Rhododendron MYB transcription factor RhMYB8 and application thereof

Abstract

The invention relates to the technical field of plant genetic engineering, and discloses an azalea MYB transcription factor RhMYB8 and application thereof. The nucleotide sequence of the rhododendron MYB transcription factor RhMYB8 is as shown in SEQ ID NO. 1, and the amino acid sequence coded by the rhododendron MYB transcription factor RhMYB8 is as shown in SEQ ID NO. 2. The rhododendron MYB transcription factor RhMYB8 is over-expressed in the rhododendron, so that the cold resistance of the rhododendron is improved. By adopting the rhododendron MYB transcription factor RhMYB8 and the application thereof, a new functional gene and a core target are provided for horticultural plant cold-resistant molecular breeding, and theoretical and technical foundations are laid for improving the low-temperature stress resistance of plants and cultivating new varieties of cold-resistant plants by using molecular means; the method has an important application value in quality and efficiency improvement and cold-resistant variety breeding in the azalea industry.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the MYB transcription factor of rhododendron. RhMYB8 And its applications. Background Technology

[0002] Low temperature stress is one of the important environmental factors limiting the growth and geographical distribution of garden plants. (Azalea) Rhododendron As an important ornamental plant, rhododendrons have a wide range of applications in landscaping. However, some evergreen rhododendron varieties are quite sensitive to low temperatures. Low winter temperatures can easily cause leaves to freeze, cell membranes to be damaged, and plant growth to be inhibited, thus affecting their ornamental value and promotion.

[0003] Plants activate complex molecular regulatory networks to adapt to adverse environments under low-temperature stress, with transcription factors playing a crucial role in regulating these responses. MYB transcription factors, one of the largest families of transcription factors in plants, play important functions in plant growth and development, secondary metabolism regulation, and abiotic stress responses. However, currently, there are no reports on the function of rhododendron MYB genes in regulating plant cold tolerance, nor are there any publicly available applications of related rhododendron genes in breeding cold-resistant plant varieties. Therefore, identifying and utilizing MYB genes related to cold tolerance in rhododendrons can provide important candidate genes for regulating the cold tolerance of rhododendrons and other important crops using molecular methods. Summary of the Invention

[0004] The purpose of this invention is to provide rhododendron MYB transcription factors. RhMYB8 Its application, including overexpression of rhododendron MYB transcription factor in rhododendrons. RhMYB8 It can improve the cold resistance of azaleas; it provides new functional genes and core targets for molecular breeding of cold-resistant horticultural plants, and lays a theoretical and technical foundation for improving plant resistance to low temperature stress and cultivating new cold-resistant plant varieties using molecular methods. It has important application value for improving the quality and efficiency of the azalea industry and for the breeding of cold-resistant varieties.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a rhododendron MYB transcription factor. RhMYB8 , RhMYB8 The nucleotide sequence is shown in SEQ ID NO.1.

[0006] Secondly, this invention provides a rhododendron MYB transcription factor. RhMYB8 Application in improving the cold resistance of rhododendrons: Overexpression of rhododendron MYB transcription factor in rhododendrons. RhMYB8 To improve the cold resistance of azaleas; azalea MYB transcription factor RhMYB8 The nucleotide sequence is shown in SEQ ID NO.1.

[0007] Thirdly, this invention provides azaleas. RhMYB8 Egg white, rhododendron RhMYB8 The amino acid sequence of the protein is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.1.

[0008] Fourthly, the present invention provides azaleas. RhMYB8 Application of protein in improving the cold resistance of rhododendrons RhMYB8 The amino acid sequence of the protein is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.1; overexpression of rhododendron in rhododendron RhMYB8 The gene encoding the protein enhances the cold resistance of azaleas.

[0009] Fifthly, the present invention provides a rhododendron MYB transcription factor comprising the above-mentioned rhododendron MYB transcription factor. RhMYB8 The recombinant expression vector, wherein the recombinant expression vector is a plant overexpression vector, namely pHB-YFP- RhMYB8 .

[0010] Furthermore, the present invention provides a rhododendron MYB transcription factor comprising the above-mentioned rhododendron MYB transcription factor. RhMYB8 Transgenic cell lines.

[0011] Furthermore, the present invention provides a rhododendron MYB transcription factor comprising the above-mentioned rhododendron MYB transcription factor. RhMYB8 Genetically modified recombinant bacteria.

[0012] In a sixth aspect, the present invention provides the application of the above-mentioned recombinant expression vector, the above-mentioned transgenic cell line, or the above-mentioned transgenic recombinant bacteria in improving the cold resistance of rhododendrons.

[0013] In a seventh aspect, the present invention provides a method for cultivating cold-resistant rhododendrons, wherein the rhododendron MYB transcription factor with the nucleotide sequence shown in SEQ ID NO. 1 is used. RhMYB8 By overexpressing the gene in rhododendron plants, cold-resistant rhododendrons were obtained through screening and cultivation.

[0014] Furthermore, a method for cultivating cold-resistant rhododendrons includes the following steps: incorporating rhododendron MYB transcription factors containing nucleotide sequences as shown in SEQ ID NO. 1. RhMYB8 The recombinant expression vector was transformed into rhododendron plant cells, tissues or organs through Agrobacterium-mediated transformation, gene gun, vacuum permeation or microinjection. The transformed plant material was then cultivated into complete plants, and rhododendron plants with enhanced cold resistance were screened.

[0015] The rhododendron MYB transcription factor described in this invention RhMYB8 The advantages and positive effects of its application are: 1. This invention is the first to clone and identify the R2R3-MYB transcription factor gene in rhododendrons that positively regulates cold resistance. RhMYB8 This study clarified the cold resistance function and regulatory mechanism of rhododendrons, filled the gap in the research on cold resistance genes of rhododendrons, and provided a new research direction for the analysis of the molecular mechanism of cold resistance in rhododendrons.

[0016] 2. This invention verifies RhMYB8 Overexpression significantly enhances the cold resistance of rhododendron leaves. Under low temperature stress, the frost damage symptoms of overexpressing lines are significantly reduced, the malondialdehyde (MDA) content is significantly reduced, and the accumulation of flavonols is significantly increased, providing a highly efficient functional target for the molecular improvement of cold resistance traits in rhododendrons.

[0017] 3. This invention clarifies that RhMYB8 By direct activation FLS1 , COR2 The downstream cold-response genes play a role in cold resistance, providing important evidence for elucidating the molecular regulatory network of rhododendron's response to low-temperature stress.

[0018] 4. The present invention provides RhMYB8 Genes can be widely used in molecular breeding for cold resistance in horticultural plants. They can be used not only for the cultivation of new cold-resistant varieties of azaleas, but also for the improvement of the cold resistance of other ornamental plants and economic crops through heterologous expression, which has broad application prospects and industrial value.

[0019] 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

[0020] Figure 1 In Embodiment 1 of the present invention RhMYB8 Electrophoresis diagram of PCR amplification of gene CDS sequence; Figure 2 In Embodiment 3 of the present invention RhMYB8 Subcellular localization results of the protein; Figure 3 For the low-temperature stress in Example 4 of the present invention, RhMYB8 Phenotypic comparison of rhododendron leaves overexpressing the gene and the unexpressed control; Figure 4 For the period before and after low-temperature stress in Example 4 of the present invention, RhMYB8 The results of physiological index measurements in the overexpression group and the control group are shown in the following figures: (a) is a comparison of total flavonol content; (b) is a comparison of MDA content. Figure 5 For the period before and after low-temperature stress in Example 4 of the present invention, RhMYB8 The results of the relative expression levels of downstream target genes are shown in Figure (a). RhMYB8 Relative expression level; (b) is FLS1 Relative expression level; (c) isCOR2 Relative expression level; Figure 6 This is to verify the dual-luciferase experiment in Example 5 of the present invention. RhMYB8 The results of the activation of the target gene promoter are shown in Figure (a). RhMYB8 right FLS1 Promoter activation verification graph; (b) is RhMYB8 right COR2 Promoter activation verification diagram. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] 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.

[0023] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0024] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0025] This invention is derived from the 'Royal Glory' azalea ( R Cloned from leaves of 'Miyo-no-Sakae' RhMYB8 The gene, whose full-length CDS sequence is shown in SEQ ID NO.1, has an open reading frame of 810 bp encoding 269 amino acids, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. Sequence analysis indicates that... RhMYB8 The encoded protein is a typical R2R3 type MYB transcription factor, with two conserved R2 and R3 repeat domains at the N-terminus. Subcellular localization results show that the protein is located in the cell nucleus, which is consistent with the nuclear localization characteristics of transcription factors.

[0026] This invention constructs RhMYB8 Multiple plant overexpression recombinant vectors for the gene, including pHB-YFP- for transient overexpression in rhododendron. RhMYB8 Vector, pCAMBIA1302-GFP for subcellular localization RhMYB8 The vector, pGreenII 0029 62-SK-, for dual-luciferase assays.RhMYB8 Vectors, and the target gene promoter reporter vector pGreenII 0800-LUC- FLS1 pro、pGreenII 0800-LUC- COR2 pro.

[0027] pCAMBIA3301 and pCAMBIA1302-GFP were purchased from Beijing TransGen Biotech Co., Ltd., while pHB-YFP, pGreenII 0029 62-SK, and pGreenII 0800-LUC vectors were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0028] This invention achieves [the desired effect] in rhododendron leaves through an Agrobacterium-mediated vacuum permeation method. RhMYB8 The transient overexpression of the gene was verified by low-temperature stress treatment. RhMYB8 It can significantly alleviate the frost damage symptoms of azalea leaves, reduce cell membrane damage under low temperature stress, enhance the ability to scavenge reactive oxygen species and resist the accumulation of secondary metabolites, and significantly enhance the cold resistance of azalea leaves.

[0029] Molecular mechanism verification shows that RhMYB8 It can directly bind to and activate key genes in downstream flavonol synthesis. FLS1 Cold response core genes COR2 The promoter of this gene significantly upregulates its transcriptional expression level, thereby regulating the plant's low-temperature stress response at the transcriptional level and enhancing its cold resistance. This provides new gene resources and technical solutions for cold-resistant molecular breeding of azaleas and other horticultural crops.

[0030] The following is a detailed explanation.

[0031] Example 1 RhMYB8 Gene cloning and bioinformatics analysis 1. Experimental Materials: The test plant material consisted of 3-year-old 'Yudai no Eri' rhododendron cuttings purchased in 2024 from Yonggen Rhododendron Planting Company in Jinhua City, Zhejiang Province, China. The cultivation conditions were: temperature 25℃, photoperiod of 14 h light followed by 10 h darkness, and light intensity of 100 μmol·m⁻¹. -2 ·s -1 The substrate was a mixture of peat, vermiculite, perlite, and fermented fiber in a ratio of 2:1:1:1. Healthy, disease-free, fresh, young leaves were selected, sampled, and immediately flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0032] 2. Total RNA Extraction and cDNA Synthesis. Total RNA was extracted from rhododendron leaves using the RNA prepPure Polysaccharide-Polyphenol Plant Total RNA Extraction Kit (Beijing Tiangen Biotech Co., Ltd., catalog number DP441). Quality-compliant total RNA was collected and synthesized using PrimeScript.TM The RT reagent kit with gDNA Eraser (Perfect Real Time, TaKaRa Biotechnology Co., Ltd., catalog number: RR047A) was used for reverse transcription to synthesize first-strand cDNA.

[0033] 3. RhMYB8 PCR amplification and cloning of the gene. Based on the CDS sequence of the Rhododendron reference genome and the Nco I restriction site of the pCAMBIA3301 vector, specific amplification primers with homologous arms were designed: RhMYB8 -p3301-F and RhMYB8 -p3301-R; RhMYB8 -p3301-F (SEQ ID NO.3): gggggactcttgaccatggATGGGAAGGGCTCCCTGC; RhMYB8 -p3301-R (SEQ ID NO.4): ttaccctcagatctaccatggTCAGATCAATAACGATTCGGCA.

[0034] Using the synthesized cDNA as a template, PrimeSTAR was employed. ® PCR amplification was performed using Max DNA Polymerase (Takara Biotech Ltd., catalog number R045A). The following PCR system was prepared on ice: Table 1 PCR system

[0035] The PCR amplification program was set as follows: 98℃ pre-denaturation for 1 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ total extension for 10 min.

[0036] PCR amplification products were detected by 1% agarose gel electrophoresis. Figure 1 As shown in the figure, the target band of the expected size (810 bp) was excised and purified using a DNA gel extraction kit. The purified target fragment was ligated into the vector pCAMBIA3301, then transformed into DH5α Escherichia coli competent cells, plated on LB solid medium containing kanamycin, and incubated overnight at 37°C inverted.

[0037] Single colonies were selected for positive identification via culture PCR. Positive colonies were then sent to a sequencing company for Sanger sequencing. The sequencing results were verified by BLAST alignment, confirming that the obtained sequence was substantially identical to the reference sequence, indicating successful cloning. RhMYB8 The full-length CDS sequence of the gene, its nucleotide sequence is shown in SEQ ID NO.1.

[0038] SEQ ID NO.1: .

[0039] 4. Bioinformatics analysis. Sequence analysis results showed that... RhMYB8 The gene’s open reading frame (ORF) is 810 bp in length and encodes 269 amino acids, as shown in SEQ ID NO.2.

[0040] SEQ ID NO.2: MGRAPCCAKVGLHRGPWTAREDSLLSKYIQVHGEGNWRSLPKKAGLFRCGKSCRLRWMNYLRPDIKRGNIGPDEEDLIVRMHALLGNRWSLIAGRLPGRTDNEIKNYWNTHLSKRLRDQGTDPSTHKKLSEYPN EPPPRKRRNSNRKQNKSNSEAQKQKVHNPKPTRITSLNSLTMSRNNSFDWTPNEALNGVPWSSSLKGDVDRVGFLIGDDQDPDMVNGSDLECQSGVPLSDHNTTEKLYEDYLQLLIRTEGLDLLQLDSFAESLLI .

[0041] Domain analysis of the encoded protein using the NCBI Conserved Domain Database (CDD) revealed that the protein has two highly conserved SANT domains (R2 and R3 repeat motifs) at its N-terminus, belonging to a typical R2R3-MYB transcription factor family member.

[0042] Example 2 RhMYB8 Construction of plant expression vectors 1. Vector linearization. Based on the restriction enzyme sites of different vectors, double digestion with corresponding restriction endonucleases is used for linearization. The vectors and corresponding restriction enzyme sites are as follows: Table 2 Vectors and corresponding enzyme sites

[0043] The 25 μL double enzyme digestion reaction system consisted of: 1 μg of vector plasmid, 1 μL each of restriction enzyme 1 and restriction enzyme 2, 2.5 μL of 10×QuickCut Buffer, and enzyme-free water to a final volume of 25 μL. Digestion was performed at 37℃ for 30 min. After detection by 1% agarose gel electrophoresis, the linearized vector fragment was recovered and purified, and stored at -20℃ for later use.

[0044] 2. Amplification of the target fragment and promoter fragment. Based on the terminal sequence of the linearized vector, design specific primers with homologous arms: RhMYB8 -SK-F (SEQ ID NO.5) RhMYB8-SK-R(SEQ ID NO.6); RhMYB8 -YFP-F(SEQID NO.7), RhMYB8 -YFP-R(SEQ ID NO.8); RhMYB8 -GFP-F(SEQ ID NO.9), RhMYB8 -GFP-R(SEQID NO.10); FLS1 p-LUC-F(SEQ ID NO.11), FLS1 p-LUC-R(SEQ ID NO.12); COR2 p-LUC-F(SEQID NO.13), COR2 p-LUC-R(SEQ ID NO.14)。

[0045] RhMYB8 -SK-F(SEQ ID NO.5): cgctctagaactagtggatcccgaATGGGAAGGGCTCCCTGC; RhMYB8 -SK-R(SEQ ID NO.6): gtcgacggtatcgataagcttcgaGATCAATAACGATTCGGCAAAGGA; RhMYB8 -YFP-F(SEQ ID NO.7): cttggatcctcgagctgcagATGGGAAGGGCTCCCTGC; RhMYB8 -YFP-R(SEQ ID NO.8): cccttgctcaccatactagtGATCAATAACGATTCGGCAAAGGAATCC; RhMYB8 -GFP-F(SEQ ID NO.9): gagaacacgggggacgagctcATGGGAAGGGCTCCCTGC; RhMYB8 -GFP-R(SEQ ID NO.10): gctcaccatgtcgactctagaGATCAATAACGATTCGGCA; FLS1 p-LUC-F(SEQ ID NO.11): tcgacggtatcgataagcttACCCACCACTCATACAAGATGGT; FLS1 p-LUC-R (SEQ ID NO.12): gctctagaactagtggatccCCTGAGAATACGAAGAAACTTATCACTTCAGTG; COR2 p-LUC-F (SEQ ID NO.13): tcgacggtatcgataagcttACATTGAGCTCGAATCATCGAAATTCG; COR2 p-LUC-R (SEQ ID NO.14): gctctagaactagtggatccTCTCCAAGCAGTATCCCAAAATCAATC.

[0046] by RhMYB8 Using a positive clone plasmid of the gene as a template, the target gene fragment with homologous arms was amplified by PCR; using rhododendron genomic DNA as a template, amplification was performed... FLS1 and COR2 The promoter fragment of the gene. PCR amplification, product recovery and purification methods are the same as in Example 1.

[0047] 3. Seamless Cloning and Transformation. Using a seamless cloning kit, the purified target fragment was homologously ligated with the corresponding linearized vector. The 20 μL reaction mixture consisted of: 4 μL of 5×CE II Buffer, 2 μL of Exnase II, and the target gene fragment and linearized vector were added at a molar ratio of 2:1. Enzyme-free water was then added to bring the volume to 20 μL. The reaction was carried out at 37°C for 30 min to complete the recombination ligation. The ligation product was transformed into DH5α E. coli competent cells, plated on LB agar plates with the corresponding antibiotics, and incubated overnight at 37°C. Single clones were picked for colony PCR identification. Positive clones were sent for sequencing verification. Recombinant vectors whose sequencing results were completely identical to the target sequence were considered successfully constructed. Plasmids from positive clones were extracted and stored at -20°C for later use.

[0048] 4. Agrobacterium transformation. Using the freeze-thaw method, the successfully constructed recombinant vector plasmid and the empty control plasmid were transformed into Agrobacterium GV3101 competent cells (the dual-luciferase assay vector needs to carry the pSoup helper plasmid). The specific steps are as follows: (1) Take 1-2 μg of plasmid and add it to 50 μL of Agrobacterium competent cells, mix gently, and incubate on ice for 30 min.

[0049] (2) Quickly place it in liquid nitrogen to freeze for 5 min, then in a 37°C water bath for 5 min, and then in an ice bath for 2 min.

[0050] (3) Add 500 μL of antibiotic-free LB liquid medium and culture at 28℃ and 200 rpm for 2-4 h with shaking.

[0051] (4) Centrifuge at 4000 rpm for 2 min, discard part of the supernatant, resuspend the bacterial cells and spread them on LB solid medium containing the corresponding antibiotic, and incubate in the dark at 28℃ for 36-48 h.

[0052] (5) Select a single clone for bacterial culture PCR positive verification. The positive strain is the Agrobacterium engineered strain required for subsequent experiments. Add glycerol and store at -80℃ for later use.

[0053] Example 3 RhMYB8 Subcellular localization analysis of proteins 1. Experimental materials. Seeds of Tobacco Benzoinus were sown in nutrient soil (peat:vermiculite = 2:1) and cultured in a growth chamber at 25℃ with a photoperiod of 16 h light and 8 h darkness for 5-6 weeks. Healthy plants free from pests and diseases and with 4 functional leaves were selected for the experiment.

[0054] 2. Preparation of Agrobacterium infection solution. Take the preserved pCAMBIA1302-GFP- RhMYB8 The recombinant vector Agrobacterium strain and the empty vector control strain were inoculated into LB liquid medium containing the corresponding antibiotics and cultured at 28°C with shaking at 200 rpm until OD. 600 Centrifuge at 6000 rpm for 5 min to collect bacterial cells, resuspend the cells in tobacco infection solution (10 mM MES, 10 mM MgCl2, 200 μM acetylsalicylic acid, pH 5.6), adjust OD600 to 1.0, and incubate at room temperature in the dark for 3 h for later use.

[0055] 3. Tobacco Infection and Microscopic Observation. Using a needleless syringe, Agrobacterium infection solution was slowly injected into the lower epidermis of tobacco leaves. The injection area was marked, with three biological replicates per group. The injected tobacco plants were placed in a 25℃ artificial climate chamber and incubated in the dark for 48 h. Leaves from the marked areas were harvested, and 0.5 cm × 0.5 cm mesophyll tissue samples were cut to prepare temporary slides. Cell nuclei were stained with 10 μg / mL DAPI staining solution and incubated at room temperature in the dark for 5 min. Fluorescence observation was performed using a Zeiss LSM880 confocal laser scanning microscope with the following parameters: GFP excitation wavelength 488 nm, emission wavelength 510 nm; DAPI excitation wavelength 358 nm, emission wavelength 461 nm; chlorophyll autofluorescence excitation wavelength 640 nm, emission wavelength 675 nm.

[0056] 4. Results. The GFP fluorescence signal in the empty vector control group was widely distributed in the cell membrane, cytoplasm, and nucleus; while in the 35S:: RhMYB8 The green fluorescence signal of the -GFP fusion protein is specifically distributed only in the cell nucleus and completely colocalizes with the DAPI nuclear staining signal. The results indicate that... RhMYB8 The protein is a nuclear localized protein ( Figure 2 (As shown).

[0057] Example 4 RhMYB8 Functional verification of overexpression enhancing cold resistance of rhododendron leaves 1. Preparation of Agrobacterium infection solution. Take pHB-YFP- RhMYB8 Agrobacterium-positive strains from the recombinant vector (experimental group) and the pHB-YFP empty vector control (control group) were inoculated into LB liquid medium and cultured at 28°C with shaking at 200 rpm until OD. 600 The bacterial cells were collected by centrifugation at 9000 rpm for 5 min, resuspended in tobacco infection solution, and the OD600 was adjusted to 1.0. The cells were then incubated at room temperature in the dark for 3 h for later use.

[0058] 2. Instantaneous transformation of azalea leaves. Mature functional leaves of the 'Oyo no Ei' azalea variety, with uniform leaf age, free from pests and diseases, and fully expanded, were selected. The experimental group was set up as follows (35S:: RhMYB8 Three biological replicates were set up for each group, along with an empty control group. Micro-wounds were created on the underside of leaves using a sterile parallel needle roller. The leaves were then completely immersed in the prepared Agrobacterium infection solution and placed in a vacuum pump for vacuum infiltration: a vacuum was drawn to 0.1 MPa at room temperature and maintained for 30 min, then slowly released to atmospheric pressure until the leaves completely settled, indicating sufficient extraction. The leaves were removed, gently rinsed with sterile water, and dried. They were then placed in sterile water containing 0.02% plant gel and incubated in the dark at 25°C for 48 h to complete transient transformation.

[0059] 3. Low temperature stress treatment. Leaves from the experimental group and control group after dark culture were taken. Leaves with uniform growth and size were taken from each group and wrapped in sterile gauze with uniform moisture. They were first pre-cooled in a 4℃ refrigerator for 2 h. Then they were transferred to a -8℃ low temperature constant temperature water bath circulation device for cold stress treatment for 2 h. After the treatment, they were wrapped in moist sterile gauze and placed in a 4℃ refrigerator for gradient slow freezing for 30 min.

[0060] 4. Phenotypic observation and physiological index determination. Immediately after the slow freezing was completed, the leaf phenotype was observed and photographed for recording. Subsequently, leaf samples were taken, flash-frozen in liquid nitrogen, and stored at -80℃ for subsequent physiological index and gene expression level determination.

[0061] (1) Phenotypic observation results: Before low temperature treatment, there was no significant phenotypic difference between the experimental group and the control group; after freezing at -8℃, the leaves of the unloaded control group showed large-area water-soaked necrosis, with obvious extravasation of cytoplasm from leaf veins and leaf margins, exhibiting typical symptoms of severe frost damage; while RhMYB8 The leaves of the overexpression group showed intact structure with only slight inoculation marks, and no obvious signs of frost damage, water stains, or necrosis, indicating that... RhMYB8 Overexpression significantly enhanced the tolerance of rhododendron leaves to low-temperature stress. Figure 3 (As shown).

[0062] (2) Physiological index measurement results: The total flavonol content and malondialdehyde (MDA) content were measured separately, with three technical replicates for each index. The measurement results showed that ( Figure 4 As shown in the figure): Before low-temperature treatment, there was no significant difference in MDA content between the experimental group and the control group; after freezing treatment, the MDA content in the control group increased significantly, while the MDA content in the experimental group remained at a low level, significantly lower than that in the control group, indicating that... RhMYB8 Overexpression significantly alleviated cell membrane lipid peroxidation damage induced by low-temperature stress. Regarding total flavonol content, there was no significant difference between the two groups before treatment; after low-temperature treatment, both groups showed significant accumulation, with the experimental group showing a significantly higher content than the control group. These results indicate that... RhMYB8 It can promote the synthesis and accumulation of flavonols.

[0063] 5. Gene expression analysis. qRT-PCR was used to determine the gene expression levels in the leaves of the experimental and control groups before and after low-temperature treatment. RhMYB8 Downstream target genes FLS1 and COR2 The relative expression level. (Using rhododendrons) 18S As an internal reference gene, 2 -△△Ct The relative expression levels of genes were calculated using a method with three technical replicates per group.

[0064] (1) Before low-temperature treatment, the experimental group RhMYB8 The relative expression level of the experimental group was approximately 6.5 times that of the control group, achieving transient overexpression; after freezing treatment, the experimental group... RhMYB8 The expression level was approximately 7.4 times that of the control group, still maintaining a significantly high expression level. Figure 5 (as shown in (a)).

[0065] (2) Downstream target gene expression results showed that before low-temperature treatment, the two groups FLS1 , COR2 There was no significant difference in expression levels in the experimental group; after freezing treatment, the expression levels in the experimental group... FLS1 and COR2 The expression levels of all three were significantly upregulated, and were significantly higher than those of the control group ( Figure 5 As shown in (b) and (c), where FLS1The expression level was approximately 9.4 times that of the control group. The results indicate that... RhMYB8 It can significantly activate downstream cold-response genes under low-temperature stress. FLS1 and COR2 Transcriptional expression.

[0066] Example 5 RhMYB8 Verification of transcriptional activation of downstream target genes 1. A dual-luciferase reporter gene assay was used to verify... RhMYB8 right FLS1 and COR2 The direct activation of gene promoters involves the following steps: 2. Preparation of Agrobacterium infection solution: Take pGreenII 0029 62-SK- RhMYB8 (Effect vector), pGreenII0800-LUC- FLS1 pro (report carrier), pGreenII 0800-LUC- COR2 The pro (reporter vector) and the corresponding empty vector control Agrobacterium strain were used to prepare the infection solution, and the method was the same as in Example 3, with the OD600 adjusted to 1.0.

[0067] 3. Infection solution mixing: The infection solutions of the effect vector and the reporter vector were mixed uniformly at a volume ratio of 10:1. An experimental group and an empty control group were set up, with 3 biological replicates in each group.

[0068] 4. Tobacco Injection and Culture: The mixed infection solution was injected into the leaves of 5-6 week old Nicotiana benthamiana plants. The injection area was marked to avoid vein contamination and cross-contamination. The injected tobacco plants were then cultured in the dark for 48 hours under the same conditions as in Example 3.

[0069] 5. Fluorescence signal detection: After dark incubation, 1 mM D luciferase substrate was injected into the injection area of ​​the leaf and placed at room temperature in the dark for 10 min. Fluorescence signals were acquired using a CCD imaging device with an exposure time of 10 min, and the fluorescence intensity was quantitatively analyzed using ImageJ software.

[0070] 6. Results: Compared with the unloaded control group, RhMYB8 and FLS1 pro COR2 tobacco leaves co-transformed with pro all showed significantly enhanced fluorescence signals, with fluorescence intensity being significantly higher than that of the control group. Figure 6 As shown). The results indicate that, RhMYB8 The protein can significantly activate FLS1 and COR2 Transcriptional activity of gene promoters.

[0071] Therefore, this invention utilizes the aforementioned rhododendron MYB transcription factor. RhMYB8 Its applications, through cloning rhododendronsRhMYB8 Genes were used to construct plant recombinant expression vectors, achieving transient overexpression in rhododendron leaves; biological function verification was then performed. RhMYB8 Compared to the empty vector control group, the overexpression material significantly reduced freezing damage symptoms under low-temperature stress, significantly decreased cell membrane lipid peroxidation damage, and significantly increased flavonol accumulation. It also significantly activated downstream cold-response target genes. FLS1 , COR2 Transcriptional expression, Indicating RhMYB8 Genes have the function of positively regulating the cold resistance of plants. This invention provides new functional genes and core targets for molecular breeding of cold-resistant horticultural plants, laying a theoretical and technical foundation for improving plant resistance to low-temperature stress and cultivating new cold-resistant plant varieties using molecular methods. It has important application value for improving the quality and efficiency of the rhododendron industry and for the breeding of cold-resistant varieties.

[0072] 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. Rhododendron MYB transcription factor RhMYB8 Its features are: RhMYB8 The nucleotide sequence is shown in SEQ ID NO.

1.

2. Rhododendron MYB transcription factor RhMYB8 Its application in improving the cold resistance of azaleas is characterized by: Overexpression of rhododendron MYB transcription factor in rhododendrons RhMYB8 To improve the cold resistance of azaleas; azalea MYB transcription factor RhMYB8 The nucleotide sequence is shown in SEQ ID NO.

1.

3. Azalea RhMYB8 Protein, characterized by: azalea RhMYB8 The amino acid sequence of the protein is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.

1.

4. Azalea RhMYB8 The application of protein in improving the cold resistance of rhododendrons is characterized by: azalea RhMYB8 The amino acid sequence of the protein is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.1; overexpression of rhododendron in rhododendron RhMYB8 The gene encoding the protein enhances the cold resistance of azaleas.

5. Containing the rhododendron MYB transcription factor as described in claim 1 RhMYB8 The recombinant expression vector is characterized by: The recombinant expression vector is a plant overexpression vector, namely pHB-YFP- RhMYB8 .

6. Containing the rhododendron MYB transcription factor as described in claim 1 RhMYB8 Transgenic cell lines.

7. Containing the rhododendron MYB transcription factor as described in claim 1 RhMYB8 Genetically modified recombinant bacteria.

8. The application of the recombinant expression vector of claim 5, the transgenic cell line of claim 6, or the transgenic recombinant bacteria of claim 7 in improving the cold resistance of rhododendrons.

9. A method for cultivating cold-resistant rhododendrons, characterized in that: The rhododendron MYB transcription factor with the nucleotide sequence shown in SEQ ID NO.1 RhMYB8 By overexpressing the gene in rhododendron plants, cold-resistant rhododendrons were obtained through screening and cultivation.

10. The method according to claim 9, characterized in that, The steps include: including the use of rhododendron MYB transcription factor containing the nucleotide sequence shown in SEQ ID NO.

1. RhMYB8 The recombinant expression vector was transformed into rhododendron plant cells, tissues or organs through Agrobacterium-mediated transformation, gene gun, vacuum permeation or microinjection. The transformed plant material was then cultivated into complete plants, and rhododendron plants with enhanced cold resistance were screened.

Images