Chrysanthemum transcription factor cmwrky33 and application thereof in resisting chrysanthemum black spot disease

Abstract

This invention discloses chrysanthemum transcription factors CmWRKY33 This invention relates to the field of molecular biology technology and its application in combating chrysanthemum black spot disease. The invention screened chrysanthemum transcription factors through the chrysanthemum transcriptome. CmWRKY33 Furthermore, the chrysanthemum transcription factor was discovered for the first time. CmWRKY33 Significantly induced by the pathogen of black spot disease and SA, its expression was upregulated, making it a positive regulator of chrysanthemum's resistance to black spot disease. Furthermore, CmWRKY33 By increasing disease-related genes CmNPR1 , CmTGA2 and SA synthesis key enzyme genes CmPAL、CmICS The expression of [a specific substance] increases the content of SA in plants, thereby enhancing the plant's resistance to black spot disease. This invention, through analysis... CmWRKY33 It participates in and regulates the molecular mechanism of chrysanthemum's resistance to black spot disease, providing a basis for its application. CmWRKY33 This provides a theoretical and technical basis for solving the problem of black spot disease in chrysanthemum production.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, and particularly relates to chrysanthemum transcription factors. CmWRKY33 And its application in combating black spot disease in chrysanthemums. Background Technology

[0002] chrysanthemum( Chrysanthemum morifolium Chrysanthemums (Ramat.) belong to the genus Chrysanthemum in the family Asteraceae. They are perennial herbaceous plants with a cultivation history of over 3,000 years in China and are one of the world's most famous ornamental plants. Chrysanthemums come in many varieties and have diverse flower shapes, making them widely used in cut flowers, potted plants, and landscaping, thus possessing high economic value. Furthermore, some varieties of chrysanthemums also have medicinal and tea-making value, and are commonly used traditional Chinese medicines included in the Chinese Pharmacopoeia, showing broad application prospects.

[0003] Because chrysanthemums are typically propagated through cuttings and division, they are susceptible to various pathogens during their growth stages, leading to diseases. This directly increases the production cost of chrysanthemum products and restricts their sales and export. The inventors' preliminary investigations revealed that black spot disease is one of the leading diseases affecting chrysanthemums and has gradually become a limiting factor in chrysanthemum cultivation and production.

[0004] The main pathogen of chrysanthemum black spot disease is Alternaria. Alternaria Fungi, such as Alternaria, were isolated and identified as the pathogen causing black spot disease on Chrysanthemum morifolium. Alternaria sp.). Alternaria sp. is a typical necrotrophic pathogen. Plants typically defend against necrotrophic pathogens through JA and Eth-mediated pathways. SA-mediated signaling pathways are more effective in protecting plants against vivitrophic or semi-vivitrophic pathogens, and SA and JA-mediated signaling pathways have antagonistic effects. Exogenous application of SA inhibits JA response genes (…). LOX2 , PDF1.2 and VSP The expression of SA (Sarcoptes Acid) and JA (Januarylex JA) makes plants more susceptible to necrotrophic fungi. However, with further research, increasing evidence suggests that the SA and JA signaling pathways are not entirely opposed in their defense against pathogens, but rather overlap, forming a complex defense network. Studies have shown that the SA signaling pathway can participate in the response to necrotrophic pathogens. For example, Nováková et al. found that after rapeseed was inoculated with the necrotrophic Sclerotinia sclerotiorum, the SA content in the leaves of infected plants increased and the expression of SA marker genes was upregulated; similar results were obtained in tomatoes. Chrysanthemum CmNPR1 The SA signaling pathway is involved in the response of chrysanthemum to necrotrophic black spot pathogen. Alternaria Sp. defense. Complete SA signaling defense of potatoes against necrotrophic fungi. Alternaria solaniThe invasion of pathogens is necessary. Because the plant immune system is so complex, the molecular mechanisms by which plants respond to various hormone signaling pathways to defend against pathogen invasion require further in-depth research.

[0005] Currently, the most studied transcription factors related to biotic stress in plants include the WRKY, bZIP, NAC, AP2 / ERF, MYB, and bHLH families. Among them, WRKY transcription factors are a plant-specific class of transcription factors that participate in the regulation of multiple signaling pathways. WRKY transcription factors generally have a conserved heptapeptide structure at the N-terminus. When plants face biotic and abiotic stresses, WRKY transcription factors can regulate gene expression by activating related hormone signaling pathways, such as SA, ABA, and JA, thereby prompting the plant to make corresponding responses. WRKY33 belongs to Group I of the WRKY transcription factor family. It has one zinc finger domain at the C-terminus and two WRKY domains at the N-terminus. It is an important regulator in the pathway of plant resistance to necrotrophic pathogens. In Arabidopsis thaliana... wrky33 In mutants, SA-related genes PAD4 and PR1 , PR2 , PR5 Upregulation of JA signaling pathway was found to be severely impaired, leading to increased susceptibility to necrotrophic pathogens. This was observed when exposed to *Botrytis cinerea* (…). Botrytis cinerea During infection, the Arabidopsis CCCH protein C3H14 activates the WRKY33-ORA59-PAD3 cascade, thereby regulating the JA / ET and Camalexin-mediated defense responses. Overexpression in apples... MdWRKY33 It can improve resistance to leaf spot disease.

[0006] Depend on Alternaria Chrysanthemum black spot disease caused by *Sp. sp.* is a problem that urgently needs to be solved in chrysanthemum production. Although WRKY33 has been reported in some plants, its regulatory effects on the disease are both positive and negative. CmWRKY33 The role of chrysanthemum in resisting black spot disease is not yet clear. Summary of the Invention

[0007] To address the aforementioned problems in production, this invention provides a chrysanthemum transcription factor. CmWRKY33 This invention relates to chrysanthemum transcription factors and their application in combating chrysanthemum black spot disease. The invention screened chrysanthemum transcription factors through chrysanthemum transcriptome analysis. CmWRKY33 Furthermore, the chrysanthemum transcription factor was discovered for the first time. CmWRKY33 Significantly induced by the pathogen of black spot disease and SA, its expression was upregulated, making it a positive regulator of chrysanthemum's resistance to black spot disease. Furthermore, CmWRKY33 By increasing disease-related genes CmNPR1 , CmTGA2and SA synthesis key enzyme genes CmPAL, CmICS The expression of [a specific substance] increases the content of SA in plants, thereby enhancing the plant's resistance to black spot disease. This invention, through analysis... CmWRKY33 It participates in and regulates the molecular mechanism of chrysanthemum's resistance to black spot disease, providing a basis for its application. CmWRKY33 This provides a theoretical and technical basis for solving the problem of black spot disease in chrysanthemum production.

[0008] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One of the objectives of this invention is to provide CmWRKY33 The application of genes in regulating resistance to chrysanthemum black spot disease, the aforementioned CmWRKY33 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the regulation is positive regulation.

[0009] The CmWRKY33 The gene is 1655 bp in length, including coding and non-coding regions. The first 46 bps are the 5' UTR of the non-coding region, the 47th to 1510 bps are the coding region, and the 1511th to 1655th bps are the 3' UTR of the non-coding region.

[0010] Furthermore, the positive regulation method includes overexpression CmWRKY33 Gene.

[0011] A second objective of this invention is to provide a primer combination, wherein the primer combination is used for overexpression and / or silencing of the [specific term / object]. CmWRKY33 Genes; among which are used for overexpression CmWRKY33 The primer nucleotide sequences for the gene are shown in SEQ ID NO.21~SEQ ID NO.22, and are used for silencing. CmWRKY33 The primer nucleotide sequences of the gene are shown in SEQ ID NO.25~SEQ ID NO.30.

[0012] A third objective of this invention is to provide a kit comprising the aforementioned primer combination.

[0013] The fourth objective of this invention is to provide a recombinant expression vector containing the aforementioned... CmWRKY33 Gene.

[0014] The fifth objective of this invention is to provide a host bacterium containing the aforementioned... CmWRKY33 The gene may contain the recombinant expression vector.

[0015] The sixth objective of this invention is to provide the application of the recombinant expression vector and / or the host bacteria in improving the resistance of chrysanthemum to black spot disease.

[0016] Furthermore, the application includes its use in the breeding of new varieties and / or lines of chrysanthemums resistant to black spot disease.

[0017] The seventh objective of this invention is to provide a method for improving the resistance of chrysanthemum to black spot disease, wherein the method involves overexpressing [a specific gene] in the target chrysanthemum material. CmWRKY33 Genes are being processed.

[0018] The eighth objective of this invention is to provide a method for breeding new varieties and / or lines of chrysanthemums resistant to black spot disease, the method comprising overexpressing in target chrysanthemum material CmWRKY33 The steps to obtain transgenic plants from genes.

[0019] Furthermore, the chrysanthemums mentioned above are Huai chrysanthemums.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention has discovered that chrysanthemum transcription factors CmWRKY33 Significantly induced by the pathogen of black spot disease and SA, its expression is upregulated, making it a positive regulator of chrysanthemum's resistance to black spot disease; and CmWRKY33 By increasing disease-related genes CmNPR1 , CmTGA2 and SA synthesis key enzyme genes CmPAL, CmICS The expression of SA increases the content of SA in plants, thereby enhancing the plant's resistance to black spot disease.

[0021] (2) This invention analyzes CmWRKY33 It participates in and regulates the molecular mechanism of chrysanthemum's resistance to black spot disease, providing a basis for its application. CmWRKY33 This provides a theoretical and technical basis for solving the problem of black spot disease in chrysanthemum production. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating the breeding process of 'Huaiju No. 2' in Embodiment 1 of the present invention. Figure 2 This is the variety identification certificate for 'Huaiju No. 2' in Embodiment 1 of the present invention; Figure 3 In Embodiment 1 of the present invention CmWRKY33 Evolutionary tree analysis diagram; Figure 4 In Embodiment 1 of the present invention CmWRKY33 Genes in Alternaria sp. expression level analysis under inoculation and SA treatment; Figure 5 In Embodiment 2 of the present invention CmWRKY33 Expression analysis diagram in different tissues of Chrysanthemum indicum; Figure 6 This is an analysis of the transcriptional activation activity and subcellular localization of the CmWRKY33 protein in Example 3 of the present invention; Figure 7 In Embodiment 4 of the present invention CmWRKY33 Overexpression of Huai chrysanthemum strains in Alternaria Morphological diagram under sp. infection; Figure 8 In Embodiment 4 of the present invention Alternaria sp. processing CmWRKY33 Overexpression chrysanthemum strains CmNPR1 , CmPR1 , CmTGA2 A diagram illustrating the impact of gene expression levels; Figure 9 In Embodiment 5 of the present invention CmWRKY33 Image showing expression levels in transiently silenced plants; Figure 10 In Embodiment 5 of the present invention CmWRKY33 Instantaneous silence of detached leaves of plants Alternaria Morphological diagram under sp. infection; Figure 11 In Embodiment 5 of the present invention CmWRKY33 A graph showing the detection of SA content in transiently silenced plants; Figure 12 In Embodiment 5 of the present invention CmWRKY33 Instantaneous silence in the plant CmCM , CmICS , CmPAL , CmNPR1 , CmPR1 , CmTGA2 A diagram illustrating the impact of gene expression levels. Detailed Implementation

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0024] The materials and methods used in the experiments of this invention are as follows: I. Experimental Materials Plant materials: The chrysanthemum plant material used in the experiment was the Huai chrysanthemum variety 'Huaiju No. 2', whose tissue culture seedlings were propagated in our laboratory. The material used for protein localization and other experiments was wild-type tobacco, Nicotiana benthamiana (…). Nicotiana benthamiana ).

[0025] Test strains and vectors: Alternaria alternata, the pathogen of black spot disease in Chrysanthemum morifolium ( Alternariasp. (see the paper "Isolation and Identification of the Pathogen of Black Spot Disease of Chrysanthemum" published by Zhao Xiting et al.), Escherichia coli DH5α, Agrobacterium GV3101, cloning vector pMD18-T, and overexpression vector Super1300-GFP(C).

[0026] Instruments: UV-2600 ultraviolet spectrophotometer, ultra-micro ultraviolet spectrophotometer, pH meter, ultraviolet gel imaging system, YJ-1450A clean bench, gradient PCR instrument, high-speed refrigerated centrifuge, LightCycler 96 real-time fluorescence quantitative PCR instrument, biosafety cabinet, autoclave, analytical balance, stereo microscope, upright intelligent electric microscope, electrophoresis apparatus, constant temperature shaker, metal bath, electric thermostatic incubator, constant temperature water bath, etc.

[0027] Reagents: 2×Es Taq MasterMix (Dye) and 2×Pfu MasterMix (Dye) reagents were purchased from Kangwei Company; total RNA extraction reagent (RNAiso Plus) and T4 ligase were purchased from TaKaRa. Xba I. Kpn I. Spe I. Asc I and Pac Grade I reagents were purchased from NEB (Beijing) Co., Ltd.; Evans blue, agarose, nitro blue tetrazolium (NBT), diaminobenzidine (DAB), agar, kelp polysaccharide, and aniline blue were purchased from Beijing Solarbio Co., Ltd.; LB medium was purchased from Shanghai Sangon Biotech Co., Ltd.; ampicillin (Amp) was also purchased. β All biochemical reagents, including galactoside (X-gal), isopropyl thiogalactoside (IPTG), rifampin (Rif), kanamycin (Kan), chloramphenicol (Chl), cephalosporin (Cef), hygromycin (HmB), salicylic acid (SA), acetylsuccinone (AS), 2-(N-morpholino)ethanesulfonic acid (MES), methyl jasmonic acid (MeJA), and guaiacol, were of analytical grade or higher. Primers and sequencing used in the experiment were performed by Sangon Biotech (Shanghai).

[0028] The main reagent kits used in the experiment included: plasmid extraction kit, microcolumn DNA concentration gel recovery kit, SE seamless cloning and assembly kit, and DNA marker, all purchased from Beijing Zhuangmeng Company; the SYBR fluorescence quantitative kit and reverse transcription kit were purchased from Novizan (Nanjing) Company. Except for yeast culture medium, all commonly used culture media were prepared by autoclaving at 121℃ for 20 min, while yeast culture medium was autoclaved at 115℃ for 15 min.

[0029] The culture media used in the experiment are shown below: Rooting solid medium: 1 / 2 MS + 0.01 mg / L NAA + 7.3 g / L agar powder, pH 5.93; LB liquid medium: 25 g / L LB powder, pH 7.0; LB solid medium: 25 g / L LB powder + 15 g / L agar powder, pH 7.0; PDA medium: 200 g / L potato + 20 g / L glucose + 10 g / L agar powder.

[0030] II. Test Methods 1. Total RNA extraction and cDNA synthesis RNA was extracted according to the RNAiso Plus (TaKaRa, Beijing) instructions. RNA degradation and integrity were detected by 1% agarose gel electrophoresis and UV irradiation. RNA concentration was measured using a micro UV spectrophotometer. RNA of higher quality was used for subsequent reverse transcription. The extracted total RNA was reverse transcribed into cDNA according to the reverse transcription kit instructions. The reaction system and conditions are shown in Table 1.

[0031] Table 1. Reaction system and conditions for cDNA synthesis

[0032] Note: X indicates that, except for 4×gDNA wiper Mix and RNA, the other components were made up with RNase-free ddH2O to bring the total volume to 16µL.

[0033] 2. Real-time quantitative PCR The expression levels of relevant genes were detected using a real-time PCR kit, and chrysanthemums were selected. Ubiquitin Gene( CmUBI EU86XX25 was used as an internal control. Three replicates were set up for each sample. Specific primers are shown in Table 2. The qRT-PCR reaction system is shown in Table 3.

[0034] Table 2 CmbHLHs Gene qRT-PCR primers

[0035] Table 3. qRT-PCR reaction system

[0036] 3. CmWRKY33 Gene cloning According to the established CmWRKY3� The transcript sequences were designed using Primer 6.0 software at the 5' UTR and 3' UTR of the ORF region.CmWRKY33 Specific primers for gene cloning were used for PCR amplification with *Chrysanthemum indicum* cDNA as a template. The specific primers are shown in Table 4, and the reaction system is shown in Table 5.

[0037] Table 4 CmbHLH18 Gene PCR primers

[0038] Table 5 PCR reaction system

[0039] 4. CmWRKY33 Construction of overexpression vectors Based on what has been obtained CmWRKY^3 The cDNA sequence was obtained, and appropriate restriction enzyme sites were selected and corresponding protective bases were added using the Super1300-35S-GFP vector. Primers were designed at both ends of the ORF region using Primer 6.0 software. CmWRKY33 - Xba IF and CmWRKY33 - Kpn IR. PCR amplification was performed using cDNA from *Chrysanthemum indicum* as a template. Primers. CmWRKY33 - Xba IF and CmWRKY33 - Kpn The specific sequences of IR are shown in Table 6. The PCR products were recovered and purified using a kit. After detecting the concentrations of the purified products and the Super1300-35S-GFP plasmid, they were respectively... Xba I and Kpn I. Perform double digestion, and then ligate the digested products with T4 ligase. The digestion and ligation system and conditions are shown in Tables 7 and 8.

[0040] Table 6 Primers CmWRKY33-XbaI -F and CmWRKY33-Kpn I -R

[0041] Table 7. Double enzyme digestion reaction system and conditions

[0042] Table 8 Connection System and Conditions

[0043] 5. CmWRKY^3 Subcellular localization Remove the Agrobacterium Super1300-35S-GFP empty vector and Super1300- from the -80℃ freezer. CmWRKY33The -GFP vector was activated by streaking on LB solid medium (Rif + Kan) and cultured at 28 ℃ for 2 days. Single colonies were picked and cultured in 5 mL of LB liquid medium (5 µL Rif + 5 µL Kan) at 28 ℃ with shaking at 180 r / min until turbidity was reached. Then, the culture was inoculated into 20 mL of LB solution (20 µL Rif + 20 µL Kan), and 8 µL AS (0.1 mol / L) and 200 µL MES (1 mol / L) were added for expansion culture until OD was reached. 600 =0.6-0.8. Place the bacterial culture in a 50 mL centrifuge tube, incubate at 4℃, 4000 r / min for 15 min, discard the supernatant, resuspend the bacteria in 10 mmol / L MgCl2, and adjust OD... 600 =1.0, add 16 µL AS (0.1 mol / L) and mix well. Inject the infection solution into the back of the tobacco plant using a sterile syringe without a needle, injecting 3 leaves per plant. Use uninjected tobacco plants as a control. Then place the plants in darkness (25 ℃, 100% relative humidity) for 1 day, and then transfer them to normal light for 2 days before harvesting. Immerse the infected tobacco leaves in DAPI for 10 min and observe them using an upright intelligent fluorescence microscope. CmWRKY33 Subcellular localization of proteins in tobacco cells.

[0044] 6. CmWRKY33 Transcriptional self-activation activity assay Select pGBKT7 vector BamH I and EcoR I As a double restriction site, according to CmWRKY33 The sequence and BamH I and EcoRIPrimers were designed based on the sequence; primer sequences are shown in Appendix Table 9. The transcription activation analysis vector pGBKT7-CmBBX32 was obtained. 100 μL of AH109 competent cells thawed on ice were taken, and 2-5 μg of the pre-chilled constructed vector plasmid, 10 μL of carrier DNA (incubated at 95-100 ℃ for 5 min, then rapidly chilled on ice, repeated once), and 500 μL of PEG / LiAc were added sequentially. The mixture was then pipetted several times to mix thoroughly and incubated at 30 ℃ for 30 min (twisting 6-8 times at 15 min). The tube was then placed in a 42 ℃ water bath for 15 min (twisting 6-8 times at 7.5 min). The tube was centrifuged at 5000 rpm for 40 s, the supernatant was discarded, and the cells were resuspended in 400 μL of ddH2O. The resuspended cells were centrifuged for 30 s, the supernatant was discarded, and the cells were resuspended in 50 μL of ddH2O. The cells were then plated on SD / -Trp medium and incubated at 30 ℃ for 48-96 h. Single yeast colonies grown on SD / -Trp medium were transferred to SD / -Trp-His and SD / -Trp-His+X-α-gal medium for screening and color development. The colonies were incubated at 30 ℃ for 5 days, and colony growth and color development were observed.

[0045] Table 9 Primers CmWRKY33-BamHI -F and CmWRKY33-EcoRI -R

[0046] 7. Chrysanthemum tissue culture seedlings Alternaria sp. vaccination method When the 'Huaiju No. 2' seedlings grow to 8-10 leaves, they will be stored at 4℃. Alternaria sp. activation: Under aseptic conditions, spores were picked up with a hook and inoculated onto PDA medium. The medium was then inverted and placed in a dark incubator at 24°C. After 5 days, sterile distilled water (containing 0.01% Tween) was added, and spores were scraped off with a sterile pipette tip to prepare a spore suspension. The spores were counted using a hemocytometer, and the spore concentration was continuously adjusted to 1×10⁻⁶ under an optical microscope. 7 Count / mL. The needle-pricking method was used to prick 3-5 leaves of *Chrysanthemum indicum* from top to bottom. Alternaria sp. inoculation was performed, and after inoculation, the leaves were covered with plastic wrap and a plastic shell, and incubated in the dark for 48 h (100% relative humidity and 24 ℃), after which they were transferred to a normal culture room. At 0, 1, 3, and 5 days after inoculation, 0.1 g of leaf samples were taken, flash-frozen in liquid nitrogen, and stored at -80 ℃. Each treatment was repeated in triplicate.

[0047] 8. CmWRKY33 Construction of transient silence carrier Artificial microRNAs (amiRNAs) were designed using the online website Web MicroRNA Designer (WMD), available at http: / / wmd3.weigelworld.org / cgi-bin / webapp.cgi. Target sequences were screened using the Chrysanthemum Transcriptome Database. Sequences completely identical to a portion of the target gene's CDS sequence were selected as candidates. These candidate sequences were validated using an online website (http: / / www.icugi.org / chrysanthemum / ?tdsourcetag=s_pcqq_aiomsg). Primers were designed using "WMD Oligo," resulting in four specific primers (I, II, III, and IV); primers A and B were designed based on the pRS300 sequence at its 5' and 3' ends. The amiRNA gene was amplified using six primers (four specific primers I, II, III, and IV, and two universal primers A and B) and the plasmid pRS300. The recovered fragment was ligated into the pCE2TA / Blunt-Zero vector, mixed thoroughly, and incubated at 25°C for 5 min. The ligation product was then transformed into *E. coli* DH5α competent cells using a heat shock method. White single colonies were selected from overnight LB agar plates and propagated by shaking. The bacterial culture obtained from the above steps was subjected to colony PCR, and five positive single colonies were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0048] Based on the pRS 300 sequence, appropriate restriction enzyme sites Kpn I and Xba I were selected. The final PCR product was digested with Kpn I and Xba I and then ligated into... Kpn I- Xba The silencing vector primers in the pCVA vector after I enzyme digestion are shown in Table 10, and the enzyme digestion system is shown in Table 11.

[0049] Table 10 Primers for silencing vectors

[0050] Table 11. Double enzyme digestion reaction system and conditions

[0051] The ligation product was transformed into competent *E. coli* cells DH5α. After 12 h, single colonies were picked from Amp-resistant culture dishes using a sterile pipette tip and cultured in liquid LB medium containing Amp (37 ℃, 180 r / min) with shaking for colony PCR verification. The recombinant plasmid was extracted and transformed into *Agrobacterium* GV3101. The *Agrobacterium* colonies that had been correctly verified by PCR were further cultured, and after adding 80% glycerol, stored at -80 ℃.

[0052] 9. Determination of SA content Take 0.2 g of plant leaves, grind them with liquid nitrogen, add 1 mL of pre-cooled PBS buffer (pH 7.4), and centrifuge at 12,000 rpm for 10 min. Detect the SA content according to the Plant Salicylic Acid (SA) ELISA Research Kit (TransGen Biotech).

[0053] 10. Gene expression level detection Samples were taken on days 0, 1, 3, 5, and 7 after inoculation, flash-frozen in liquid nitrogen, and stored at -80°C for subsequent determination of defense gene expression levels. Each sample was replicated three times.

[0054] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0055] Example 1: Response to black spot pathogen-induced CmWRKY33 Initial selection of transcription factors (1) Using the above experimental method, the breeding process of 'Huaiju No. 2' is as follows: Figure 1 As shown, the obtained variety identification certificate is as follows: Figure 2 As shown, using this chrysanthemum as material, one CmWRKY-type transcription factor was identified and analyzed, and a phylogenetic analysis was performed. The results are as follows. Figure 3 As shown.

[0056] The results showed that the discovered CmWRKY protein sequence, analyzed using NCBI software, possesses a relatively conserved functional domain as a WRKY transcription factor. Figure 3 ).

[0057] (2) Furthermore, the present invention obtains gene coding region sequences from the chrysanthemum transcriptome database, and performs PCR amplification and sequencing using cDNA from the leaves of 'Huaiju No. 2' tissue culture seedlings as templates. CmWRKY33 The sequencing results of the genes are shown in Table 12.

[0058] Table 12 CmWRKY33 nucleotide and amino acid sequences

[0059] The results showed that chrysanthemum CmWRKY33The gene is 1655 bp in length (see SEQ ID NO. 1), with the first 46 bp being the 5' UTR (non-coding region), the 47th to 1510th bp being the coding region (1464 bp in total), and the 1511th to 1655th bp being the 3' UTR (non-coding region), encoding a total of 172 amino acids (see SEQ ID NO. 2). A phylogenetic tree was constructed using MEGA 6.0, comparing its amino acid sequence with that of WRKY-like genes in Arabidopsis thaliana. Figure 3 As shown, the results indicate that it is similar to Arabidopsis thaliana. WRKY33 It has the highest homology, so it was named CmWRKY33 .

[0060] (3) In order to determine CmWRKY33 It is a response Alternaria sp. and SA induction, this invention studied CmWRKY33 Responding in 'Huaiju No. 2' Alternaria Changes in sp. expression after inoculation and SA treatment. Specifically, three treatment groups and corresponding control groups were set up using tissue culture seedlings of 'Huaiju No. 2'. Treatment group 1 used... Alternaria sp. infection was performed. The control group was treated with sterile water (CK), while treatment group 2 was treated with a 2 mmol / L SA solution for root soaking. The control group was treated with sterile water (CK). The results are as follows: Figure 4 As shown.

[0061] The results showed that in treatment group 1, when seedlings were treated with sterile water (CK), CmWRKY33 No significant change in gene expression levels, inoculation with pathogens Alternaria After sp., CmWRKY33 The relative expression level showed a trend of first increasing and then decreasing from 0 days to 7 days. The gene expression level peaked at 3 days after inoculation, being more than 6 times higher than before inoculation, and then decreased to the same level as the control (CK) at 7 days. After treatment with SA... CmWRKY33 Gene expression also showed a trend of first increasing and then decreasing, with the highest gene expression level at 4 h of treatment being 5.8 times that before treatment.

[0062] Therefore, it can be concluded that when subjected to... Alternaria sp. after infection CmWRKY33 In response to stress, expression levels rise rapidly, and the response to SA is also observed.

[0063] Example 2 CmWRKY33 Tissue-specific expression analysis in 'Huaiju No. 2' 0.1 g of different tissues from Huaijuhua were taken and subjected to qRT-PCR to detect... CmWRKY33 The expression levels of [something] in different tissues, as shown in the following results: Figure 5 As shown.

[0064] Depend on Figure 5 It can be seen that, CmWRKY33 It is expressed in all tissues, with the highest expression in leaves and the lowest in stem tips during the vegetative growth stage. The relative expression level from high to low is leaf > root > stem > stem tip. Therefore, it can be concluded that... CmWRKY33 The expression in chrysanthemum is tissue-specific.

[0065] Example 3 Transcription Factors CmWRKY33 Biochemical characteristic detection In order to determine CmWRKY33 Subcellular localization, CmWRKY33 The ORF was constructed into pSuper1300-GFP to create Super1300- CmWRKY33 -GFP, to carry out tobacco infection; in order to determine CmWRKY33 The transcriptional self-activation activity was detected using a yeast system, and the results were as follows: Figure 6 As shown.

[0066] Depend on Figure 6 It can be seen that pSuper1300- CmWRKY33 -GFP only shows color in the cell nucleus in tobacco, indicating that CmWRKY33 Located within the cell nucleus, consistent with the characteristics of transcription factors functioning within the nucleus. On SD / -Trp solid medium, all three yeast strains transformed with the plasmid grew normally; on SD / -Trp-His solid medium, yeast transformed with the pGBKT7 plasmid failed to grow normally, while yeast transformed with the pGBKT7-... CmWRKY33 Yeast containing the pGBKT7-VP16 plasmid could grow normally and turn blue on SD / -Trp-His solid medium containing X-α-gal. The results indicate that... CmWRKY3 Protein 3 has transcriptional activation activity.

[0067] Example 4 CmWRKY33 Overexpression of chrysanthemum Alternaria resistance testing of sp. (1) In order to study CmWRKY33 Overexpression of chrysanthemum lines Alternaria To assess resistance to *Sp.*, this invention selects overexpression lines. CmWRKY33-OX-3 , CmWRKY33-OX-5 and CmWRKY33-OX-13 As the treatment group, and with the non-overexpressing strain as the control group, they were respectively subjected to Alternaria sp. infection treatment, each treatment group and the control group in Alternaria The morphological diagram under sp. infection is as follows: Figure 7 As shown.

[0068] The results showed that in the control plants, lesions appeared and the yellowing area expanded on day 3. By day 5, the lower 2-3 leaves of the plants began to show symptoms, with more obvious lesions and a tendency to wither. In contrast, CmWRKY33 Plants that overexpressed the gene showed milder disease symptoms, among which... CmWRKY33-OX-5 Small lesions began to appear on the plant on the 3rd day, and the leaf edges turned yellow. By the 5th day, the lesions were more obvious, and the leaves turned yellow further. CmWRKY33-OX-3 No obvious lesions were found on the plants throughout the observation period; only yellowing of the leaf edges appeared on the 5th day, indicating a relatively mild disease overall. CmWRKY33-OX- 13 The plants with the mildest disease were... Alternaria After the sp. fungal infection, the leaves remained healthy and grew well for 5 days, without any lesions or yellowing of the leaves.

[0069] (2) To further explore the expression CmWRKY33 Chrysanthemum improves the body Alternaria This invention investigates the mechanism of resistance to *Sp.* and examines genes related to the *SA* resistance signaling pathway. CmNPR1 , CmPR1 and CmTGA2 The expression, the result is as follows Figure 8 As shown.

[0070] The results show that, CmWRKY33 Chrysanthemum plants CmNPR1 Gene expression levels were higher than in WT plants, and on day 0, CmWRKY33 -OX-5、 CmWRKY33 -OX-3 strain CmNPR1 Gene expression levels were significantly different from those of WT. CmWRKY33 The OX-13 strain differed significantly from the WT strain. After infection, CmNPR1 Gene expression levels initially increased, showing a trend of high at first and then low, peaking on day 3 and declining on day 5. Different time periods were observed. CmWRKY33 Overexpression plants and WT plants CmNPR1 Gene expression levels also differ, in the absence of... Alternaria Before sp. infection, i.e., on day 0, CmWRKY33 -OX-5、 CmWRKY33 -OX-3、 CmWRKY33 -In each strain of OX-13 CmNPR1Gene expression levels were 1.52, 1.95, and 2.24 times that of WT, respectively; on day 1, they were 1.51, 1.48, and 2.03 times that of WT, respectively; on day 3, they were 1.38, 1.54, and 1.89 times that of WT, respectively; and on day 5, they were 1.18, 1.49, and 1.68 times that of WT, respectively. On day 3, when expression levels were highest, WT, ... CmWRKY33 -OX-5、 CmWRKY33 -OX-3、 CmWRKY33 -OX-13 strains CmNPR1 The expression levels were 5.31, 4.8, 4.16, and 4.46 times that of day 0, respectively. CmPR1 The changes in gene expression levels still show a trend of first increasing and then decreasing, and CmWRKY33 The expression levels in overexpressing plants were all higher than those in WT plants. Specifically, at day 0, CmWRKY33 -OX-5、 CmWRKY33 -OX-3、 CmWRKY33 -In each strain of OX-13 CmPR1 Gene expression levels were 1.71, 1.87, and 2.58 times that of WT, respectively; on day 1, they were 1.4, 1.92, and 2.02 times that of WT, respectively; on day 3, they were 1.58, 1.56, and 2.06 times that of WT, respectively; and on day 5, they were 1.38, 1.69, and 2.2 times that of WT, respectively. On day 3, when expression levels were highest, WT and... CmWRKY33 -OX-5、 CmWRKY33 -OX-3、 CmWRKY33 -OX-13 strains CmPR1 The gene expression levels were 2.68, 2.49, 2.22, and 2.14 times that of day 0, respectively. CmTGA2 Changes in gene expression levels and CmNPR1 , CmPR1 They exhibit the same trend. Specifically, at day 0, CmWRKY33 -OX-5、 CmWRKY33 -OX-3、 CmWRKY33 -In each strain of OX-13 CmTGA2 The gene expression levels were 1.44, 1.57, and 1.88 times that of WT, respectively; on day 1, they were 1.58, 1.94, and 2.36 times that of WT, respectively; on day 3, they were 2.14, 2.4, and 2.88 times that of WT, respectively; and on day 5, they were 1.48, 1.27, and 1.58 times that of WT, respectively. On day 3, when expression levels were highest, WT, ... CmWRKY33 -OX-5、 CmWRKY33 -OX-3、 CmWRKY33 -OX-13 strains CmTGA2 The gene expression levels were 3.23, 4.79, 4.95, and 4.97 times that of day 0, respectively.

[0071] Example 5 CmWRKY33 Instant silence chrysanthemum Alternaria resistance testing of sp. (1) In order to study silence CmWRKY33 Chrysanthemum Alternaria The effects of sp. resistance were investigated by transiently silencing 37 *Chrysanthemum indicum* plants, and then selecting 6 of these silent plants for in vitro testing. Alternaria sp. processing. Results are as follows: Figure 9 and Figure 10 As shown.

[0072] The results showed that the leaves of the control group did not show obvious lesions and remained healthy and green overall, indicating that the control group plants had good disease resistance; the leaves of the experimental group turned yellow and showed obvious lesions, and the lesion area was large, indicating that the experimental group plants were more susceptible to disease. CmWRKY33 Gene silencing significantly reduces resistance to black spot disease. This indicates... CmWRKY33 Genes may play an important role in the disease resistance mechanism of chrysanthemum, and the loss of their function will make the plant more susceptible to infection by pathogens.

[0073] (2) In order to study CmWRKY33 To investigate whether SA (sulfate-induced chlorophyll) regulates resistance to chrysanthemum black spot disease by affecting SA biosynthesis, the SA content in transiently silenced plants was measured, and the results were as follows: Figure 11 As shown.

[0074] The results showed that the SA content in the leaves of the experimental group was significantly lower than that in the control group. This indicates that... CmWRKY33 The silence of SA leads to a decrease in SA content.

[0075] (3) In order to study CmWRKY33 The effects on SA biosynthesis and SA signaling pathways were investigated, and disease-related genes were examined. CmNPR 1 、CmTGA 2. CmPR1 ) and key enzyme genes for SA synthesis (CmCM, CmPAL, CmICS ) Expression level. Results are as follows Figure 12 As shown.

[0076] The results showed that, compared with the control group, transiently silenced plants had significantly higher mortality rates. CmPAL The expression level decreased by 3.1 times; CmICS It decreased by 3.2 times; CmNPR1 It decreased by 5.1 times; CmTGA2 It decreased by 3.7 times.

[0077] This indicates CmWRKY33 It may be possible through regulation CmPAL, CmICS, CmNPR1 and CmTGA2 The expression of genes such as [unspecified gene] is involved in the regulation of resistance to chrysanthemum black spot disease.

[0078] In summary, black spot disease is one of the primary diseases affecting chrysanthemums, severely impacting chrysanthemum production. This invention obtained a chrysanthemum CmWRKY transcription factor through transcriptome analysis, and its response to pathogen treatment and SA treatment induction was found. After cloning its sequence, it was named [name missing] after sequence analysis. CmWRKY33 Expression vectors were constructed and their localization was confirmed using tobacco. CmWRKY33 The protein is located in the cell nucleus. This was verified through homologous transformation of chrysanthemum. CmWRKY33 Regulatory role in resistance to the pathogen of black spot disease. Phenotypic observation revealed that overexpression... CmWRKY33 It significantly improved the resistance of chrysanthemums to black spot disease; conversely, transient silencing... CmWRKY33 This reduces the resistance of chrysanthemums to black spot disease. SA content determination revealed... CmWRKY33 The changes in SA levels were positively correlated, therefore disease-related genes were detected. CmNPR 1 、CmTGA 2. CmPR1 ) and SA synthesis key enzyme genes ( CmCM , CmPAL, CmICS ) Expression level. Finding CmWRKY33 Overexpression or silence can significantly affect CmPAL, CmICS, CmNPR and CmTGA2 The expression.

[0079] In short, this invention discovers chrysanthemum transcription factors. CmWRKY33 Its expression was significantly induced by the pathogen of black spot disease and SA, and it is upregulated, making it a positive regulator of chrysanthemum's resistance to black spot disease; CmWRKY33 By increasing disease-related genes CmNPR1 , CmTGA2 and SA synthesis key enzyme genes CmPAL, CmICS The expression of SA increases the content of SA in plants, thereby enhancing the plant's resistance to black spot disease.

[0080] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. CmWRKY33 The application of genes in regulating resistance to chrysanthemum black spot disease is characterized by, The CmWRKY33 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; and the regulation is positive regulation.

2. The application according to claim 1, characterized in that, The method of positive regulation comprises overexpression CmWRKY33 of the gene.

3. A primer combination, characterized in that, for overexpression and / or silencing in the use of claim 1 CmWRKY33 gene; wherein the primer nucleotide sequence for overexpression CmWRKY33 of the gene is shown in SEQ ID NO. 21 ~ SEQ ID NO. 22, and the primer nucleotide sequence for silencing CmWRKY33 of the gene is shown in SEQ ID NO. 25 ~ SEQ ID NO.

30.

4. A reagent kit, characterized in that, This includes the primer combination described in claim 3.

5. A recombinant expression vector, characterized in that, It contains the application described in claim 1. CmWRKY33 Gene.

6. A host bacterium, characterized in that, It contains the application described in claim 1. CmWRKY33 The gene or contains the recombinant expression vector as described in claim 5.

7. The application of the recombinant expression vector of claim 5 and / or the host bacteria of claim 6 in improving the resistance of chrysanthemum to black spot disease.

8. The application according to claim 7, characterized in that, The applications include the development of new varieties and / or lines of chrysanthemums resistant to black spot disease.

9. A method for improving the resistance of chrysanthemum to black spot disease, characterized in that, The method involves overexpression in target chrysanthemum material. CmWRKY33 Genes are being processed.

10. A method for cultivating new varieties and / or new lines of chrysanthemums resistant to black spot disease, characterized in that, The method involves overexpression in target chrysanthemum material. CmWRKY33 The steps to obtain transgenic plants from genes.

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