CN116064575B

CN116064575B - Chrysanthemum transcription factor CmbHLH18 and application thereof in resisting chrysanthemum black spot

Info

Publication number: CN116064575B
Application number: CN202211012726.8A
Authority: CN
Inventors: 赵喜亭; 王添乐; 刘波; 王丹丹; 王晓萌; 崔惠; 王云鹏; 耿志先
Original assignee: Xinxiang City Garden Greening Center; Xinxiang Municipal Design And Research Institute Co ltd; Henan Normal University
Current assignee: Xinxiang City Garden Greening Center; Xinxiang Municipal Design And Research Institute Co ltd; Henan Normal University
Priority date: 2022-08-23
Filing date: 2022-08-23
Publication date: 2023-08-22
Anticipated expiration: 2042-08-23
Also published as: CN116064575A

Abstract

The invention provides a chrysanthemum transcription factor CmbHLH18 and application thereof in resisting chrysanthemum black spot, belonging to the field of molecular biology. The chrysanthemum transcription factor CmbHLH18 is screened by the chrysanthemum transcriptome, and the chrysanthemum transcription factor CmbHLH18 is found to be obviously induced by black spot pathogenic bacteria and SA for the first time, and the expression is up-regulated, so that the chrysanthemum transcription factor CmbHLH18 is a forward regulation factor for resisting black spot; cmbHLH18 enhances plant resistance to melasma by increasing the expression of the defensin genes CmPAL, cmPOD and the antioxidant enzyme genes CmSOD, cmCAT, cmAPX and CmGR, reducing the level of active oxygen in the plant. Based on the function of the presently discovered transcription factor CmbHLH18, the gene can be applied to the resistance regulation of plants to black spot pathogenic bacteria.

Description

Chrysanthemum transcription factor CmbHLH18 and application thereof in resisting chrysanthemum black spot

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a chrysanthemum transcription factor CmbHLH18 and application thereof in resisting chrysanthemum black spot.

Background

Flos Chrysanthemi (Chrysanthemum morifolium Ramat.) belongs to the genus Chrysanthemum of Compositae, is a perennial herb, has a cultivation history of over three thousand years in China, and is one of the famous ornamental plants in the world. The flower type of the plant is various, the flower type is varied, the plant is widely applied to cut flowers, potted plants, landscaping and the like, and the plant has high economic value. Meanwhile, some varieties of chrysanthemum have the functions of tea and medicine, are common traditional Chinese medicine varieties recorded in Chinese pharmacopoeia, and have wide application prospects.

Because chrysanthemum adopts cuttage propagation and separated plant propagation throughout the year, the chrysanthemum is easy to be infected by various pathogenic bacteria in the growth stage, thereby causing diseases, directly increasing the production cost of chrysanthemum products and restricting the sales and export of the products. Through the early investigation of the research laboratory, it is found that the black spot is one of the primary diseases of the chrysanthemum, and the black spot becomes a limiting factor for chrysanthemum cultivation production.

The main pathogenic bacteria of chrysanthemum black spot is Alternaria (Alternaria) fungi, and the black spot pathogenic bacteria which are separated and identified on chrysanthemum are Alternaria sp. Alternaria sp. Plants are generally protected against dead nutritional pathogens by JA and Eth mediated pathways. The SA-mediated signaling pathway is more effective in protecting plants against living or semi-living nutritional pathogens, and the SA and JA-mediated signaling pathways are antagonistic. Exogenous spraying of SA suppresses expression of the JA response genes (LOX 2, PDF1.2 and VSP), making the plant more susceptible to the dead vegetative fungi. However, with the intensive research, there is increasing evidence that the SA and JA signaling pathways are not completely contradictory to pathogen defenses, but rather cross each other, forming a complex defensive network. Studies have shown that SA signaling pathways are involved in responses to dead-body trophic pathogens, e.g., novA kov A et al found that similar results were obtained in tomato with increased SA content and up-regulated expression of the SA marker gene in infected plant leaves after the rape was inoculated with dead-body trophic sclerotinia. The chrysanthemum CmNPR1 response SA signaling pathway is involved in the defense of chrysanthemum against the dead body trophic black spot pathogen Alternaria sp. Complete SA signal defense is necessary for potato attack by the dead-body vegetative fungus Alternaria solani. Because the immune system of plants is too complex, there is still a need for intensive research into the molecular mechanisms by which plants respond to various hormonal signaling pathways to defend against pathogen invasion.

At present, a large number of transcription factors related to biotic stress are studied in plants, and the families of WRKY, bZIP, NAC, AP/ERF, MYB, bHLH and the like are included. Wherein bHLH (basic Heix-Loop-Heix, bHLH) transcription factors are named for having highly conserved bHLH domains. Because bHLH transcription factors can interact with DNA and protein at the same time, the bHLH transcription factors have DNA binding and dimerization capacity, so that the bHLH transcription factors play a direct or indirect role in the aspects of transmission of various complex signal path networks, protection of plants against various stresses, plant growth regulation and the like. The plant bHLH transcription factor plays an important role in responding to biological stress processes such as infection of pathogenic bacteria, and has different action modes in different plants, and two modes of positive and negative regulation exist. The finding that 28 bHLH genes are significantly up-regulated in tobacco after infection by phytophthora, possibly related to their defense against phytophthora; the sensitivity of arabidopsis to Pst.DC3000 is obviously enhanced by the heterologous overexpression of the wheat TabHLH060 gene; overexpression of the IIId subgroup AtbHLH17 and AtbHLH13 in Arabidopsis reduces resistance to Botrytis cinerea via the JA-mediated pathway; overexpression of GhbHLH171 in cotton can activate the synthesis and signal path of JA, and improve the resistance of cotton to verticillium.

Chrysanthemum black spot caused by Alternaria sp. Is a problem to be solved in chrysanthemum production. However, the role of bHLH transcription factors in the resistance of chrysanthemum to black spot is not yet clear.

Disclosure of Invention

In order to solve the problems in the production, the invention provides a chrysanthemum transcription factor CmbHLH18 and application thereof in resisting chrysanthemum black spot. The chrysanthemum transcription factor CmbHLH18 is screened by the chrysanthemum transcriptome, and the chrysanthemum transcription factor CmbHLH18 is found to be obviously induced by black spot pathogenic bacteria and SA for the first time, and the expression is up-regulated, so that the chrysanthemum transcription factor CmbHLH18 is a forward regulation factor for resisting black spot; cmbHLH18 enhances the resistance of the chrysanthemum to melasma by increasing the expression of the defensin genes CmPAL, cmPOD and the antioxidant enzyme genes CmSOD, cmCAT, cmAPX and CmGR, and reducing the level of active oxygen in the chrysanthemum. The invention provides theoretical and technical basis for solving the problem of black spot disease in chrysanthemum production by applying CmbHLH18 by analyzing the molecular mechanism of CmbHLH18 to participate in and regulate the black spot resistance of the chrysanthemum.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the invention provides a gene for resisting black spot, which is chrysanthemum CmbHLH18, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.

Further, the whole length of the gene is 1025bp, and the gene comprises a coding region and a non-coding region, wherein the 1 st to 285 th bp is the 5'UTR of the non-coding region, the 286 th to 803 th bp is the coding region, and the 804 th to 1025 th bp is the 3' UTR of the non-coding region.

The invention also provides a vector containing the gene.

The invention also provides a protein coded by the gene, and the amino acid sequence of the protein is shown as SEQ ID No.2.

The invention also provides a product containing the carrier or the protein.

The invention also provides application of the gene, the vector, the protein or the product in resisting plant black spot.

Further, the plant is a plant of the family Compositae.

Still further, the asteraceae plant is chrysanthemum.

Still further, the chrysanthemum is Huai chrysanthemum.

Still further, the application is to utilize agrobacterium-mediated expression vector transformation to enable chrysanthemum to overexpress the CmbHLH18 gene so as to obtain the disease resistance to black spot.

Compared with the prior art, the invention has the following technical effects:

(1) The invention discovers that the chrysanthemum transcription factor CmbHLH18 is obviously induced by black spot pathogenic bacteria and SA, and the expression is up-regulated, thus being a forward regulation factor for resisting the black spot of chrysanthemum; cmbHLH18 reduces the level of active oxygen in the chrysanthemum by improving the expression of defensive enzyme genes CmPAL, cmPOD and antioxidant enzyme genes CmSOD, cmCAT, cmAPX and CmGR so as to enhance the resistance of the chrysanthemum to melasma;

(2) The invention provides theoretical and technical basis for solving the problem of black spot disease in chrysanthemum production by applying CmbHLH18 by analyzing the molecular mechanism of CmbHLH18 to participate in and regulate the black spot resistance of the chrysanthemum.

Drawings

FIG. 1 is a flowchart of the breeding of 'Huai chrysanthemum No. 2' in example 1;

FIG. 2 is a variety identification certificate of 'Huai chrysanthemum No. 2' in example 1;

FIG. 3 is a graph of the analysis of the evolutionary tree of CmbHLH18 of example 1;

FIG. 4 is a graph showing the analysis of the expression levels of 5 CmbHLH genes in example 1 under Alternaria sp. Inoculation, SA and MeJA treatment;

FIG. 5 is a graph showing the analysis of CmbHLH18 expression in different tissues of Chrysanthemum morifolium in example 2;

FIG. 6 is a subcellular localization map of the CmbHLH18 protein of example 3;

FIG. 7 shows the measurement of the gene expression level in the CmbHLH18 overexpressing Huai chrysanthemum strain in example 4;

FIG. 8 is a morphology of the CmbHLH18 overexpressing Huai chrysanthemum strain in example 5 under Alternaria sp.

FIG. 9 is a view of leaf dead cells under Alternaria sp. Infestation of the CmbHLH18 overexpressing Huai strain of example 5, wherein the scale in the figure represents 1cm; dpi represents days post inoculation;

FIG. 10 shows the detection of O in leaf blades of CmbHLH18 overexpressing pocket chrysanthemum after inoculation with Alternaria sp. NBT staining in example 5 ₂ · ^- Wherein the scale in the figure represents 1cm; hpi represents the hours after inoculation;

FIG. 11 shows H in leaf blades of CmbHLH18 overexpressing pocket chrysanthemum after DAB staining assay inoculation with Alternaria sp. In example 5 ₂ O ₂ Wherein the scale in the figure represents 1cm; hpi represents the hours after inoculation;

FIG. 12 is a graph showing the effect of Alternaria sp. Treatment on the expression level of CmbHLH18 over-expressed chrysanthemum strain CmSOD, cmPOD, cmCAT, cmAPX, cmPAL and CmgR gene in example 5.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The reagents, kits and instruments used in the following examples are commercially available, and the methods used in the examples are consistent with the methods conventionally used unless otherwise specified.

1. Test materials

Plant material: the chrysanthemum plant material used in the test is Huai chrysanthemum variety 'Huai chrysanthemum No. 2', and the tissue culture seedling is propagated in the laboratory. The test material for protein localization and the like is wild tobacco Benshi tobacco (Nicotiana benthamiana).

Test strain and vector: huai Huangju Blackspot pathogen strain Alternaria sp.) A2 strain (see Zhao Xiting et al, paper "isolation and identification of feverfew Blackspot pathogen"), E.coli DH 5. Alpha., agrobacterium GV3101, cloning vector pMD18-T, over-expression vector Super1300-GFP (C).

Instrument: UV-2600 ultraviolet spectrophotometer, ultra-micro ultraviolet spectrophotometer, pH meter, ultraviolet gel imager, YJ-1450A ultra-clean bench, gradient PCR instrument, high-speed refrigerated centrifuge, lightCycler 96 real-time fluorescence quantitative PCR instrument, biosafety cabinet, autoclave, analytical balance, stereoscopic microscope, overhead intelligent electric microscope, electrophoresis instrument, constant temperature shaking table, metal bath, electrothermal constant temperature incubator, constant temperature water bath pot, etc.

Reagent: 2 XEs Taq Master mix (Dye) and 2 Xpfu Master mix (Dye) reagents were purchased from Kangda; total RNA extraction reagent (RNAiso Plus), T4 ligase was purchased from TaKaRa; xba I, kpn I, speI, asc I, pac I, etc. are available from NEB (Beijing); evans blue dye liquor (Evans blue), agarose, azablue tetrazolium (NBT), diaminobenzidine (DAB), agar, laminarin, and aniline blue were purchased from Beijing Soy Bao; LB medium was purchased from Shanghai Ind; biochemical reagents such AS ampicillin (Amp), β -galactoside (X-gal), isopropylthiogalactoside (IPTG), rifampicin (Rif), kanamycin (Kan), chloramphenicol (Chl), cephalosporin (Cef), hygromycin (HmB), salicylic Acid (SA), acetosyringone (AS), 2- (N-morpholin) ethanesulfonic acid (MES), methyl jasmonate (MeJA), and guaiacol are all analytically pure or higher. Primers and sequencing used in the experiments were done by the manufacturer (Shanghai).

The main kits used in the test are mainly: plasmid extraction kit, microcolumn concentrated DNA gel recovery kit, SE seamless cloning and assembly kit and dnagarker purchased from beijing bang company; SYBR fluorescence quantification kit and reverse transcription kit were purchased from Norwezan (Nanjing) Inc. The preparation of common culture medium is 121 deg.C, 20min high temperature high pressure sterilization, 115 deg.C, 15min high temperature high pressure sterilization except yeast culture medium.

The media used for the experiments are shown below:

rooting solid culture medium: 1/2MS+0.01mg/LNAA+7.3g/L agar powder, pH 5.93;

LB liquid medium: 25g/L LB powder, pH 7.0;

LB solid medium: 25g/L LB powder+15 g/L agar powder, pH 7.0;

PDA medium: 200g/L potato, 20g/L glucose and 10g/L agar powder.

2. Test method

1. Total RNA extraction and cDNA Synthesis

RNA extraction was performed according to RNAiso Plus (TaKaRa, beijing) instructions, 1% agarose gel electrophoresis and UV-gel detection for RNA degradation and integrity, and ultra-micro UV spectrophotometry for RNA concentration for subsequent reverse transcription. The total RNA extracted was reverse transcribed into cDNA according to the reverse transcription kit instructions, and the reaction system and conditions are shown in Table 1.

TABLE 1 reaction System and conditions for cDNA Synthesis

2. Real-time fluorescent quantitative PCR

The expression level of CmbHLHs under different stresses is detected by using a fluorescent quantitative PCR kit, a chrysanthemum Ubiquitin gene (CmUBI, EU86XX 25) is selected as an internal reference, 3 repeats are arranged for each sample, specific primers are shown in Table 2, and a qRT-PCR reaction system is shown in Table 3.

TABLE 2 CmbHLHs Gene qRT-PCR primers

TABLE 3 reaction System for qRT-PCR

3. CmbHLH18 Gene cloning

According to the determined transcript sequence of CmbHLH18, the specific primers of CmbHLH18 gene clone are designed in the 5'UTR and 3' UTR of the ORF region by using software Primer 6.0, PCR amplification is carried out by using chrysanthemum cDNA as a template, the specific primers are shown in Table 4, and the reaction system is shown in Table 5.

TABLE 4 CmbHLH18 Gene PCR primers

TABLE 5 PCR reaction System

Note that: x represents the cDNA content in X. Mu.L < 0.5. Mu.g

4. CmbHLH18 over-expression vector construction

According to the cDNA sequence of the CmbHLH18 which is obtained, a proper enzyme cutting site is selected in combination with the Super1300-35S-GFP vector, corresponding protective bases are added, and primers CmbHLH18-Xba I-F and CmbHLH18-Kpn I-R are designed at two ends of an ORF region by using Primer 6.0 software. PCR amplification was performed using cDNA of Huai chrysanthemum as a template. The specific sequences of the primers CmbHLH18-Xba I-F and CmbHLH18-Kpn I-R are shown in Table 6. The PCR products were recovered and purified using a kit, and after detecting the concentrations of the purified products and Super1300-35S-GFP plasmid, double cleavage was performed with XbaI and KpnI, respectively, and then the cleaved products were ligated with T4 ligase, and the cleavage and ligation systems and conditions were as shown in tables 7 and 8.

TABLE 6 primers CmbHLH18-Xba I-F and CmbHLH18-Kpn I-R

TABLE 7 double cleavage reaction System and conditions

Table 8 connection system and conditions

5. CmbHLH18 subcellular localization

Taking out stored Agrobacterium Super1300-35S-GFP empty load and S from-80 ℃ refrigeratorThe uper1300-CmbHLH18-GFP vector was streaked on LB solid medium (Rif+Kan) and incubated at 28℃for 2d. Single colonies were picked up in 5mL LB liquid medium (5. Mu.L of Rif+5. Mu.L of Kan), shake-cultured at 28℃and 180r/min until turbid, inoculated into 20mL of LB liquid (20. Mu.LRif+20. Mu.L of Kan), and expanded by adding 8. Mu.LAS (0.1 mol/L) and 200. Mu.LMES (1 mol/L) to OD ₆₀₀ =0.6-0.8. Placing the bacterial liquid in a 50mL centrifuge tube at 4 ℃ for 15min at 4000r/min, discarding the supernatant, and using 10mmol/LMgCl ₂ Resuspension of the cells and OD adjustment ₆₀₀ =1.0, 16 μlas (0.1 mol/L) was added and mixed well. The infection solution was injected from the back of tobacco using a needleless sterile syringe, 3 leaves were injected per plant of tobacco, empty tobacco was injected as a control, then the plant was placed in darkness (25 ℃ C., 100% relative humidity) for 1d, and after 2d incubation under normal light, the material was obtained. The infected tobacco leaves were immersed in DAPI for staining for 10min, and subcellular localization of CmbHLH18 protein in tobacco cells was observed using a positive intelligent fluorescence microscope.

6. Inoculation method of chrysanthemum tissue culture seedling Alternaria sp

Activating Alternaria sp when seedlings of 'Huai chrysanthemum No. 2' grow to 8-10 leaves, picking spores with a crochet hook under aseptic operation, inoculating to PDA culture medium, inverting in a dark incubator at 24deg.C, adding sterile distilled water (containing Tween 0.01%), scraping spores with a sterile gun head, preparing spore suspension, counting with a blood cell counter, and continuously adjusting spore concentration to 1×10 under an optical microscope ⁷ And each mL. Inoculating 3-5 pieces of Chrysanthemum leaf from top to bottom by needle punching, covering with fresh-keeping film and plastic shell, culturing in dark environment for 48 hr (100% relative humidity and 24 deg.C), and transferring to normal culture room. 0.1g of leaf was taken as sample at 0, 1, 3, 5d of inoculation, and after quick freezing with liquid nitrogen, stored at-80℃for 3 replicates per treatment.

7. Evanskia staining

Soaking plant leaves inoculated with pathogenic bacteria in 0.5% Evanskia dye solution for 24 hr, removing chlorophyll in 95% alcohol, replacing 95% alcohol for several times, and storing completely decolorized leaves in 50% glycerol. And (5) placing the treated leaves of each strain under a stereoscopic microscope for observation, and carrying out microscopic photographing on the distribution condition of dead cells.

8. Blue Tetrazolium (NBT) staining

0.1g of NBT powder was weighed, and 100mL of 10mmol/L phosphate buffer (pH=7.8) was added thereto, followed by shaking to prepare 1g/L NBT solution. The leaves of the plants 12h after inoculation were placed in a petri dish and the prepared NBT solution was poured into the petri dish. Irradiating under light for 2h until spots appear on the leaves, clamping the leaves into a 50mL centrifuge tube containing 75% alcohol, placing into a water bath kettle at 95 ℃ to remove chlorophyll, replacing 75% alcohol for multiple times during the period, and finally storing the leaves completely decolorized in 50% glycerol. The treated leaves were observed under a stereoscopic micromirror and photographed.

9. Diaminobenzidine (DAB) staining

0.1g of DAB powder was weighed, and 100mL of 10mmol/L phosphate buffer (pH=7.8) was added thereto, and the mixture was shaken well to prepare 1g/L DAB solution. Leaves 12h after inoculation were placed in a petri dish and the DAB solution that had been prepared was poured into the petri dish. Irradiating under light for 8h until spots appear on the leaves, clamping the leaves into a 50mL centrifuge tube containing 75% alcohol, placing into a water bath at 95 ℃ to remove chlorophyll, replacing 75% alcohol for multiple times during the period, finally storing the leaves completely decolorized in 50% glycerol, and placing under a stereoscopic microscope for observation and photographing.

10. Gene expression level detection

After inoculation, samples were taken on days 0, 1, 3, 5, 7, respectively, and after liquid nitrogen flash freezing were stored at-80 ℃ for subsequent determination of defensive gene expression, each sample was replicated three times.

The technical scheme of the invention is further elaborated in the following in conjunction with examples.

Example 1 preliminary selection of bHLH-like transcription factors in response to Induction by Blackspot pathogens

(1) By using the test method, the culture flow chart of 'Huai chrysanthemum No. 2' is shown in fig. 1, the obtained variety identification certificate is shown in fig. 2, 71 CmbHLH transcription factors are analyzed and identified by taking chrysanthemum as a material, and the result is shown in fig. 3.

The results show that: a total of 71 CmbHLH protein sequences were found, one of which was shown by NCBI software analysis to have a functional domain that is relatively conserved as a bHLH transcription factor and belongs to the bHLH transcription factor subfamily IV (FIG. 3).

(2) Furthermore, the invention obtains the gene coding region sequence in the chrysanthemum transcriptome database, uses the 'Huai chrysanthemum No. 2' tissue culture seedling leaf cDNA as a template to carry out PCR amplification and sequencing, and the sequencing result of the CmbHLH18 gene is shown in Table 9.

Table 9 CmbHLH18 nucleotide and amino acid sequences

The result shows that the total length of the chrysanthemum CmbHLH18 gene is 1025bp, see SEQ ID NO.1, wherein 1-285 bp is the 5'UTR of the non-coding region, 286-803 bp is the coding region, 516bp is the total length, 804-1025 bp is the 3' UTR of the non-coding region, and the total code is 172 amino acids, see SEQ ID NO.2. The amino acid sequence of MEGA6.0 was used to build a evolutionary tree with the amino acid sequence of the bHLH-like gene in Arabidopsis thaliana, as shown in FIG. 3, which shows the highest homology with bHLH18 in Arabidopsis thaliana, so it was designated as CmbHLH18.

(3) In order to determine that CmbHLH18 is the most potent response to Alternaria sp and responds to SA, a member of CmbHLH that is insensitive to MeJA, the present invention investigated the changes in CmbHLH18 expression in' wyoming No.2 in response to Alternaria sp. The method comprises the following steps: 3 treatment groups and corresponding control groups are respectively arranged on tissue culture seedlings of 'Huai chrysanthemum No. 2', the treatment group 1 is infected by Alternaria sp, the control group is treated by sterile water (CK), the treatment group 2 is treated by 2mmol/L SA solution for root dipping, the control group is treated by sterile water (CK), the treatment group 3 is treated by 2mmol/LMeJA for root dipping, and the control group is treated by sterile water (CK), and the result is shown in figure 4.

The result shows that: in treatment group 1, there was no significant change in CmbHLH gene expression levels when seedlings were treated with sterile water (CK), and after inoculation with the pathogen Alternaria sp, each CmbHLH gene exhibited a tendency to rise and then fall, with CmbHLH16, cmbHLH28, cmbHLH30, and CmbHLH60 reaching the highest expression levels at day 3, 6.72 fold, 5.61 fold, 6.58, and 3.93 fold, respectively, of the control. Whereas the CmbHLH18 gene peaked earlier (1 d) than the control by a factor of 9.12. In treatment group 2, the expression levels of the 5 genes all showed a trend of up-regulated expression after SA treatment, wherein CmbHLH18, cmbHLH16, and CmbHLH28 reached a maximum at 8h post-treatment, approximately 19.23-fold, 48.66-fold, and 27.36-fold, respectively, of the control; cmbHLH30, cmbHLH60 reached a maximum 2h after treatment, approximately 49.88 and 6.76 times that of the control, respectively. In treatment group 3, after MeJA treatment, the expression of the four genes was significantly up-regulated except for CmbHLH18, with the maximal up-regulation having CmbHLH28 and CmbHLH30, each reaching a maximum at 2h post-treatment, approximately 18.75 and 31.55 times that of the control, respectively.

Thus, it is available that CmbHLH18 responds most strongly to Alternaria sp.

EXAMPLE 2 tissue expression specific analysis of CmbHLH18 in' Huai chrysanthemum No.2

0.1g of different tissues of Huai chrysanthemum are respectively taken, qRT-PCR is carried out, and the expression level of CmbHLH18 in the different tissues is detected, and the result is shown in figure 5.

As can be seen from FIG. 5, cmbHLH18 is expressed in various tissues, where the expression level in roots is highest, the expression level in Yu She, stems, flowers is significantly higher, and the expression level is about 158 times that in the lowest tissue organ (flowers). Thus, it is available that the expression of CmbHLH18 in chrysanthemum is tissue specific.

Example 3 Biochemical Property detection of transcription factor CmbHLH18

To determine subcellular localization of CmbHLH18, the ORF of CmbHLH18 was constructed into pSuper1300-GFP to make Super1300-CmbHLH18-GFP, while tobacco infestation was performed separately using pSuper1300-GFP empty as a control group, and the results are shown in fig. 6.

As can be seen from FIG. 6, the nuclear membrane of the control pSuper1300-GFP in tobacco is shown to be bright, while the pSuper1300-CmbHLH18-GFP only develops color in the tobacco, indicating that CmbHLH18 is located in the nucleus and accords with the functional characteristics of the transcription factors in the nucleus.

EXAMPLE 4 screening of CmbHLH18 overexpressing Chrysanthemum strains

(1) The constructed over-expression vector Super1300-CmbHLH18-GFP is transferred into 'Huai-ju No. 2' by using an agrobacterium-mediated leaf disc transformation method, and HmB resistance screening is carried out, so that a regenerated plant is finally obtained. The vector construction primers CmbHLH18-Xba I-F and CmbHLH18-Kpn I-R are used for carrying out common PCR amplification detection on the genomic DNA of the leaves of the 7 transgenic plants of 'Huai chrysanthemum No. 2', and simultaneously carrying out detection on the expression quantity of CmbHLH18 genes in the leaves of the 7 transgenic plants, and the result is shown in figure 7.

The results show that: the 7 resistant plants all have a target band of 750bp, and the expression of CmbHLH18 in different transgenic lines is higher than that of a control line.

Example 5 detection of resistance of CmbHLH18 overexpressed Chrysanthemum to Alternaria sp.

(1) In order to study the resistance of the CmbHLH18 over-expressed chrysanthemum strain to Alternaria sp, the invention selects the over-expressed strain CmbHLH18-81, cmbHLH18-65 and CmbHLH18-61 as treatment groups, and simultaneously uses the non-over-expressed strain as control groups to carry out Alternaria sp infection treatment, the morphological diagram of each treatment group and the control group under Alternaria sp infection is shown in figure 8, the disease index grading standard is shown in table 10, and the disease index results of each treatment group and the control group are shown in table 11.

TABLE 10 grading Standard of disease after inoculation of Spot-containing pathogen

TABLE 11 disease index of CmbHLH18 overexpressing Huai chrysanthemum lines after Alternaria sp

The result shows that: the vigor of CK and the over-expressed strain did not differ significantly before pathogen infestation did not occur. On day 5 after inoculation with Alternaria sp. The leaves of CK appeared to be more severe in the symptoms than the transgenic lines, with yellow wilted leaves significantly more than the overexpressing lines, where the overexpressing plant leaves were only slightly chlorosis and sporadic lesions. And the disease index of the leaf is counted, and the disease index of the leaf of the over-expression plant is obviously lower than that of a control group.

(2) After the dead body nutritional pathogenic bacteria Alternaria sp are inoculated, host leaf cells can be killed along with the prolongation of the infection time of the pathogenic bacteria, and leaf tissues can not effectively resist the invasion of the pathogenic bacteria, so that the leaf cells can die. To examine the dead and alive status of the chrysanthemum leaf, the present invention further performed Evanskian staining of leaves of the above CK and over-expressed strain, and the results are shown in FIG. 9.

As a result, it was found that before Alternaria sp. Inoculation, dead cells were sporadically distributed in each strain, but there was no obvious difference, and it was hypothesized that the cells could be the result of normal growth metabolism; on day 4 after inoculation with Alternaria sp, the inoculated leaves of CK were almost all blue necrotic cells, whereas the dead cells in the leaves of the overexpressing plants were all significantly smaller in area than CK. The over-expression of CmbHLH18 is shown to maintain more cell activity when the leaf is infected by the black spot pathogenic bacteria, and enhances the resistance of chrysanthemum to Alternaria sp.

(3) To further analyze how CmbHLH18 increased resistance to chrysanthemum following Alternaria sp. The invention detects superoxide anion radical (O) in the leaves by NBT dyeing and DAB dyeing respectively ₂ · ^- ) And hydrogen peroxide (H) ₂ O ₂ ) To observe the level of active oxygen in the leaves of different strains and to judge the ability of the plants to scavenge active oxygen, the results are shown in FIG. 10.

The results showed that after NBT staining, the degree of staining was shallow in each strain and there was no significant difference before the infection with Alternaria sp. Indicating that each strain O before the infection with Alternaria sp. Was ₂ · ^- Is similar at the original level, and leaves of the CmbHLH18 overexpressing plants were stained to a significantly higher degree than CK at 12 hours after treatment. This indicates that a certain amount of O has accumulated in leaves of Alternaria sp.post-treatment over-expressed CmbHLH18 plants ₂ · ^- It is hypothesized that a certain amount of active oxygen may be required to inhibit or kill the invading pathogenic bacteria in order to combat the pathogenic bacteria infestation.

(4) To further verify the above results, the present invention also performed DAB staining treatment, and the results are shown in fig. 11.

The result shows that: the DAB staining results are consistent with the NBT staining results, the staining degree in each strain is light and has no obvious difference before the infection by Alternaria sp, and the dark brown spot area on the leaves of the over-expressed CmbHLH18 plant is obviously more than CK after 12h of the Alternaria sp. This demonstrates that Alternaria sp.post-treatment over-expresses H in leaves of the CmbHLH18 transgenic line ₂ O ₂ The content is obviously higher than CK. This further illustrates that the CmbHLH 18-overexpressed transgenic lines accumulate a certain amount of active oxygen for combating pathogens after infection by them.

(5) To further explore the mechanism of action of over-expressing CmbHLH18 chrysanthemum to increase resistance to Alternaria sp the present invention examined the expression of antioxidant enzyme related genes CmSOD, cmCAT, cmAPX and CmGR, and the expression of defensin enzyme related genes CmPOD and CmPAL, the results are shown in figure 12.

The results showed that after inoculation with the pathogenic fungus Alternaria sp, the expression level of CmSOD of the leaves of each strain was drastically increased at day 5 after the decrease at days 1 and 3, and slightly decreased at day 7. In the whole process, the expression level of CmSOD of the over-expression strain is higher than CK. The CmCAT expression level showed a tendency to rise and then fall throughout the period, reaching a maximum at day 5, and the CmCAT expression level of the over-expressed strain began to be significantly higher than CK after day 3 of inoculation. The expression level of CmAPX showed a tendency to increase and decrease as a whole, and also started to be significantly higher than CK after day 3 of inoculation. The CmGR expression levels all showed a trend of increasing and then decreasing on the whole, and the overexpressed plants began to be significantly higher than CK after day 1 of inoculation. The expression level of CmPOD as a whole showed a tendency to rise and then fall, reaching a maximum at day 5, and the over-expressed plants began to be significantly higher than CK after day 3 of inoculation. The expression level of CmPAL in the over-expressed plants showed a tendency to rise and then fall throughout the period, and gradually decreased after the rapid rise reached the maximum on day 1, whereas the expression level of CmPAL in CK showed a peak only on day 5. The results show that over-expression of CmbHLH18 increases the expression of the relevant enzyme gene, either before or after infection by the pathogenic bacteria. After inoculation with Alternaria sp, cmPAL responds earliest and is significantly upregulated, and other genes also successively peak expression after the appearance of lesions (day 5 of inoculation). The effect of over-expressing CmbHLH18 chrysanthemum on improving the resistance to Alternaria sp. Is achieved by regulating the genes related to antioxidant enzymes and genes related to defensin enzymes, thereby affecting the level of active oxygen in chrysanthemum leaves.

It can be seen from the above that: black spot is one of the primary diseases of chrysanthemum, and seriously affects the production of chrysanthemum. The invention obtains 71 chrysanthemum CmbHLH transcription factors through transcriptional analysis, and detects that five members can respond to pathogen treatment and induction of SA and MeJA treatment, wherein CmbHLH18 is the most obvious. After cloning to obtain the CmbHLH18 sequence, an expression vector was constructed and localization of the CmbHLH18 protein to the nucleus was confirmed by tobacco localization. And the regulation and control effect of CmbHLH18 on the resistance of the black spot pathogenic bacteria is verified through the homologous transformation of chrysanthemum. Through phenotypic observation, it was found that over-expression of CmbHLH18 significantly increased the resistance of the plants to melasma. Over-expression of CmbHLH18 was found to reduce the number of dead cells in plant leaves by leaf dead cell staining, and by leaf NBT, DAB staining, there was some accumulation of reactive oxygen species in the leaves of CmbHLH 18-overexpressed chrysanthemum, but these reactive oxygen species did not destroy the activity of the plant's own cells, presumably these appropriate amounts of reactive oxygen species were present to inhibit or kill pathogenic bacteria. The detection of the related enzyme gene expression proves that the over-expression of the CmbHLH18 significantly improves the expression of the defense enzyme genes CmPAL and CmPOD, in particular the expression of the CmPAL gene. Meanwhile, the over-expression of CmbHLH18 also improves the expression of antioxidant enzyme genes CmSOD, cmCAT, cmAPX and CmGR, and improves the activity of related enzymes in the middle and later period of pathogenic bacteria infection, so as to remove excessive active oxygen and enable the active oxygen to be at a proper level.

In short, the invention discovers that the chrysanthemum transcription factor CmbHLH18 is obviously induced by black spot pathogenic bacteria and SA, and the expression is up-regulated, thus being a forward regulation factor for resisting the black spot of chrysanthemum; cmbHLH18 enhances plant resistance to melasma by increasing the expression of the defensin genes CmPAL, cmPOD and the antioxidant enzyme genes CmSOD, cmCAT, cmAPX and CmGR, reducing the level of active oxygen in the plant.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. A gene for resisting black spot is characterized in that the gene is chrysanthemumCmbHLH18The nucleotide sequence is shown as SEQ ID No. 1.

2. The gene of claim 1, wherein the gene is 1025 to bp in its entire length and comprises a coding region and a non-coding region, wherein 1 to 285 to bp are the non-coding region 5'utr, 286 to 803 to bp are the coding region, and 804 to 1025 to bp are the non-coding region 3' utr.

3. A vector comprising the gene of claim 1.

4. A protein encoded by the gene of claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID No.2.

5. A product comprising the vector of claim 3 or the protein of claim 4.

6. The use of a gene, vector, protein or product according to any one of claims 1 to 5 for combating chrysanthemum black spot.

7. The use according to claim 6, wherein the chrysanthemum is feverfew.

8. The use according to any one of claims 6 to 7, wherein the use is for over-expressing a chrysanthemum in a plant by transformation with an agrobacterium-mediated expression vectorCmbHLH18The gene further enables the plant to obtain disease resistance to black spot.