A coding gene of chrysanthemum transcription factor, the transcription factor and its application

Abstract

The invention discloses a coding gene of a chrysanthemum transcription factor, the transcription factor and an application thereof. The coding gene comprises a gene sequence as shown in Seq_1, and the sequence of the chrysanthemum transcription factor is as shown in Seq_2. The coding gene or the transcription factor is applied to the cultivation of heat-resistant chrysanthemum varieties, and the influence of temperature on the fading of petals of the heat-resistant chrysanthemum varieties is improved by regulating the expression level of the coding gene. The invention starts from the research on the regulation of chrysanthemum flower color phenotype by HSFs gene, studies the mechanism of heat shock transcription factor CmHSFA2b for regulating chrysanthemum flower color, and obtains a cultivation method for chrysanthemum flower color cultivated in a high temperature environment.

Description

Technical Field

[0001] The invention relates to a coding gene of a chrysanthemum transcription factor, the transcription factor and application thereof, and belongs to the technical field of molecular biology. Background Art

[0002] Chrysanthemum (Chrysanthemum×morifolium Ramat.) is one of the four major cut flowers in the world, and is also a traditional famous flower that is both medicinal and edible. It is widely used in the ornamental flower, tea, food, Chinese medicinal materials and other industries. South China has the advantage of a warm climate in winter and spring, and is one of the main production areas of flowers in winter and spring in my country. At present, most of the mainstream commercial chrysanthemum varieties on the market still come from the high-latitude cold regions of Europe, America and Japan. These varieties cannot adapt well to the high temperature climate environment in South China and Southwest China. Most varieties generally have the phenomenon of fading due to high temperatures. In addition, the global greenhouse effect and urban heat island effect have caused the petals of most red and purple chrysanthemum varieties in high-temperature areas to fade, which seriously affects the market value and ornamental value of chrysanthemums. Chrysanthemums are classified by color into pink, purple, orange, yellow, white, and complex colors. The petal pigments of red and purple chrysanthemum varieties are mainly anthocyanins (Anthocyanin, also known as anthocyanin glycosides). Compared with the very detailed research on the synthesis pathway of anthocyanins and its regulation, the current research on the regulation of anthocyanin degradation pathways is still very rare, and the regulatory mechanism of plant anthocyanin metabolism (synthesis or degradation) pathways under high temperature stress is still unclear. Existing studies have shown that high temperature stress often inhibits the accumulation of plant anthocyanins and forms a faded phenotype, but the specific molecular mechanism is still unclear, so it is impossible to improve the effect of temperature on chrysanthemum color, limiting the market value of flowers. Summary of the invention

[0003] In order to overcome the deficiencies of the prior art, the first object of the present invention is to provide a gene encoding a chrysanthemum transcription factor.

[0004] The second object of the present invention is to provide a chrysanthemum transcription factor, which has a direct influence on regulating the flower color of chrysanthemum.

[0005] The third object of the present invention is to provide an application of a coding gene and a chrysanthemum transcription factor to a method for cultivating chrysanthemum flower color in a high temperature environment.

[0006] The fourth object of the present invention is to provide a breeding agent for heat-resistant chrysanthemum varieties.

[0007] The first object of the present invention can be achieved by adopting the following technical scheme: a gene encoding a chrysanthemum transcription factor (CmHSFA2b), the encoding gene includes a gene sequence shown in Seq_1.

[0008] The second object of the present invention can be achieved by adopting the following technical scheme: a chrysanthemum transcription factor, the encoding gene of the chrysanthemum transcription factor is as described above; the sequence of the chrysanthemum transcription factor is shown in Seq_2.

[0009] The third object of the present invention can be achieved by adopting the following technical scheme: an application of a coding gene or a chrysanthemum transcription factor, and using the coding gene or the chrysanthemum transcription factor for the cultivation of heat-resistant chrysanthemum varieties.

[0010] Furthermore, by regulating the expression level of the coding gene, the effect of temperature on the petal fading of heat-resistant chrysanthemum varieties can be improved.

[0011] Further, the temperature is ≥25°C at night and ≥34°C during the day.

[0012] Furthermore, the breeding of heat-resistant chrysanthemum varieties includes the step of infecting the chrysanthemum leaf disk or the head inflorescence at the bud stage with a vector carrying the coding gene through a mediated method.

[0013] Furthermore, the vector is pB 2 GW 7 carrier.

[0014] Furthermore, the regulation is to increase the expression level, and the tool for regulating the encoding gene is an overexpression tool.

[0015] Furthermore, the tool for regulating the coding gene is a gene silencing, gene editing or gene knockout tool.

[0016] The fourth object of the present invention can be achieved by adopting the following technical scheme: a cultivation reagent for heat-resistant chrysanthemum varieties, the reagent regulating the expression level of the encoding gene.

[0017] Furthermore, the regulated expression level is overexpression; the cultivation reagent is a proliferation medium; and the proliferation medium includes MS, 3 mg / L 6-BA, 0.5 mg / L NAA and 0.2 mg / L PVP.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention provides a gene encoding CmHSFA2b protein, an amino acid sequence of chrysanthemum heat shock transcription factor CmHSF A2b protein, and applications, such as cultivation methods and reagents, recombinant vectors and cells carrying a gene sequence encoding CmHSFA 2b protein, and cultivation reagents;

[0020] 2. The present invention starts with the study of HSFs gene regulation of chrysanthemum flower color phenotype, studies the mechanism of heat shock transcription factor CmHSFA2b in regulating chrysanthemum flower color, and obtains a method for cultivating chrysanthemum flower color in a high temperature environment;

[0021] 3. The research results of the present invention can be used for the molecular breeding of heat-resistant chrysanthemums, laying a theoretical foundation for molecular breeding for cultivating domestic chrysanthemum varieties with stable flower color and non-fading in high temperature areas, which is of great significance to promoting the high-quality development of my country's floriculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The relative expression level of the CmHSFA2b gene in the capitula of chrysanthemum during the S1-S4 period;

[0023] Figure 2 Phenotype of 'Zihongtuogui' chrysanthemum, which is easy to fade under high temperature, after spraying with glufosinate ammonium;

[0024] Figure 3 Cloning the amplified band of CmHSFA2b gene of Chrysanthemum 'Purple Windmill';

[0025] Figure 4 Map of the entry cloning vector Gateway pDONR 221;

[0026] Figure 5 Overexpression vector pB 2 GW 7 Spectrum of

[0027] Figure 6 DNA identification of the overexpression plant of chrysanthemum 'Zihongtuogui' that is prone to fading under high temperature;

[0028] Figure 7 Analysis of gene expression levels in leaves of 'Zihongtuogui' CmHSFA2b overexpressing plants;

[0029] Figure 8 Flower color phenotype analysis of CmHSFA2b overexpressing plants in 'Zihongtuogui';

[0030] Fig. 9 Map of the silencing expression vector pNC-TRV2;

[0031] Fig.10 Analysis of inflorescence phenotype of the heat-resistant chrysanthemum 'Purple Windmill' after silencing of the CmHSFA2b gene;

[0032] Fig.11 The expression level of CmHSFA2b gene in gene-silenced plants of 'Purple Windmill'. DETAILED DESCRIPTION

[0033] Below, the present invention is further described in conjunction with the accompanying drawings and specific embodiments:

[0034] Example 1

[0035] 1. High temperature treatment and collection of chrysanthemum experimental materials

[0036] Chrysanthemum experimental materials selected chrysanthemum varieties 'Zihongtuogui' that are easy to fade under high temperature and 'Zifengche' that does not fade under high temperature. All chrysanthemum test materials were collected from the chrysanthemum base in Zhuliaoliao Village, Baiyun District, Guangzhou (113.23°E, 23.16°N). Potted chrysanthemums with head inflorescences in four developmental stages (S1 is the bud color development stage, S2 is the initial flowering stage, S3 is the early flowering stage, and S4 is the middle flowering stage) were selected. According to the day and night temperature characteristics of winter and spring in South China, a normal temperature control group (24℃, 10h, 20000Lx during the day; 15℃, 14h, 0Lx at night) and a high temperature experimental group (35℃, 10h, 20000Lx during the day; 26℃, 14h, 0Lx at night) were set up. After 7 days of treatment, the head inflorescences of each developmental stage were collected and quickly frozen with liquid nitrogen, and then stored in a -80℃ ultra-low temperature refrigerator.

[0037] 2. Extraction of total RNA from chrysanthemum and expression analysis of CmHSFA2b gene

[0038] The ligulate petals of the capitula at four developmental stages were taken for RNA extraction and purification using the FastPure Universal Plant Total RNA Isolation Kit (Nanjing Novogene Biotech Co., Ltd., Nanjing), and the specific steps were carried out according to the instructions. 0.1 μg of total RNA was taken and reverse transcribed into cDNA using the HifairⅢ1st Strand cDNA Synthesis SuperMix for qPCR Kit (Yisheng Biotech Co., Ltd., Shanghai), and the cDNA product obtained by reverse transcription was diluted 3 times and used for qRT-PCR.

[0039] Download the feverfew HSFA2b as the reference sequence from the feverfew reference genome database (http: / / mum-garden.kazusa.or.jp / ), and design primers online through the NCBI-BLAST website (http: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ):

[0040] Upstream primer qCmHSFA2b-F: 5′-AAAGAGCACCTTCGCAGTCA-3′ (Seq_3);

[0041] Downstream primer qCmHSFA2b-R: 5′-TGGGCTCGTGTGTTTTGTTG-3′ (Seq_4);

[0042] The internal reference gene is CmEF1ɑ gene (Genbank accession number: KF305681.1):

[0043] Upstream primer qCmEF1ɑ-F: 5′-TTTTGGTATCTGGTCCTGGAG-3′ (Seq_5);

[0044] Downstream primer qCmEF1ɑ-R: 5′-CCATTCAAGCGACAGACTCA-3′ (Seq_6).

[0045] Primer verification: The standard curve method was used to detect the amplification efficiency and correlation coefficient of the primers. The experiment used the CF XConnect Real-Time Fluorescence Quantitative PCR System (BIO-RAD, USA), and the reaction system was 50 μL, which contained 25 μL Hieff qPCR SYBR Green Master Mix (Yisheng Biotechnology Co., Ltd., Shanghai), 1 μL upstream / downstream primers (10 μM), 1 μL cDNA template and appropriate amount of ddH 2 O. The following procedure was used for the reaction: 95°C pre-denaturation for 5 min; 95°C denaturation for 10 s, 60°C annealing for 20 s, 72°C extension for 20 s, 40 cycles; and finally 72°C final extension for 5 min. After amplification, the reaction products were subjected to gel electrophoresis to confirm that the amplified strips were clear and single.

[0046] Expression analysis of CmHSFA2b gene in chrysanthemum:

[0047] The reaction procedure is divided into two steps: the first step is pre-denaturation at 95℃ for 5min; the second step is denaturation at 95℃ for 10s and annealing at 60℃ for 30s, which is repeated for 40 cycles; the melting curve is made by the default setting of the fluorescence quantitative PCR instrument. The cDNA concentration is adjusted according to the Δct cycle value, with a fold change of: 2 -ΔΔct For reference, dd H 2 The cDNA was diluted with 10% HO, and then qRT-PCR was performed to detect whether the cycle number Δct was between 20 and 21. The specific CmHSFA2b gene primers, the internal reference CmEF1ɑ gene primers and the 3-fold diluted cDNA were subjected to real-time fluorescence quantitative detection reaction (the same cDNA template was repeated 3 times, the same treatment group was repeated 3 times, and the internal reference gene was used as a control in each reaction).

[0048] The relative expression of genes was calculated using 2 -ΔΔCT The calculation result is as follows Figure 1The results showed that the expression level of CmHSFA2b gene in the inflorescence of 'Zifengche (ZFC)' at each stage after high temperature treatment was significantly higher than that at normal temperature. The expression level of CmHSFA2b gene in 'Zihongtuogui (ZHTG)' was the highest at the early flowering stage (S3) under normal temperature conditions, and at this stage, the expression level of CmHSFA2b gene in the control group of normal temperature plants and the high temperature treatment group was quite different, that is, the expression level of this gene in the inflorescence at the early flowering stage was significantly reduced after high temperature treatment.

[0049] Example 2

[0050] 1. Hormone orthogonal experiment on the proliferation culture medium of chrysanthemum 'Zihongtuogui' that is easy to fade under high temperature

[0051] The explants were selected from the fully expanded young leaves of the 30-day-old 'Zihong Tuogui' chrysanthemum sterile seedlings. The leaves were cut into 0.5*0.5 cm thick leaves with flat wounds on all sides using a scalpel. 2 Leaf discs of different sizes were inoculated in M1 to M9 proliferation medium, as shown in Table 1:

[0052] Table 1 Chrysanthemum leaf disc proliferation culture medium, induction rate and bud number data

[0053]

[0054] Ten leaf discs were inoculated in each culture medium, and the results were repeated five times, with a total of 50 leaf discs inoculated. After the culture medium after the leaf discs were inoculated was cultured for 30 days at 25°C and 14h / 10h (light / dark) conditions, the callus induction rate and the number of buds of the leaf discs were counted, wherein the basic formula of the proliferation culture medium was: MS+3wt% sucrose+0.7wt% agar powder, on which different proportions of 6-benzylaminopurine (6-BA) and naphthaleneacetic acid (NA A) were added; the calculation formula of the callus induction rate was: the number of explants producing callus / the number of explants inoculated*100%. The experimental results show that the more suitable proliferation culture medium formula for 'Zihongtuogui' leaf disc tissue culture is: MS+3mg / L 6-BA+0.5mg / L NAA+0.2mg / L PVP+3wt% sucrose+0.7wt% agar. This culture medium formula is the key formula affecting the transformation success rate of 'Zihongtuogui' overexpression plants, which indirectly affects the flower color phenotype of the overexpression plants.

[0055] 2. Experiment on the concentration of glufosinate spraying on chrysanthemum 'Zihongtuogui' that is easy to fade under high temperature

[0056] Glufosinate (also known as glufosinate) belongs to organophosphorus herbicides and is the active ingredient of non-selective broad-spectrum herbicide Basta. Glufosinate strongly inhibits the activity of glutamine synthetase in plants, thereby causing nitrogen metabolism disorder in plants, excessive accumulation of ammonium, disintegration of chloroplasts, and inhibition of photosynthesis, which ultimately leads to yellowing and death of plants. The glufosinate aqueous solution (PPT content of 10wt%) for spraying experiment was purchased from Shenggong Bioengineering (Shanghai) Co., Ltd., and was diluted with pure water 1000 times (PPT concentration of 100mg / L, 0.01wt%), 2000 times (PPT concentration of 50mg / L, 0.005wt%), 3000 times (PPT concentration of 33mg / L, 0.0033wt%), 4000 times (PPT concentration of 25mg / L, 0.0025wt%), and 5000 times (PPT concentration of 20mg / L, 0.002wt%) for standby use. Chrysanthemum seedlings of 'Zihong Tuogui' with similar growth were sprayed with different concentrations of glufosinate solution. Three pots were treated with each concentration, and pure water was sprayed as a control. The spraying was done once every 3 days, for a total of 4 times. A commercially available 100mL miniature plastic spray bottle with good atomization effect should be used, and it should be ensured that both sides of all chrysanthemum leaf discs are sprayed evenly. During the treatment, the soil matrix humidity and air humidity (relative humidity of about 80%) were maintained at a high level. After 3 days of the fourth treatment, the growth status of the plants was recorded. The experimental results showed that when the PPT concentration in the sprayed glufosinate solution was 0.01wt%, 0.005wt% and 0.0033wt%, the chrysanthemum plants of 'Zihong Tuogui' showed varying degrees of yellowing and wilting. When the PPT concentration was 0.005wt%, the yellowing degree of the plants was the highest. The results are as follows: Figure 2 As shown, this PPT concentration is the key concentration for the subsequent successful screening of 'Zihongtuogui' overexpression positive plants, which indirectly affects the flower color phenotype of the overexpression plants.

[0057] Example 3

[0058] 1. Cloning of Chrysanthemum CmHSFA2b Gene

[0059] The nucleotide sequence of CmHSFA2b of 'Purple Windmill' was cloned and the length was in line with the expected value. The length of its coding sequence was 1029 bp. Figure 3 The sequencing results showed that the nucleotide sequence of the CmHSFA2b gene of 'Zi Feng Che' was shown in Seq_1, and the amino acid sequence of the encoded protein was shown in Seq_2.

[0060]

[0061] MDPFYSVVKEEYPSSGSGSGDGRGARQMIMSVQIPQPMEGLHDAGPPPFLTKVYDMVEDKSIDDIIRWSRGGQSFVVLDPQAFSTNLLPRYFKHNNFSSFVRQLNTYGFRKIDPDIWEFANETFVRGQRHVLKNIKRKRAPSQSLSPQKTQSPGSETATLGSDEVGRLKHEKEVL MMELVKLRQKQQNTRAQIQAMEVRLQGTEEKQRKMMRFLAKAMQNPDFIRKLVKHGKGKELQEAFMNQIGESSGGSKLIKAEPEDFTDASTFEVSELEALALEMQGFGRNQRNQEEESNKLEFKGGERELDDEFWEELFSERVDDKSGVEDVNFLAEKLDFLGSSPK(Seq_2).

[0062] 2. Gene Function Analysis of Overexpressed CmHSFA2b in Chrysanthemum

[0063] Gene overexpression experiments were performed using the Gateway BP ClonaseII Enzyme Mix and Gateway LR Clonase II Enzyme Mix kits from Thermo Fisher Scientific (China) Co., Ltd. The Gateway recombination can clone the PCR product into a donor vector containing attP sites through the BP recombination reaction to generate an entry clone; then the entry clone is recombined into a destination vector containing attR sites through the LR recombination reaction to generate an expression clone. The Gateway pDONR 221 vector was purchased from Thermo Fisher Scientific (China) Co., Ltd. The vector map is shown in Figure 4 As shown; pB 2 GW 7 The vector is preserved by the Key Laboratory of Tropical and Subtropical Flowers and Garden Plants of Guangdong Provincial Colleges and Universities, Zhongkai University of Agriculture and Engineering. The vector map is as follows Figure 5 .

[0064] According to the cloned 'Purple Windmill' CmHSFA2b sequence and the HSFA2b reference sequence downloaded from the feverfew reference genome database, primers were designed online through the NCBI-BLAST website (according to the primer design requirements of the Gateway recombination kit, attB adapter sequences were added at both ends of the primers, which are underlined below):

[0065] Upstream primer attB-CmHSFA2b-F: 5'- ggggacaagtttgtacaaaaaagcaggcttcATGGATCCTTTTTACTCCGTTG-3'(Seq_7);

[0066] Downstream primer attB-CmHSFA2b-R: 5'- ggggaccactttgtacaagaaagctgggtc TTACTTTGGGCTTGAACCCAAG-3'(Seq_8);

[0067] Using chrysanthemum cDNA as a template, the designed adapter primers were used according to the instructions of 2×Hieff Canace Plus PCR Master Mix (Yisheng Biotechnology Co., Ltd., Shanghai) to configure the target gene amplification system and set the reaction program. The PCR product was detected by 2m / v% agarose gel electrophoresis to confirm that the band size was correct, and then the EZNAGel Extraction Kit D2500 kit of Omega Bio-Tek was used for gel excision, recovery and purification. The specific steps were carried out according to the instructions.

[0068] 2.1BP recombination reaction (entry cloning vector construction)

[0069] The purified gel recovery product was mixed with the Gateway pDONR221 vector to form a 10 μL system, including adding 15-150 ng of gel recovery product, 150 ng of Gateway pDONR221 vector, 2 μL of BP Clonase II EnzymeMix, and TE buffer (pH 8.0) to 10 μL. After fully mixing, ordinary PCR was used to react at 25°C for 1 hour. This experiment used DH5αFast Chemically Competent Cell F (11803ES80) from Yisheng Biotechnology Co., Ltd. and was transformed by heat shock method. The specific steps were carried out according to the instructions. After the reaction product was transformed into competent cells, positive transformants were screened using LB medium containing kanamycin, and the bacterial solution was verified by PCR using universal primers M13F and M13R. The bacterial solution whose agarose gel electrophoresis band results were consistent with the expected size was sequenced for verification (all sequencing work in this experiment was performed by Sangon Biotech (Shanghai) Co., Ltd.). The sequencing results showed that the bacterial solution into which the target gene was successfully transferred was extracted using the EZNAPlasmid Mini Kit I (D6943) kit from Omega Bio-Tek to obtain the recombinant plasmid containing the entry clone. The specific steps were carried out according to the instructions.

[0070] 2.2LR recombination reaction (expression cloning vector construction)

[0071] The plasmid containing the entry clone was fused with pB 2 GW7 The vector mixture was prepared into a 10 μL system, including 50-150 ng of the plasmid containing the entry clone, 150 ng of pB 2 GW 7 Vector, 2μL LR Clonase II Enzyme Mix, TE buffer (pH8.0) to 10μL, mix thoroughly and use ordinary PCR to react at 25℃ for 1h, and the reaction product is transformed into DH5αFast Chemically Competent Cell F by heat shock method. After the reaction product is transformed into competent cells, LB medium containing spectinomycin is used to screen positive transformants, and attB1 and attB2 primers are used for bacterial liquid PCR verification. The primer sequences are as follows:

[0072] attB1: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3'(Seq_9);

[0073] attB2: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3'(Seq_10);

[0074] The bacterial solution whose agarose gel electrophoresis band results were consistent with the expected size was sequenced for verification. The sequencing results showed that the bacterial solution into which the target gene was successfully transferred was extracted using the EZNAPlasmid Mini KitⅠ kit to obtain the recombinant plasmid containing the expression clone.

[0075] 2.3 Transformation of Agrobacterium tumefaciens with recombinant plasmid

[0076] This experiment used GV3101 Chemically Competent Cell (CAT#: AC1001) from Shanghai Weidi Biotechnology Co., Ltd. and transformed by freeze-thaw method. The specific steps were carried out according to the instructions. After the recombinant plasmid containing the expression clone was transformed into competent cells, positive transformants were screened using LB medium containing rifampicin and spectinomycin, and the bacterial solution was verified by PCR using attB1 and attB2 primers. The verified correct bacterial solution was mixed with 50% v / v glycerol in a 1:1 ratio for seed preservation and stored in a -80°C ultra-low temperature refrigerator.

[0077] 2.4 Acquisition of receptor materials

[0078] The explants were selected from the young and tender stem segments of healthy 'Zihongtuogui' plants, and the apical buds and leaves were removed and rinsed in running water for 30 minutes. In a clean bench, the chrysanthemum stem segments were soaked in 75% v / v alcohol (50 μL Tween-20 was added) for 30 seconds and rinsed with sterile water 5 times. Then, they were soaked in 2wt% NaClO solution for 6 minutes (50 μL Tween-20 was added) and rinsed with sterile water 5 times. The chrysanthemum stem segments were placed on sterile filter paper to dry the surface liquid, and cut into about 1 cm stem segments with bud points with a sterile scalpel, and the morphological lower end was inoculated into the differentiation medium. When the small buds differentiated from the stem segments in the differentiation medium grew to 1 cm, they were cut off and inoculated into the antibiotic-free rooting medium for rooting induction. If the plants had good rooting, the sterile seedlings of 'Zihongtuogui' were obtained. The differentiation medium formula is: MS + 3mg / L 6-BA + 0.5mg / L NAA + 3wt% sucrose + 0.7wt% agar + 200mg / L carbenicillin; the antibiotic-free rooting medium formula is: 1 / 2MS + 0.1mg / L NAA + 3wt% sucrose + 0.7wt% agar.

[0079] 2.5 Agrobacterium-mediated genetic transformation of leaf discs of 'Zihongtuogui'

[0080] 2.5.1 Pre-culture of receptor material

[0081] Take the leaves of the 30-day-old 'Zihong Tuogui' sterile seedlings and cut them into 0.5*0.5cm squares with flush wounds on all sides. 2 The leaf discs of different sizes are placed flat on the pre-culture medium with the back of the leaves facing downwards and cultured in the dark for 48 hours. According to Example 2, the formula of the pre-culture medium, i.e., the proliferation culture medium, is: MS+3mg / L 6-BA+0.5mg / L NAA+0.2mg / L PVP+3wt% sucrose+0.7wt% agar. Adding PVP to the culture medium in this step and the subsequent steps can effectively prevent the chrysanthemum leaf discs from browning after Agrobacterium infection.

[0082] 2.5.2 Preparation of infection solution

[0083] Take out the stored Agrobacterium culture liquid from the -80℃ ultra-low temperature refrigerator, streak it on the YEB medium containing rifampicin and spectinomycin, culture it in a 28℃ incubator for 48h, pick out a single colony and put it in 4mL YEB liquid medium containing the corresponding antibiotics for initial shaking, culture it in a constant temperature shaker at 28℃ and 240rpm for 24h, take 1mL of it and add it to 100mL YEB liquid medium containing the corresponding antibiotics, and culture it in a constant temperature shaker under the same conditions until OD 600nm The bacterial solution was transferred to a sterile 50 mL centrifuge tube and collected in a refrigerated centrifuge at 4 °C, 6000 rpm, 5 min. After centrifugation, the supernatant was removed in an ultra-clean workbench and MS resuspension solution was added to adjust the OD to 0.6. 600nmWhen the concentration is 0.6, it can be used for infection. The formula of MS resuspension is: MS + 3wt% sucrose + 100μM acetosyringone.

[0084] 2.5.3 Infection

[0085] Pour the infection solution into a sterile container and soak the pre-cultured leaf disc in the infection solution for 8 minutes, shaking it continuously to ensure full contact between the bacterial solution and the leaf disc. After infection, remove the leaf disc from the infection solution and dry the surface liquid with sterile filter paper.

[0086] 2.5.4 Co-culture

[0087] The infected leaf disk was inoculated with the back of the leaf facing downward on the pre-culture medium and cultured in the dark at 25°C for 48 h.

[0088] 2.5.5 Delayed culture

[0089] The co-cultivated explants were immersed in sterile water containing 250 mg / L timentin for 10 min, and the liquid was shaken continuously to make full contact with the leaf disc, and then rinsed with sterile water twice, and the surface liquid was dried with sterile filter paper, and the leaf disc was inoculated on the delay medium with the back of the leaf facing down, and cultured in the dark at 25°C for 48 h. The delay medium formula is: MS + 3 mg / L 6-BA + 0.5 mg / L NAA + 0.2 mg / L PVP + 3wt% sucrose + 0.7w / v% agar + 250 mg / L timentin.

[0090] 2.5.6 Decarboxylation culture

[0091] The leaves after delayed culture were inoculated with the back side facing downward on the decarboxylation medium for 7 days. The decarboxylation medium formula was: MS + 3 mg / L 6-BA + 0.5 mg / L NAA + 0.2 mg / L PVP + 3 wt% sucrose + 0.7 w / v% agar + 500 mg / L carbenicillin.

[0092] 2.5.7 Induction culture

[0093] The explants after decarboxylation culture were immersed in sterile water containing 500 mg / L carbenicillin for 10 minutes, and the liquid was shaken continuously during the period to make full contact with the leaf disc, and then rinsed with sterile water twice, and the surface liquid was dried with sterile filter paper, and the leaf disc was inoculated on the induction medium with the back of the leaf facing down, and cultured under the environmental conditions of 25°C and 14h / 10h (light / dark), and a new induction medium was replaced every 10 days. The induction medium formula is: MS+3mg / L 6-BA+0.5mg / L NAA+0.2mg / L PVP+3wt% sucrose+0.7w / v% agar+250mg / L carbenicillin.

[0094] 2.5.8 Rooting induction

[0095] When the buds differentiated from the leaf disc in the induction medium grow to 1-2 cm, they are cut off and inoculated into the rooting medium for rooting induction. The formula of the rooting medium is: 1 / 2MS+0.1mg / L NAA+3wt% sucrose+0.7w / v% agar+250mg / L carbenicillin.

[0096] 2.5.9 Hardening and transplanting

[0097] After 15 days of rooting induction, the plants with good root growth can open the culture bottle cap and harden for 7 days. Then the plants can be removed from the culture bottle, the excess agar in the roots can be washed off, and they can be transplanted into a clean general horticultural substrate to maintain normal field flowering culture.

[0098] 2.6 Screening and identification of transgenic chrysanthemum

[0099] The pB2GW7 vector contains the glufosinate-resistant Bar gene, which encodes phosphinothricin acetyltransferase PAT. PAT can acetylate the free amino group of PPT, so that the activity of glutamine synthetase is not inhibited. Plants containing this gene have resistance to glufosinate herbicides. Use pure water to dilute 2000 times of glufosinate aqueous solution (PPT concentration is 0.005wt%) to spray the 'Zihong Tuogui' plants obtained by screening and transformation. The specific spraying process refers to Example 2. Take the third leaf from the top bud of the plant that grows normally after treatment and quick-freeze it in liquid nitrogen. The collected samples are extracted with the FastPure Plant DNA Isolation Mini Kit of Nanjing Novozyme Biotechnology Co., Ltd., and PCR identification is performed with attB1 and attB2 primers. The plant corresponding to the DNA of the target band obtained by agarose gel electrophoresis is a transgenic positive plant. A total of three transgenic positive plants were obtained in this experiment. The results are detailed in Figure 6 .

[0100] 2.7 Expression analysis of transgenic chrysanthemum

[0101] Total RNA was extracted and purified from leaves of transgenic chrysanthemum plants, and then reverse transcribed to synthesize cDNA. The cDNA product obtained by reverse transcription was diluted 3 times and used for qRT-PCR, using qCmHSFA2b-F / qCmHSFA2b-R as CmHSFA2b gene primers and qCmEF1ɑ-F / qCmEF1ɑ-R as internal reference gene primers, and the reaction procedure was referred to Example 1.

[0102] The relative expression of genes was calculated using 2 -ΔΔCT The calculation results are shown in Figure 7 The results showed that the overexpression plants (pB 2 GW 7-HSFA2b) in wild-type plants (WT) and empty-type control plants (pB 2 GW 7 ), among which the expression level of CmHSFA2b in overexpression plant 1 was significantly increased. This indicates that pB 2 GW 7 The 35S promoter in the vector can effectively increase the transcription level of CmHSFA2b, thereby enhancing the expression of this gene in 'Zihongtuogui' plants.

[0103] 2.8 Phenotypic identification of transgenic chrysanthemum

[0104] Chrysanthemum overexpressing plants and empty control plants were cultivated under a short-day environment of 10h / 14h (light / dark) until flowering, and the phenotypes of the plants and inflorescences were observed. The results showed that the color of the ligulate petals of the inflorescence of the 'Zihongtuogui' plant overexpressing CmHSFA2b was significantly darker. Figure 8 .

[0105] Example 4

[0106] 1. Validation of virus-induced gene silencing (VIGS) of CmHSFA2b in chrysanthemum

[0107] The gene silencing experiment was performed using the Nimble Cloning kit of Hainan Yitian Biotechnology Co., Ltd. Nimble Cloning (NC cloning) can clone the PCR product into the circular expression vector of the NC system through Nimble Mix. The pNC-TRV2 vector was donated by Dr. Yan Pu (Yan et al., 2020). The vector map can be found at Fig. 9 .

[0108] According to the cloned 'Purple Windmill' CmHSFA2b sequence and the HSFA2b reference sequence downloaded from the feverfew reference genome database, primers were designed online through the NCBI-BLAST website (according to the primer design requirements of the Nimble Cloning kit, 20 bp universal adapter sequences were added to both ends of the primers, which are marked with underscores below):

[0109] Upstream primer NC-CmHSFA2b-F: 5'- agtggtctctgtccagtcct TACTCCGTTGTTAAAGAAGAATACCAATC-3'(Seq_11);

[0110] Downstream primer NC-CmHSFA2b-R: 5'- ggtctcagcagaccacaagt ATAAGTATTGAGCTGCCTGACAAAG-3'(Seq_12).

[0111] The designed adapter primers were used according to the instructions for use of 2×Hieff Canace Plus PCR Master Mix to configure the target gene amplification system and set the reaction program. The obtained PCR products were purified using the EZNACycle-Pure Kit (D6492) from Omega Bio-Tek. Since the target fragments were all less than 200 bp, 6 times CP buffer was added. The specific steps were carried out according to the instructions.

[0112] 1.1 Construction of TRV2-HSFA2b recombinant vector

[0113] The purified PCR product was mixed with the NC system expression vector TRV2 to prepare a 10 μL system, including 10-80 ng of PCR product, 20-120 ng of NC system expression vector, 5 μL Nimble Mix, ddH 2 0 to 10 μL, mix thoroughly and use common PCR to react at 50°C for 45 minutes, and transform the reaction product into DH5αFast ChemicallyCompetent Cell F by heat shock method. After the reaction product is transformed into competent cells, use LB medium containing kanamycin to screen positive transformants, and use PE GFP-C5 and NosR primers to verify the bacterial liquid PCR. The primer sequences are as follows:

[0114] PEGFP-C5: 5'-catggtcctgctggagttcgtg-3'(Seq_13);

[0115] NosR: 5'-accggcaacaggattcaatc-3' (Seq_14).

[0116] The bacterial solution whose agarose gel electrophoresis band results were consistent with the expected size was sequenced for verification. The sequencing results showed that the bacterial solution into which the target gene was successfully transferred was extracted using the EZNAPlasmid Mini KitⅠ200 kit to obtain the plasmid containing the recombinant vector.

[0117] 1.2 Transformation of recombinant plasmid into Agrobacterium tumefaciens

[0118] The plasmid containing the recombinant vector was transformed into GV3101 Chemically Competent Cell by freeze-thaw method. Positive transformants were screened using LB medium containing rifampicin and kanamycin, and bacterial liquid PCR verification was performed using PEGFP-C5 and NosR primers. The verified TRV2-HSFA2b bacterial liquid was mixed with 50% v / v glycerol in a 1:1 ratio for seed preservation and stored in a -80°C ultra-low temperature refrigerator.

[0119] 1.3 Agrobacterium-mediated infection of chrysanthemum plants

[0120] This experiment referred to the method of Wang Yuhan et al. (2018) and used a modified vacuum infection method to infect the plants of the high temperature non-fading chrysanthemum 'Purple Windmill'. A total of three groups of experiments were conducted, namely the pure water control group (CK), the empty control group (TRV2), and the VIGS experimental group (TRV2-HSFA2b).

[0121] 1.3.1 Preparation of infection solution

[0122] Take out the stored TRV2, TRV2-HSFA2b, and TRV1 (GenBank: AF406990, i.e., pTRV1 vector without exogenous gene transfer) Agrobacterium culture liquid from -80℃ ultra-low temperature freezer, streak on YEB medium containing rifampicin and kanamycin respectively, culture in 28℃ incubator for 48h, pick out single colony and put it in 10mL YEB liquid medium containing corresponding antibiotics for initial shaking, culture in constant temperature shaker at 28℃, 240rpm for more than 24h until the culture liquid is saturated, take 5mL of it and add 500mL YEB liquid medium containing corresponding antibiotics, and culture in constant temperature shaker under the same conditions until OD 600nm The bacterial solution was transferred to a sterile 50 mL centrifuge tube several times, and the cells were collected in a refrigerated centrifuge at 4 ° C, 6000 rpm, 5 min. After centrifugation, the supernatant was removed in an ultra-clean workbench, and the MMA solution was added and mixed thoroughly to adjust the OD 600nm When the concentration is 1.0, it can be used for infection. The formula of MMA solution is: 10mM MgCl 2 +10mM MES (pH5.6) +100μM acetosyringone. After all three bacterial solutions are resuspended in MMA solution to meet the requirements, mix TR V1 bacterial solution with TRV2 and TRV2-HSFA2b bacterial solutions in equal volumes. The prepared infection solution needs to be kept at room temperature and away from light for 4 hours before use.

[0123] 1.3.2 Infection

[0124] The plant material was a plant of chrysanthemum 'Purple Windmill' that had differentiated into flower buds. The plant material was deprived of water 2 days in advance and kept in the dark for 4 hours before infection. After marking, the plant pot was covered with a bag to prevent the flower mud from pouring out. After the infection solution was left to stand at room temperature and away from light, 30μL of Silwet L-77 surfactant was added and mixed thoroughly. The plant material was inverted so that the flower buds of the plant were immersed in the infection solution for 1 minute (the pure water control group was immersed in ultrapure water). Then the plant material was placed in a vacuum pump and evacuated to 0.7atm. After 10 minutes of negative pressure treatment, the air was slowly exhausted and the plant was taken out. The plant was then infiltrated with the bacterial solution and the negative pressure treatment was repeated once. After the plant material was infected, it was fully watered and cultured away from light for 24 hours. After the end of the light-proof period, normal field flowering culture was resumed.

[0125] 1.4 Phenotypic identification and expression analysis of CmHSFA2b gene-silenced plants

[0126] After 21 days of infection, the plant materials were placed in an intelligent light incubator for high temperature treatment for 5 days (35°C, 10h, 20000Lx during the day; 26°C, 14h, 0Lx at night). The humidity in the incubator was about 70%, and water was applied every 2 days to keep the soil moist. After the treatment, the plant materials were photographed and recorded. The head inflorescences at the early stage of flowering were taken and quick-frozen with liquid nitrogen, and then stored in a -80°C ultra-low temperature refrigerator. Total RNA was extracted from the ligulate petals of the chrysanthemum samples, and cDNA was obtained by reverse transcription. CmEF1ɑ was used as the internal reference gene, and the expression level of CmHSFA2b in the three groups of experimental plants was verified by qRT-PCR experiments. The melting curve and standard curve of the experiment were analyzed using relative quantitative software, and the relative expression level was calculated using 2 -ΔΔCt Comparative analysis.

[0127] The results showed that after silencing CmHSFA2b in the buds of Chrysanthemum 'Purple Windmill' and subjecting them to high temperature for 5 days (35℃, 10h, 20000Lx during the day and 26℃, 14h, 0Lx at night), the ligulate petals of the 'Purple Windmill' inflorescence showed obvious fading, which was in sharp contrast to the pure water control group and the empty control group. Fig.10 The ligulate petals of the three treatment groups at the early stage of flowering were taken for expression analysis by qRT-PCR. It was found that the expression level of the CmHSFA2b gene after silencing treatment was significantly reduced compared with the pure water control group and the empty control group. The results are as follows Fig.11 shown.

[0128] The present invention first discovered that the expression of a heat shock transcription factor encoding gene CmHSFA2b in chrysanthemum can stabilize the color of the chrysanthemum tongue-shaped petals from fading at high temperatures. When the gene is overexpressed, the tongue-shaped petals of the chrysanthemum variety that is easy to fade can not fade or fade not obviously after high temperature treatment; when the expression of the gene is inhibited, the tongue-shaped petals of the chrysanthemum variety that does not fade at high temperature fade from purple-red to pink-purple under high temperature environment, indicating that the transcription factor plays an important role in stabilizing the color of chrysanthemums from fading at high temperatures, laying a theoretical foundation for the research on the color regulation of high-temperature chrysanthemums and molecular improvement breeding, and has important application value.

[0129] For those skilled in the art, various other corresponding changes and modifications can be made according to the technical solutions and concepts described above, and all of these changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. A gene encoding a chrysanthemum transcription factor, characterized in that: The coding gene is the gene sequence shown in SEQ ID NO.

1.

2. A chrysanthemum transcription factor, characterized in that The coding gene of the chrysanthemum transcription factor is as described in claim 1; the sequence of the chrysanthemum transcription factor is shown in SEQ ID NO.

2.

3. An application of a gene encoding a chrysanthemum transcription factor, characterized in that: The expression level of the coding gene as claimed in claim 1 is increased by an overexpression tool, thereby improving the effect of temperature on the fading of petals of heat-resistant chrysanthemum varieties.

4. An application of a chrysanthemum transcription factor, characterized in that: The expression level of the gene encoding the chrysanthemum transcription factor as claimed in claim 2 is increased by an overexpression tool, thereby improving the effect of temperature on the fading of petals of heat-resistant chrysanthemum varieties.

5. The use according to claim 3 or 4, characterized in that The temperature is ≥ 25°C at night and ≥ 34°C during the day.

6. The use according to claim 3 or 4, characterized in that The breeding of the heat-resistant chrysanthemum variety comprises the steps of infecting the chrysanthemum leaf disk or the head inflorescence at the bud stage with the vector carrying the coding gene through a mediated method.

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