Random coils are unstable coding regions, where protein structure is prone to change. Therefore, it can be inferred that the proportion of random coils is positively correlated with protein function, suggesting that the functions of *Polygonum sambac* MYB-related gene family proteins exhibit greater variability and diversity.

[0035] Table 2. Prediction of secondary structure of MYB-related gene family proteins in Polygonum sambac.

[0036] Tertiary structure prediction of proteins from the *Polygonum sambac* MYB-related gene family revealed that proteins clustered within the same subgroup exhibited similar three-dimensional models. All *Polygonum sambac* MYB-related gene family proteins contained numerous random coils, with some proteins exhibiting a high proportion of α-helices, a result consistent with secondary structure prediction. Therefore, the tertiary structure of the *Polygonum sambac* MYB-related gene family is primarily composed of random coils and α-helices.

[0037] (5) Spatiotemporal expression characteristics of the MYB-related gene family under drought stress: Total RNA was extracted from the roots, stems, and leaves of *Polygonum sambac* at different time points after sorbitol-simulated drought treatment using the Total RNA Extractor (Trizol) kit (B511311), and then reverse transcribed into cDNA. Using the obtained cDNA as a template, qRT-PCR was employed to detect the spatiotemporal expression patterns of six genes representing different subfamilies within the MYB-related gene family under drought stress.

[0038] The results showed that under mild and severe drought stress, the expression levels of all six genes were higher at 24 h than at 6 h and 0 h. Furthermore, at 24 h, the gene expression levels showed a root-stem-leaf pattern (except for PB-MIX_transcript_3955). In addition, the expression levels of these genes in roots and stems were higher under severe drought stress than under mild stress, while the expression levels of most genes in leaves were higher under mild stress than under severe stress. This indicates that members of the MYB-related gene family play an important regulatory role at 24 h under different levels of drought stress, and the expression levels of the MYB-related gene family show tissue specificity with different levels of drought.

[0039] Example 2 Cloning and Analysis of Gene AbMYB01 Based on the spatiotemporal expression results of candidate genes, the gene PB-MIX_transcript_53242, which showed high expression levels in roots, stems, and leaves, was selected from multiple drought-resistant candidate genes, cloned, and named... AbMYB01 .

[0040] AbMYB01 Clones: Primers for amplification were designed using Primer Premier 6.0, as shown in Table 3.

[0041] Table 3 Primer sequences for target gene amplification

[0042] Using total RNA from Polygonum sambac leaves as a template, the gene was cloned by PCR amplification. AbMYB01; After amplification, the amplified fragments were detected by 1% agarose gel electrophoresis. The gel containing the target band was quickly excised under UV light, and the target fragment was recovered using the Magen gel recovery kit (HiPure Gel Pure DNA Mini Kit). The purified target gene was stored at -20℃ for later use.

[0043] Target gene AbMYB01 The fragment should be around 2000 bp. The size of the positive clone should be consistent with the target gene fragment. Sequencing yields the gene. AbMYB01 The CDS sequence is 2028 bp, which can encode 675 AAs, indicating that the cloning result is correct. AbMYB01 AbMYB01 Amino acid sequence (SEQ ID No.2): MDTYSSGEDLAFKTRKPYTITKQRERWTEEEHNRFLEALKLYGRAWQRIEEHIGTKTAVQIRSHAQKFFSKLEKEALVKGIPIGQAIDIDIPPPRPKRKPNNPYPRKNGAVSVVPTTQVEVKDVKQGITGSSHPGIQVLDLEKEPLPDKYIVDGGAEGRQDNSGN DLSSGSTIYPETSGISKADAKDTCNFTELMPPVKNVTNKDKSSESQLTDGAVTMDALQNNGSGHEMQGAKNFPRHVPVHILDGSARTCSDQALSSDVSYQESRPAANHHSPFHPFRGTQEEYASLLQMSSTFSSLVVSTLLQNPAAHAASMFAASFWPCPNAETSTESHP KPTNPTPSIAAVAAATVAAATAWWAAHGLLPVCPPPYHHAGFTCPPPTTANNAVNIEERKENNNNIKHFPTPEQQMDQEFSEALQAPPMSSSESGDQACPKANNILVSSNDHDKKSPEAIELKDSSKNKILKPADRSSCGSNSSSEGDADVLEKQEEEEKEENKEAEADQ AAGESISRRRSRFPSCSNDSWKEVSNGGRMAFQALFLRESLPQSFSPSHDLRKAVEQHTKKNEDRKRQEAEEMNEDRTQLDLNSNITESCSSHQETEKKRMTEDNIIEEAVANEGLLTIGLSQGKLKARRTGFKPYKRCSMEARESRMPNTTSSQEQEKGPKRLRLEEDASS AbMYB01 Subcellular localization: The PCR amplified fragment was ligated to the enzyme-digested vector pCambia1300-35S-eYFP (with EcoRI and SpeRI restriction sites) via homologous recombination. Plasmids were extracted from correctly sequenced positive clones and transformed into Agrobacterium GV3101. Colony PCR-positive clones were then confirmed by sequencing. Figure 1 Part A) is qualified and then used for subcellular localization.

[0044] Agrobacterium transformation: Positive clones were preserved; Agrobacterium injection into tobacco leaves: Overnight cultured Agrobacterium suspension was centrifuged (OD value > 0.5), suspension composition (10 mM MgCl2, 10 mM MES, 100 μM AS), and injected into 4-5 leaf stage tobacco leaves after standing at room temperature for 3 h; Confocal observation: Confocal observation was performed on tobacco leaves 24 h after injection. Candidate genes were identified by comparing nuclear markers. AbMYB01 The encoded protein is mainly located in the cell nucleus. Figure 1 (Part B), which is consistent with the prediction results of the biological information analysis of the MYB-related family of Polygonum sambac.

[0045] Bioinformatics analysis: The conserved domains of the target gene were identified using the NCBI Conserved Domain tool (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi). The protein's physicochemical properties were analyzed using the ProtParam tool in the ExPASy online software (https: / / web.expasy.org / protparam / ). Its secondary structure was predicted using SOPMA (http: / / npsa-prabi.ibcp.fr). The transmembrane domains were predicted using TMHMM 2.0 (http: / / www.cbs.dtu.dk / services / TMHMM). The signal peptide was predicted using SignalP-5.0 (https: / / services.healthtech.dtu.dk / services / SignalP / ). De novo motif prediction was performed on known promoter sequences using MEMESuite (https: / / meme suite.org / meme / ) to identify... AbMYB01 Specific binding sequence. Downloaded from the NCBI database. AbMYB01 Homologous sequences were analyzed using MEGA 12.0 software for multiple sequence alignment (ClustalW). A phylogenetic tree was constructed using the maximum likelihood method, with a bootstrap value set to 1000 replicates. Evolutionary relationships and patterns were analyzed by comparing the topological structures of different proteins.

[0046] The results show: AbMYB01The protein contains 675 amino acids, with a relative molecular mass of 76901.1, a theoretical isoelectric point of 5.72, and an instability coefficient of 62.76, classifying it as an unstable protein (proteins with an instability coefficient ≤40 are considered stable). Its adipose-weightedness index is 59, and its overall average hydrophilicity is -0.842. The lowest score for amino acid at position 566 is -3.856, while the highest score for amino acid at position 346 is 2.356, and the overall distribution is predominantly negative, suggesting hydrophilicity.

[0047] AbMYB01 Secondary structure predictions show that the sequence contains 18.23% alpha helix, 78.06% random coil, and 3.70% extended strand. AbMYB01 Tertiary structure prediction of the protein showed a GMQE value of 0.05, a QMEAN value of 0.67±0.11, and a consensus of 60.00%, indicating high model accuracy. The model's spatial conformation consisted of α-helices, random coils, and extended strands. SignalP-5.0 software prediction showed the absence of a signal peptide, indicating it is not a secretory protein. TMHMM software prediction of the protein's transmembrane sequence indicated it is a non-transmembrane protein. Further analysis using the NCBI CDD database... AbMYB01 A search of the protein's conserved domains revealed that it possesses one SANT (myb-like DNA-binding domain, SHAQKYF class) conserved domain, belonging to the MYB family.

[0048] Example 3 Gene AbMYB01 Functional verification The Agrobacterium inflorescence infection method was used to transmit the gene using Agrobacterium strain GV3101. AbMYB01 Transplanted into Arabidopsis thaliana (Columbia wild type), and genetically modified T3 generation homozygous transgenic Arabidopsis thaliana lines were used for gene transfer. AbMYB01 Functional validation was performed by determining its stress resistance indices under drought stress (simulated by a -0.2 MPa treatment). The contents of malondialdehyde and proline were determined using the thiobarbituric acid method and the acidic ninhydrin method, respectively; the contents of starch and soluble sugars were determined using the anthrone method; and the contents of soluble protein were determined using the Coomassie brilliant blue method. The activities of SOD, CAT, and POD were determined using a kit.

[0049] The transgenic expression vector carries a hygromycin (Hyg) resistance gene. The selection criterion is whether transgenic positive seedlings can grow normally on MS medium containing the resistant gene. Positive seedlings are selected on MS medium containing 25 μg / mL Hyg. Figure 2 (Part A) DNA was extracted from the leaves of positive seedlings and analyzed by PCR. The target band was consistent with the expected size. Figure 2Part B), cultivate positive seedlings and harvest seeds to obtain the T1 generation, until the T3 generation is selected. If the T3 generation seeds grow normally on MS medium containing 25 μg / mL Hyg, then a transgenic positive line is considered to have been obtained. Figure 2 (Part C) Seeds were harvested, homozygous individuals were selected, and seedlings were cultivated to verify drought resistance. Figure 2 (Part D).

[0050] Depend on Figure 3 It can be seen that overexpression AbMYB01 Arabidopsis thaliana differs significantly from the wild type phenotypically, exhibiting slenderer leaves and earlier flowering compared to the wild type; sorbitol-simulated drought treatment affects both the wild type and overexpressing Arabidopsis thaliana. AbMYB01 All Arabidopsis species were affected, exhibiting symptoms such as yellowing and drying of leaves and slowed growth, but overexpression... AbMYB01 Arabidopsis thaliana was less affected than the wild type, which still showed more signs of withering, while overexpression... AbMYB01 The leaves of Arabidopsis thaliana are turning yellow, while the leaves that are not yellow are still a tender green, indicating overexpression. AbMYB01 Arabidopsis thaliana has a stronger drought resistance than the wild type.

[0051] The results of the two-way ANOVA (Table 4) showed that the main effects of the treatments on all eight physiological indicators were highly significant. P <0.001); In the main effects of the strains, except for POD, all other indicators reached significant or highly significant levels; In the interaction between strains and treatments, except for SS, all other indicators reached significant or highly significant levels. Further simple effect analysis of different treatments on the same strain showed that drought treatment had a highly significant effect on all seven indicators of the four strains (…). P < 0.001). For SS, although the interaction was not significant, the main effect of the treatment was extremely significant, and drought treatment also significantly increased the SS content of all lines. Compared with normal irrigation, drought stress significantly increased the levels of eight physiological indicators in all lines (Table 5).

[0052] Table 4. Two-way ANOVA of treatments, strains, and their interactions on physiological parameters of Arabidopsis thaliana.

[0053] Note:* P < 0.05,** P <0.01, *** P <0.001, with degrees of freedom for treatment (1,16), strain (3,16), and interaction (3,16).

[0054] Table 5. Simple effect analysis of different treatments on physiological indicators of each strain.

[0055] Note: The values ​​in the table represent "mean value of the drought group - mean value of the control group". P <0.01, *** P <0.001. All comparisons were based on the LSD simple effects test.

[0056] Drought stress on overexpression AbMYB01 The MDA content and antioxidant enzyme activity of Arabidopsis thaliana were affected to varying degrees. Figure 4 Under normal irrigation conditions (CK), there was no significant difference in MDA content between WT and overexpression lines. Under drought treatment, the MDA content of all lines was significantly higher than that of CK, while the MDA content of all overexpression lines was significantly lower than that of WT, at 57.5%, 38.3%, and 43.0% of that of WT, respectively. Under CK conditions, the SOD activity of WT was significantly higher than that of the overexpression lines, but there was no significant difference among the overexpression lines. Under drought conditions, the SOD activity of all lines was significantly higher than that of CK, with OE1 showing the largest increase (3.00 times that of CK), OE2 and OE3 showing increases of 2.45 times and 2.39 times that of CK, respectively, and WT showing the smallest increase (0.37 times that of CK). Under CK conditions, there was no significant difference in POD activity among the lines. Under drought conditions, the POD activity of all lines increased significantly, with the POD activity of the overexpression lines significantly higher than that of WT, and there was no significant difference among the overexpression lines. Under CK conditions, except for the lack of significant differences in CAT activity between OE1 and OE2, there were significant differences in CAT activity between all lines. Under drought conditions, the CAT activity of all lines was significantly higher than that of CK, with the CAT activities of the overexpressing lines being 1.98 times, 1.10 times, and 1.34 times that of WT, respectively.

[0057] Drought stress on overexpression AbMYB01 The MDA content and antioxidant enzyme activity of Arabidopsis thaliana were affected to varying degrees. Figure 5 Under CK conditions, there was no significant difference in Pro content between WT and OE1, but their Pro content was significantly lower than that of OE2 and OE3; WT's SP content was significantly higher than that of all overexpression lines. Under drought conditions, the Pro and SP contents of all lines were significantly higher than those of CK. The Pro and SP contents of the three overexpression lines were significantly higher than those of WT, at 1.13, 1.05, and 1.08 times, and 1.10, 1.52, and 1.42 times that of WT, respectively.

[0058] Figure 6As shown, under CK conditions, the SS content of WT was significantly lower than that of the overexpressing lines, while there was no significant difference among the overexpressing lines. Drought treatment significantly increased the SS content of all lines, with OE1 showing no significant difference from WT, while OE2 and OE3 were significantly higher than WT, increasing by 25.60% and 21.96% respectively. Under CK conditions, the ST content of OE1 was significantly lower than that of WT and the other two overexpressing lines. Under drought treatment, the ST content of all lines was significantly increased compared to CK, increasing by 22.59%, 202.85%, 53.55%, and 33.69% respectively, with OE1 showing the largest increase.

[0059] Example 4 Gene AbMYB01 Promoter cloning and activity detection Genes were obtained using the Tail-PCR method. AbMYB01 The upstream promoter sequence was obtained, the amplified product was sequenced, and the resulting sequence was analyzed using PlantCARE online software. AbMYB01 The cis-acting element of the promoter. After sequencing verification, a transient transformation vector with a GUS tag was constructed, cloned, transformed, and introduced into tobacco (using the same method as gene cloning and subcellular localization as described above). The tobacco was cultured at 25°C for 2 days. Leaves were then treated with sorbitol at -0.2 MPa, with water as a control. After 24 hours, the leaves were soaked in GUS staining solution and incubated overnight at 37°C. The leaves were then destained with 70% ethanol until the background turned white before observation and photography.

[0060] Cloning genes using Tail-PCR (genome walking) method AbMYB01 The upstream promoter was first amplified using primers MIX-F1(-713) (CAATTCTGGAGAAAATAATAGTGGC, SEQ ID No. 6) and MIX-R1(+115) (GCCCGTCCATAAAGCTTCAG, SEQ ID No. 7). AbMYB01 Based on the core sequence, Tail-PCR specific primers were designed, and the *Polygonum sambac* gene was obtained through three rounds of amplification, cloning, and sequencing. AbMYB01 The upstream promoter sequence, with a length of 2540 bp, includes a 1770 bp upstream and a 770 bp downstream sequence from the transcription factor binding site. The online software PlantCARE was used to analyze... AbMYB01 Promoter sequence analysis revealed that the promoter contains 31 cis-acting elements. Besides basic promoter elements such as CAAT-box and TATA-box, it also includes functional elements such as ABRE, CGTCA-motif, MBS, TGACG-motif, and MYC, primarily involved in the regulation of ABA, MeJA, and drought, indicating that the gene... AbMYB01 These cis-acting elements primarily function during drought stress.

[0061] Gene AbMYB01 The upstream promoter sequence (SEQ ID No. 3): Will AbMYB01 The promoter transiently transformed tobacco leaves. GUS staining analysis revealed that, compared to water injection, tobacco leaves injected with the full-length promoter showed a faint blue tint, indicating promoter activity. Drought treatment resulted in significantly increased GUS signal accumulation and a deeper color compared to the control, with a marked increase in GUS content, indicating... AbMYB01 The promoter is a drought-induced promoter ( Figure 7 ).

[0062] Construct the following vectors as shown in Table 6: Table 6. Carrier-related information

[0063] MIX1 (SEQ ID No. 8): TCGCTCTCAAGTCAAGAATCATTCTCATGCATTTGTTGTATTTTTATTAGAATGGGCCCGTAAAATTGGATCCATATGTAAATCCTGGAAGGCCCAACCCATTAGTCTAGGTTTGCAATAGGCAACTTCCCACACAGCCTTTCAATAAAATTGCATCGACCCTTGTCTGTGCTTTTGTTAACATACAACACCCATGCATGCTTATTTCA CGTTTTAATCAATACTAATTACTAAGACTTTTTTTTTTTTTTTTGAGCCCAATACTAAGACATCTACAACAACTGTGAAGATATGAACTATCCCACATCGGGATTTTGAATGAAACCAAGCCTAATATATAAGCTTGGTGGGCTACTCCTCCTATTGTCAATTGATTTTAGGATGGAACCCCATCGGGCTTATGTGTTCGGCCTAACGTGC GTATGCGTATGAACCGGCGTGTGAACCCCCCACGCCATTATTGGCCCACCAGACTTTAACAACAATGGATAAACACAATCGGCGTGTGAACCCCCCACGCCATTATTGGCCCACCAGACTTTAACAACAACGGATAAACACAATTCACTCTCTATGAATCAATAGACATTACTTAAGAAACATTTACACTTTCTTATTAGTGTTTAATCAGGGTGAGCCTACGTGTACTGTAATTTAATGCATTCAAGTTGATGGCAAAGTATTGGATAAGAAATGGCTCGGTGTACTTTATTTGATAATAATTAGGTGTTTAGGTCTAATGAGACCTAAACTAGAGGCATTCATGTTTATTATTTTTAATTTAACTTACAAAAATTTAAATTAATAGACAAAAATTACCCAGAATCCATATACTGTATACA MIX2(SEQ ID No.9): GAGCCCAATACTAAGACATCTACAACAACTGTGAAGATATGAACTATCCCACATCGGGATTTTGAATGAAACCAAGCCTAATATATAAGCTTGGTGGGCTACTCCTCCTATTGTCAATTGATTTTAGGATGGAACCCATCGGGCTTATGTGTTCGGCCTAACGTGCGTATG CGTATGAACCGGGCTGTGAACCCCCCACGCCATTATTGGCCCACCAGACTTTAACAACAATGGATAAACACAATCGGGCTGTGAACCCCCCACGCCATTATTGGCCCACCAGACTTTAACAACAACGGATAAACACAATTCACTCTCTATGAATCAATAGACATTACTTAAG MIX3 (SEQ ID No. 10): TAAACACAATTCACTCTCTATGAATCAATAGACATTACTTAAGAAACATTTACACTTTCTTATTAGTGTTTAATCAGGGTGAGCTACGTGTACTGTAATTTAATGCATTCAAGTTGATGGCAAAGTATTGGATAAGAAATGGC TCGGTGTACTTTATTTGATAATAATTAGGTGTTTAGGTCTAATGAGACCTAAACTAGAGGCATTCATGTTTATTATTTTTAATTTAACTTACAAAAATTTAAATTAATAGACAAAAATTACCCAGAATCCATATACTGTATACA Self-activation assays showed that in 100 ng / mL ABA, all three MIX promoter fragments had no activating activity. Figure 8 Therefore, in the formal experiment, ABA at 100 ng / mL was used for the interaction test.

[0064] Figure 9 for AbMYB01 promoters and AbMYB01 Protein interaction results: From top to bottom, each plate shows p53p+p53, p53p+empty vector, mixp+mix, and mixp+empty vector. From right to left, each plate shows 10X serial dilutions of the bacterial culture: OD600 = 0.2, 0.02, 0.002, and 0.0002. The orange arrows indicate interacting colonies. Based on the interaction results, mix2p and... AbMYB01They have good interaction. MIX1p contains the MIX2p segment, but they do not interact, possibly because the upstream sequence of 1p contains elements that inhibit the interaction.

Claims

1. Polygonum sambac AbMYB01 Genes, characterized by, The sandwort AbMYB01 The nucleotide sequence of the gene is SEQ ID No.

1.

2. The Polygonum hydropiper according to claim 1 AbMYB01 Proteins encoded by genes.

3. The protein according to claim 2, characterized in that, The amino acid sequence of the protein is SEQ ID No.

2.

4. The Polygonum hydropiper according to claim 1 AbMYB01 The application of genes or proteins according to claim 2 in regulating plant drought resistance.

5. The application according to claim 4, characterized in that, Overexpression of Polygonum sambac according to claim 1 in the plant AbMYB01 Genes are used to enhance the drought resistance of the plant.

6. The application according to claim 4 or 5, characterized in that, The plant in question is either Polygonum sambac or Arabidopsis thaliana.

7. A drought-inducible promoter for Polygonum sambac, characterized in that, The nucleotide sequence of the drought-inducible promoter of Polygonum sambac is SEQ ID No.

3.

8. The drought-inducible promoter of Polygonum sambac according to claim 7 in regulating Polygonum sambac according to claim 1 AbMYB01 Applications in gene expression.

9. The Polygonum hydropiper according to claim 1 AbMYB01 The application of the gene, the protein according to claim 2, or the drought-inducible promoter of Polygonum sambac according to claim 7 in the breeding of drought-resistant Polygonum sambac.

10. The application according to claim 9, characterized in that, In this application, *Polygonum sambac* with high expression according to claim 1 is selected. AbMYB01 Plants that express the gene, or plants that overexpress the gene using genetic engineering techniques. According to claim 1, the Polygonum sambac AbMYB01 Breeding plants with specific genes.

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