Cloning of cold resistance gene cmmyb106 of chamaemelum nobile and application thereof

Abstract

The application discloses a cold-resistant gene CmMYB106 of chrysanthemum, wherein the nucleotide sequence is shown as SEQ ID NO:1 in a sequence listing, and the application also discloses a primer pair CmMYB106-qF and CmMYB106-qR for cloning the cold-resistant gene CmMYB106, wherein the nucleotide sequences are shown as SEQ ID NO:2 and SEQ ID NO:3 in the sequence listing. The application takes chrysanthemum as a material, clones the CmMYB106 gene, and determines the expression characteristics of the CmMYB106 gene by means of expression modes and transcription activation activities. An overexpression vector is constructed, and the chrysanthemum is genetically transformed by means of agrobacterium, and the influence of the CmMYB106 on the cold resistance of chrysanthemum is analyzed, thereby providing a theoretical basis for chrysanthemum cold resistance breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of garden plant molecular biology, and particularly relates to cloning of a Chrysanthemum coronarium cold resistance gene CmMYB106 and application thereof. BACKGROUND

[0002] The growth and development of plants cultivated in open ground cannot be separated from various environmental factors such as water, light, soil and gas, and is often limited by various environmental conditions and biological factors, affecting the growth and yield of plants. Plant growth and development are affected by biological stress and abiotic stress. Biological stress includes herbivore invasion and pathogen infection; abiotic stress mainly includes low or high temperature stress, drought or waterlogging, saline-alkali stress, heavy metal toxicity and the like. Plants are subjected to abiotic stress at all times during growth. In South China, the summer and autumn seasons are hot, the soil is dry and saline, and the absorption of water and mineral nutrients by plants is hindered. Compared with other regions in China, temperature is more important for plant growth and development in the cold northeast region. Low temperature affects the growth and development of plants, including seed germination, weak seedling growth, poor flower bud differentiation, pollen sterility, ovule abortion, fruit deformity, cold damage spots, decreased root activity, and even plant wilting or death.

[0003] Chrysanthemum morifolium is a perennial herbaceous plant of the genus Chrysanthemum in the Asteraceae family, and is one of the ten classic flowers in China and one of the four major fresh-cut flowers in the world. Chrysanthemum has rich historical and cultural heritage and double economic value, and plays an indispensable role in the international flower industry. Chrysanthemum coronarium is also a perennial herbaceous plant of the genus Chrysanthemum in the Asteraceae family, and is one of the first varieties for landscaping due to its long flowering period, diverse flower colors and shapes, and strong stress resistance.

[0004] At present, scholars at home and abroad have reported a lot on the cold resistance of Chrysanthemum coronarium, mainly focusing on low-temperature acclimation, flower frost damage and the like, and few reports on low-temperature overwintering. Due to the cold climate in the north, most Chrysanthemum coronarium cannot naturally overwinter and are frozen to death, which limits the wide application of Chrysanthemum coronarium in the northeast region and causes huge economic losses. SUMMARY

[0005] The present application relates to the field of garden plant molecular biology, and particularly relates to cloning of a Chrysanthemum coronarium cold resistance gene CmMYB106 and application thereof.

[0006] The Chrysanthemum coronarium cold resistance gene CmMYB106 of the present application, and the nucleotide sequence of the Chrysanthemum coronarium cold resistance gene CmMYB106 is shown in SEQ ID NO: 1 in the sequence listing.

[0007] A primer pair for cloning the cold resistance gene CmMYB106 of Leucanthemum vulgare, the primer pair is CmMYB106-qF and CmMYB106-qR, the nucleotide sequences are shown as SEQ ID NO:2 and SEQ ID NO:3 in the sequence listing.

[0008] A fluorescent quantitative primer pair of the cold resistance gene CmMYB106 of Leucanthemum vulgare, the nucleotide sequences are shown as SEQ ID NO:4 and SEQ ID NO:5 in the sequence listing.

[0009] A primer pair for constructing the pGBKT7-CmMYB106 plasmid, the nucleotide sequences are shown as SEQ ID NO:6 and SEQ ID NO:7 in the sequence listing.

[0010] An amino acid encoded by the cold resistance gene CmMYB106 of Leucanthemum vulgare, the sequence of the amino acid is shown as SEQ ID NO:8 in the sequence listing.

[0011] The cold resistance gene CmMYB106 of Leucanthemum vulgare is applied to improving the cold resistance of plants.

[0012] The application of the application, wherein the cold resistance gene CmMYB106 of Leucanthemum vulgare can enhance the cold resistance of plants.

[0013] The application of the application, wherein the plant is Leucanthemum vulgare.

[0014] The application differs from the prior art in that:

[0015] The application takes Leucanthemum vulgare as the material, clones the CmMYB106 gene, and determines the expression characteristics of the CmMYB106 gene such as expression mode and transcription activation activity; constructs an overexpression vector, uses Agrobacterium genetic transformation Leucanthemum vulgare, analyzes the influence of CmMYB106 on the cold resistance of chrysanthemum, and provides a theoretical basis for chrysanthemum cold resistance breeding.

[0016] The cloning of the cold resistance gene CmMYB106 of Leucanthemum vulgare and the application thereof will be further described below in combination with the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a cloning related graph of the CmMYB106 transcription factor in the application; wherein, A is a total RNA extraction graph of Leucanthemum vulgare; B is a CmMYB106 amplification band graph;

[0018] Figure 2 It is an expression amount analysis graph of CmMYB106 in different parts of Leucanthemum vulgare in the application; wherein, the error line represents the standard error, and different letters represent significant difference, P<0.05;

[0019] Figure 3 Figure 7 is a graph for expression specificity analysis of CmMYB106 under low temperature conditions in the present application;

[0020] Figure 4 Figure 8 is a graph for verification of self-activation activity of CmMYB106 transcription factor in the present application; wherein, A is the CmMYB106 sequence cutting site, B is the self-activation activity analysis of CmMYB106 transcription factor (pCL1 positive control, pGBKT7 negative control);

[0021] Figure 5 Figure 9 is a graph for growth and development process of CmMYB106 overexpression plant of ground cover chrysanthemum in the present application; wherein, A: chrysanthemum leaf disc; B: callus; C: seedling; D: rooting culture;

[0022] Figure 6 Figure 10 is a graph for identification of CmMYB106 overexpression line in the present application; wherein, A: detection of overexpression line at DNA level; B: expression amount of CmMYB106 in overexpression line; M: DNA Marker DL2000; WT: wild type; OX1, OX4, OX5, OX9: overexpression line;

[0023] Figure 7 Figure 11 is a graph for phenotype analysis of CmMYB106 overexpression line under low temperature conditions in the present application;

[0024] Figure 8 Figure 12 is a graph for antioxidant enzyme activity analysis of CmMYB106 overexpression line under low temperature conditions in the present application;

[0025] Figure 9 Figure 13 is a graph for proline and MDA content analysis of CmMYB106 overexpression line under low temperature conditions in the present application;

[0026] Figure 10 Figure 14 is a graph for H2O2 and O2- content of CmMYB106 overexpression line under low temperature conditions in the present application; wherein, A: DAB staining; B: NBT staining; -

[0027] Figure 11 Figure 15 is a graph for cold tolerance gene expression amount analysis of CmMYB106 overexpression line under low temperature treatment in the present application. DETAILED DESCRIPTION

[0028] 1. Materials and methods

[0029] 1.1 Experimental materials

[0030] 1.1.1 Strain materials

[0031] ​Agrobacterium strain: EHA105; E. coli strain: DH5a; Yeast strain: Y1H.

[0032] 1.1.2 Plant material

[0033] The ground cover chrysanthemum 'Yingjie Hongfei' was subjected to external sterilization and cultured in MS medium (sucrose 30 g / L, agar 8 g / L, pH = 5.8) under the following conditions: light cycle 14 h, day and night temperature (25 ± 2) °C. The obtained sterile seedlings were used as experimental materials for genetic transformation of the ground cover chrysanthemum.

[0034] The obtained overexpression plants and ground cover chrysanthemum seedlings were cultivated in the teaching base of the College of Agriculture of Yanbian University, with a ratio of humus soil to perlite of 1:1, a light cycle of 16 h, and day and night temperatures of (25 ± 2) °C. The seedlings were used to determine the ROS damage under subsequent low temperature treatment conditions.

[0035] 1.1.3 Experimental primer sequences

[0036] The primers used in the experiment were designed using NCBI and Primer 5.0 software and synthesized by Anhui General Biotechnology Co., Ltd. The primer sequences used are shown in the following table:

[0037] Table 1 Primer names and sequences used in this experiment

[0038]

[0039]

[0040] 1.2 CmMYB106 transcription factor cloning

[0041] 1.2.1 Extraction of RNA

[0042] (1) Material preparation: Soak the gun heads, centrifuge tubes, and mortar used in the experiment in distilled water containing proteinase K for 12 h, and then sterilize them under high temperature and pressure before drying them in an oven.

[0043] (2) Select ground cover chrysanthemum leaves, quickly freeze them in liquid nitrogen, and grind them into white powder using a mortar and pestle. Ensure that the sample remains frozen during the grinding process.

[0044] (3) Add enough Trizol (usually 1 mL of Trizol reagent for 0.2 g of tissue) to completely cover the powder sample, and continue grinding until it becomes an oily liquid.

[0045] (4) Add the ground oily liquid to a centrifuge tube, let it stand at room temperature, and then place it in a refrigerated high-speed centrifuge at 4°C and 10,000 rpm for 7 min.

[0046] (5) Take the supernatant and calculate the volume, add chloroform with one fifth of the volume of the supernatant, shake to extract evenly, then stand at room temperature for 5 min, centrifuge at 4°C, 10000 rpm for 7 min.

[0047] (6) Record the volume of the RNA supernatant after centrifugation, transfer it to a new centrifuge tube, then add the same volume of isopropanol, mix gently up and down, then place it in the -20°C refrigerator to precipitate the RNA.

[0048] (7) Centrifuge at 4°C, 10000 rpm for 7 min, discard the supernatant.

[0049] (8) Add 1 mL of 75% ethanol solution prepared in advance with DEPC water, gently invert to wash the precipitate thoroughly, centrifuge at 4°C, 10000 rpm for 7 min, discard the supernatant.

[0050] (9) Dry the RNA precipitate for 2-5 min, dissolve the RNA with an appropriate amount of RNase-free water or DEPC-treated water, and make it completely dissolved. Measure the concentration and purity of the dissolved RNA sample, and store it at -80°C.

[0051] 1.2.2 Reverse transcription to synthesize cDNA

[0052] Use FastKing one-step genomic cDNA first-strand synthesis kit (TIANGEN) to synthesize cDNA by reverse transcription.

[0053] Reaction system:

[0054]

[0055] Reaction procedure: 42°C for 15 min, 95°C for 3 min; store at -20°C for standby.

[0056] 1.2.3 Obtaining and amplifying the sequence of CmMYB106 gene

[0057] (1) Amplification of target fragment

[0058] In the chrysanthemum transcriptome database, the cDNA sequence of the ground cover chrysanthemum gene was downloaded, and specific primers (F: 5'-ATGGAGGTTACACGGGCA-3', R: 5'-TTATTTGGCTGAGGCTTCTATAA-3') CmMYB106-F / R were designed using Primer 5.0 software to amplify the CmMYB106 gene.

[0059] Reaction system:

[0060]

[0061] PCR reaction procedure: 95℃ 4 min, 95℃ denaturation 30 s, 58℃ annealing 30 s, 72℃ extension 60 s, 35 cycles, 72℃ 7 min, 4℃ preservation.

[0062] (2) PCR product recovery

[0063] According to the experimental requirements, an appropriate amount of agarose is weighed and dissolved in 0.5x TBE electrophoresis buffer, heated and treated, and after cooling, EB substitute is added, and then cooled to an appropriate temperature to solidify into a gel. After sample loading, gel electrophoresis is carried out, and after color development, the specific fragment is cut. First, the target fragment is accurately cut under the gel imaging system with a blade and placed in a 1.5 mL centrifuge tube. Then 3 times the volume of Buffer DE-A is added, and after mixing, it is placed in a 65℃ metal bath until the gel is completely dissolved (every 2 min, mix up and down). At the same time, the Eluent water is preheated to 65℃. Then 1.5 times the volume of Buffer DE-B is added and mixed up and down. Centrifuge at 12000 rpm for 2 min, discard the supernatant, add 25-30 mL of Eluent water to the prepared membrane, and let it stand at room temperature for 5 min. Centrifuge at 12000 rpm for 1 min, and elute again to improve the concentration of the recovered material. The gel recovery product is immediately placed in a -20℃ environment for subsequent use.

[0064] (3) Connection transformation

[0065] The target fragment recovered from the gel is connected to the pMD19-T vector. The reaction system is: Solution I 2.5 μL, pMD19-T 0.5 μL, and gel recovery product 2 μL (1000 ng). The reaction procedure is: 16℃ for 2h, and 4℃ preservation. The connected product is added to 100 mL of E. coli DH5α strain competent cells, completely immersed in an ice box for ice bath for 30 min. After ice bath, it is quickly taken out and placed in a 42℃ water bath for heat shock for 90 s. After heat shock, it is quickly immersed in an ice box for ice bath for 5 min. It is cultured in 700 mL of LB liquid medium at 37℃ and 180 rpm. After shaking for 1.5h, it is centrifuged at 10000 rpm for 8 min, and a small amount of supernatant is mixed with the precipitate. It is coated on LB+A (Ampicillin) medium and cultured at 37℃ for 16h.

[0066] (4) Sequencing

[0067] Samples are selected from the screened positive clone colonies, inoculated in 800 μL of LB+Amp liquid medium, cultured at 37℃ and 180 rpm for 3h, and subjected to PCR verification. The bacterial liquid successfully amplifying the target fragment is sent to Anhui General Biotechnology Company for high-precision sequencing.

[0068] 1.3 Spatial expression pattern of CmMYB106 gene

[0069] Four different tissues of wild-type C. morifolium root, stem, leaf and flower were collected, and RNA was extracted to reverse transcribe cDNA. The C-terminal sequence of CmMYB106 was used to design a fluorescent quantitative primer (F: 5'-CTCAAAGGATGGCAAGGTAT-3'; qR: 5'-ATAGAAGCCTCAGCCAAATAA-3'), and a fluorescent quantitative instrument (Agilent 3005) was used to analyze the expression level of CmMYB106 in different tissues.

[0070] The qPCR reaction system was as follows:

[0071]

[0072] The reaction program was set as follows: 95°C pre-denaturation for 2 min; 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 30 s, 35 cycles; 4°C cycle, melting curve selection of 60-95°C, and data analysis method was 2 -△△CT The experimental data was expressed as "mean ± standard error (Mean ± SE)". ΔCT (target gene) = CT (target gene) - CT (EF1α), and ΔΔCT = ΔCT - ΔCT (maximum).

[0073] 1.4 Determination of self-activation activity of CmMYB106 transcription factor

[0074] (1) The enzyme cutting sites BamH I and Xho I were found by BioXM 2.6 software, and specific primers were designed for PCR amplification to introduce the enzyme cutting sites into the CmMYB106 gene fragment. After agarose gel electrophoresis, the specific fragments were collected by gel recovery.

[0075] F: 5'-GGATCCATGGAGGTTACACGGGCA-3'

[0076] R: 5'-CTCGAGTTATTTGGCTGAGGCTTCTATAA-3'

[0077] (2) Restriction enzyme double enzyme digestion, ligation transformation and verification were the same as 1.2.3.

[0078] (3) The successfully constructed pGBKT7-CmMYB106 plasmid, pGBKT7 plasmid (negative control) and pCL1 plasmid (positive control) were respectively transformed into the competent cells of yeast Y187 strain, and the yeast bacterial liquid containing the pGBKT7-CmMYB106 plasmid and the pGBKT7 plasmid was respectively coated on SD / Trp -(Trp: tryptophan) solid medium, the yeast liquid containing pCL1 plasmid was spread on SD / Leu - (Leu: leucine) solid medium, 30℃ for 2-3d.

[0079] (4) The single colony liquid was picked, shaken and cultured, and then spotted on SD / Ade - / His - (Ade: adenine, His: histidine) medium, 30℃ for 3-4d, and the growth state of the colonies was observed.

[0080] 1.5 Screening of CmMYB106 fragments without self-activation activity

[0081] The MEME (http: / / meme-suite.org / ) online gene motif analysis website was used to input the CmMYB106 gene sequence to find the specific motif position on the gene fragment, so as to find the cutting site. According to the position of the motif, three cutting sites were selected at 350bp, 549bp and 647bp, respectively, and were named as CmMYB106 1-350 , CmMYB106 1-549 and CmMYB106 1 -647 , respectively. After enzyme digestion and ligation to the pGBKT7 vector, the self-activation activity and position were verified and determined, and the specific method was the same as 1.4. Finally, the fragment without transcriptional activation activity was screened out by observing the growth state of the colonies for subsequent experiments.

[0082] 1.6 Agrobacterium genetic transformation of C. chinensis and identification of CmMYB106 overexpression strain

[0083] (1) The Agrobacterium liquid of pORE-R4-35S-CmMYB106 was cultured in YEB liquid medium containing Kan and Rif at 28℃ and 180rpm for 16h, and the culture was incubated to OD 600 value = 0.6.

[0084] (2) After 30d of tissue culture seedling culture, the C. chinensis leaves were cut into 1×1cm square leaves in a clean bench, and were evenly arranged in MS+6-BA (6-Benzylaminopurine) 1.6mg / L+NAA (Naphthaleneacetic) 0.8mg / L+3% sucrose+8g / L agar, 25±2℃, and pre-cultured for 2-3d.

[0085] (3) The pre-cultured leaf of Ajuga pygmaea was infected by the general cultured Agrobacterium liquid, and the leaf of Ajuga reptans was soaked for 8-10 min, and then was put back into the culture medium and cultured in dark at 25°C for 3 d.

[0086] (4) After dark culture, the leaf was transferred into the co-culture medium, and the co-culture medium was MS+BA 1.6 mg / L+NAA 0.8 mg / L+3% sucrose+500 mg / L Carb medium, and was cultured at 25°C for 7 d.

[0087] (5) After co-culture, the leaf was transferred into the screening medium, and the screening medium was MS+BA 1.6 mg / L+NAA 0.8 mg / L+3% sucrose+Hygromycin 12 mg / L, and the overexpression CmMYB106 strain was screened, and the leaf disc with bud differentiation was inoculated into the rooting medium for rooting culture.

[0088] 1.6.1 Identification of overexpression strain at DNA level

[0089] (1) The DNA was extracted by CTAB method. First, the extraction liquid was prepared: extraction liquid I (50 mL) was prepared by taking sorbitol 2.685 g, Tris-HCl (1 mol / L) 5 mL, EDTA (0.25 M) 1 mL, and β-mercaptoethanol 35 μL, and adding distilled water to 50 mL; extraction liquid II (50 mL) was prepared by taking Tris-HCl (1 mol / L) 10 mL, EDTA (0.25 M) 10 mL, NaCl 5.85 g, and CTAB 1 g, and adding distilled water to 50 mL. The prepared extraction liquid was sterilized by high temperature and high pressure for 20 min, and was stored at room temperature for standby use.

[0090] (2) 0.2-0.3 g of overexpression CmMYB106 strain and Ajuga pygmaea leaf were respectively put into a mortar, and a small amount of liquid nitrogen was added for grinding the leaves to release DNA, 1.5 mL of extraction liquid I and 20 μL of β-mercaptoethanol were added, and the mixture was transferred into a 2.0 mL centrifuge tube. After mixing evenly, centrifugation was performed at 12000 rpm and 4°C for 6 min, and the supernatant was discarded.

[0091] (3) 400 μL of extraction liquid I was added to the centrifuge tube, and then 200 μL of extraction liquid II preheated to 65°C and 20 μL of β-mercaptoethanol were mixed, and the mixture was gently shaken. 120 μL of 5% sodium dodecyl cholate was added, and the mixture was incubated in a 65°C water bath for 30 min, and was shaken up and down every 10 min during the incubation.

[0092] (4) Add 600 μL of chloroform-isoamyl alcohol mixture (24:1) and shake vigorously for 15 min. Centrifuge at 12000 rpm for 6 min at 4°C. Transfer 500 μL of supernatant to a new centrifuge tube.

[0093] (5) Repeat step (4) three times. Add 600 μL of isopropanol (-20°C pre-cooled) and gently invert. DNA precipitates can be observed. Place in a -20°C refrigerator and centrifuge at 6000 rpm for 10 min at 4°C. Discard the supernatant and wash with 1 mL of 70% ethanol. Centrifuge at 6000 rpm for 10 min at 4°C. Repeat this step.

[0094] (6) Discard the supernatant and allow the sample to dry naturally at room temperature. Dissolve the DNA in RNase-free solvent and store at -80°C.

[0095] (7) Design specific primers containing overexpression vector fragments, use overexpression strain DNA as a template for PCR amplification, and wild-type DNA as a control. After amplification, perform gel electrophoresis to detect whether there are bands.

[0096] HYG-F: 5'-CTTCTACACAGCCATCGGTCCAG-3';

[0097] HYG-R: 5'-CGGAAGTGCTTGACATTGGGGAG-3';

[0098] 1.6.2 Identification of overexpression strains at RNA level

[0099] (1) Select 30-day-old overexpression strains and wild-type Chamaemelum nobile tissue culture seedlings, cut 0.3 mg of leaves with scissors, and extract their RNA. The extraction method is the same as 1.2.1. Reverse transcription to obtain corresponding cDNA, and store at -20°C for later use.

[0100] (2) Perform fluorescent quantitative PCR detection using the above-extracted cDNA as a template, and the method is the same as 1.3, 2 -△△CT Analyze the data to determine that it is an overexpression plant.

[0101] 1.7 ROS determination of CmMYB106 overexpression strains under low temperature conditions

[0102] 1.7.1 Plant materials and low temperature treatment

[0103] The wild type and overexpression CmMYB106 lines of creeping chrysanthemum were collected and inserted into the seedling pots filled with nutrient soil mixed with humus and perlite at a ratio of 1:1. The plants were cultured in a climate chamber with a temperature of 25±2℃, a light exposure time of 16h, and a relative humidity of 70%. When the seedlings developed 5-6 leaves, the plants were transferred to a culture box with a temperature of 25℃, 10℃, 5℃, and -5℃, respectively. Each temperature condition was treated for 4h. The top 3-4 leaves were immediately placed in liquid nitrogen and stored at -80℃. All treatments were repeated three times.

[0104] 1.7.2 ROS damage determination of overexpression CmMYB106 lines

[0105] (1) SOD activity was determined by the NBT colorimetric method of Li Hesheng. (2) POD activity was determined by the guaiacol colorimetric method of Li Hesheng. (3) CAT activity was determined by the ultraviolet absorption colorimetric method of Li Hesheng. (4) Pro content was determined by the sulfosalicylic acid method of Li Hesheng. (5) MDA content was determined by the TBA colorimetric method of Li Hesheng. (6) Determination of H2O2 and O2 - content. The determination of H2O2 and O2 - The qualitative test was determined by the method of Wang Guoting et al. and Qi Weiliang et al. with minor modifications. The wild type and overexpression CmMYB106 lines of creeping chrysanthemum were treated normally (25℃) and at low temperature (4℃). The 0.5mm thick leaves with the same size and intact without damage were placed in 0.1% DAB and NBT. After 8h of soaking, the reagent was discarded. The leaves were placed in 75% ethanol and washed three times in a 95℃ water bath to fade the color. The completely faded leaves were photographed and the staining was observed.

[0106] 1.7.3 Expression changes of cold resistance related genes of overexpression CmMYB106 lines

[0107] To further study the potential cold resistance mechanism of CmMYB106 overexpression, the gene expression amounts of cold related genes CmICE, CmCBF1, and CmCOR and antioxidant genes CmPOD, CmCAT, and CmSOD were determined. The WT and CmMYB106 overexpression lines with the same growth potential were selected for 4℃ low temperature treatment. The top third leaf was quickly frozen in liquid nitrogen after 3h and stored in a -80℃ ultra-low temperature refrigerator. The specific primers (Table 1) were designed, and the internal reference was CmEF1α. The method was the same as 1.3, and the data was analyzed by 2 -△△CT method.

[0108] 1.8 Data analysis

[0109] All data results of this experiment were arranged, calculated and plotted by software GraPhad Prism 8.3.0, and data processing and variance analysis were performed by SPSS17.0. All experiments were repeated at least 3 times, and the obtained data results were expressed as mean ± standard error (mean ± SEM). p < 0.05 indicated that there was a significant difference between the data results.

[0110] 2 Results and analysis

[0111] 2.1 Cloning of CmMYB106 gene

[0112] Total RNA was extracted from C. morifolium using Trizol method, and gel electrophoresis imaging detection found that there were 2 bands in the image, 28S and 18S bands were obvious, and no obvious diffusion phenomenon was found (Fig. A). It showed that the extracted RNA was not degraded and had high quality, which could be used for subsequent experiments. Figure 1 CmMYB106 gene sequence specific primer CmMYB106-OF / OR was designed (Table 1), and the target gene was amplified by PCR using C. morifolium cDNA as template. Gel imaging instrument was used for observation, and there was a bright specific band in the range of 750-1000bp (Fig. B). It was cut, recovered, and then transferred into E. coli with Solution I and pMD19-T vector. The bacterial liquid was subjected to PCR amplification, and the positive clone bacterial liquid was sent to a sequencing company for sequencing. The full length of CmMYB106 sequence was 948bp, the largest open reading frame was 945bp, and it encoded 315 amino acids.

[0113] Figure 1 B), which was cut, recovered, and then transferred into E. coli with Solution I and pMD19-T vector. The bacterial liquid was subjected to PCR amplification, and the positive clone bacterial liquid was sent to a sequencing company for sequencing. The full length of CmMYB106 sequence was 948bp, the largest open reading frame was 945bp, and it encoded 315 amino acids.

[0114] CmMYB106 gene sequence

[0115] ATGGGGAGATCATCAATTTTCGTTAAGGGTTTGAATAAAGGGTCGTGGACGTCGGAAGAAGATCAAA

[0116] AGCTTGTGACGTACATAAAAGAGCATGGTCATGGAACTTGGCGTACATTGCCTCTTAAGGCTGGGTTG

[0117] CAGAGATGTGGAAAGAGTTGTAGACTGAGATGGACGAACTATCTAAGCCCAAACATCAAAAGAGGA

[0118] ​AAGTTTAGTTTGCAAGAAGAACAAACGATCATCCAACTCCATGCGCTTCTTGGCAATAGATGGTCGAC

[0119] TATTGCAAGGCATTTACCAAAAAGAACAGATAATGAGATCAAGAATTACTGGAACACACATCTTAAGA

[0120] AGCGATTAACCAAAATGGGTATTGATCCAATCACTCACAAGTCAAAAACAGGAGCCAACACCAACGT

[0121] TAGTCATCTCACACAGTGGGAGAATGTTCGTCTCGAAGCAGAAGCTAGATCTGCTCGTGGAGGAAAG

[0122] GTTGTTTCTGACTGTTATAATCAACGAATCGATTTGACAAATATCATCAATAAGTCGTCAATGTCATCTA

[0123] TTTTAGTACCACAGCCGCTATGCTTCGATGTGCTCAAAGGATGGCAAGGTATTAATTCTTGTAAGCTCT

[0124] CCAGTGGGCTCGCGACTCTTAATGACCAAACCATGCCGATTGTGAATGTTGTTGAGAGTGTTGAACCA

[0125] CAAGCTCTAGAAGGAATGACCACAGAATATCATATGAACGACTGGAAAACATCTACAGAGTTTTTTAA

[0126] CACTATTGAAAACCCTAATAACACACTTGAAGTGAGTGACATGATATTTAAAGAGAATTATTTGGCTGA

[0127] GGCTTCTATAAATCATTTGGTTAATGGCGCGAGTAACGGAAACTGCCATGGTGATTTCGAAGACAACA

[0128] TCAGTTATTGGAGAAAAATTCTTGAAAATTTAGGGCATGGTTCATCATTTTCGTCTGTTTTCTAAC mMYB106 protein sequence

[0129] MGRSSIFVKGLNKGSWTSEEDQKLVTYIKEHGHGTWRTLPLKAGLQRCGKSCRLRWTNYLSPNIKRGKF

[0130] SLQEEQTIIQLHALLGNRWSTIARHLPKRTDNEIKNYWNTHLKKRLTKMGIDPITHKSKTGANTNVSHLTQ

[0131] WENVRLEAEARSARGGKVVSDCYNQRIDLTNIINKSSMSSILVPQPLCFDVLKGWQGINSCKLSSGLATLN

[0132] DQTMPIVNVVESVEPQALEGMTTEYHMNDWKTSTEFFNTIENPNNTLEVSDMIFKENYLAEASINHLVN

[0133] GASNGNCHGDFEDNISYWRKILENLGHGSSFSSVF

[0134] 2.2CmMYB106 transcription factor expression pattern analysis

[0135] 2.2.1CmMYB106 transcription factor expression pattern analysis in different parts

[0136] The results showed that CmMYB106 was expressed in all tissues of G. chikunguense, with the highest expression in roots, followed by flowers and leaves, and the lowest in stems. The expression level in roots was 3.58 times higher than that in stems (P < 0.05), indicating that the expression of CmMYB106 gene was tissue-specific. Figure 2

[0137] 2.2.2CmMYB106 transcription factor expression pattern analysis under low temperature conditions

[0138] To analyze the changes in CmMYB106 gene expression pattern under low temperature conditions, wild-type G. chikunguense with the same growth vigor were subjected to low temperature treatment at 4℃, and the leaf RNA was extracted and reverse transcribed into cDNA. The gene expression level of CmMYB106 was quantitatively analyzed by qRT-PCR. As shown in Fig. 2B, the expression level of CmMYB106 in wild-type G. chikunguense under low temperature conditions was significantly higher than that under normal temperature conditions (P < 0.05), indicating that the expression of CmMYB106 gene was induced under low temperature conditions. Figure 3 ​As shown, qRT-PCR results show that under low temperature stress, the expression amount of CmMYB106 gene first increases and then decreases, and under 5°C low temperature condition, the gene expression amount is 1.36 times of that under 25°C. We speculate that CmMYB106 is involved in regulating the cold tolerance of Chamaemelum nobile according to the change amount of CmMYB106 gene under low temperature condition.

[0139] 2.3 Verification of self-activation activity of CmMYB106 transcription factor

[0140] The CmMYB106 gene motif was searched by using the MEME online gene motif analysis website, and three fragments were cut according to the motif distributed on the gene sequence of CmMYB106 for self-activation activity verification. The cutting sites are shown in Figure 4 A. The three fragments are named CmMYB106 1-350 , CmMYB106 1-549 and CmMYB106 1-647 , respectively. The above gene fragments were constructed on the pGBKT7 vector and the plasmid was extracted, which was recombined with the pGBKT7-CmMYB106 plasmid and the pGBKT7 yeast expression empty vector plasmid, and then transformed into the yeast Y187 competent cells, and then cultured on the SD / -Trp / -Leu medium. It was found that the growth state was good, indicating that the recombined plasmid had successfully transformed the yeast competent.

[0141] The transformed bacteria were lightly dropped on the SD / -His,-Ade medium, and after 3 days, it was found that the yeast transformed with pGBKT7-CmMYB106 and pGBKT7-CmMYB106 1-647 plasmids successfully grew colonies, which turned blue under the condition of adding X-α-gal, which was consistent with the growth of the yeast strain transformed with pCL1 (positive control) containing self-activation activity; the colonies transformed with pGBKT7 (negative control) and the CmMYB106 1-350 and CmMYB106 1-549 plasmids were blank, and no strain grew, and the color did not change under the condition of adding X-α-gal. It is shown that CmMYB106 has self-activation activity, and the self-activation activity fragment is located at 549-647 bp of CmMYB106 Figure 4 B).

[0142] 2.4 Obtaining CmMYB106 overexpression strain

[0143] 2.4.1 Obtaining CmMYB106 overexpression strain

[0144] The pORE-R4-35S-CmMYB106 expression vector was transformed into Agrobacterium, and the transformation was confirmed by PCR using specific primers CmMYB106-OF / OR (Table 1). After Agrobacterium infection, the cells were co-cultured for 3 days. Figure 5 A), after five weeks of antibiotic selective culture, callus tissue was obtained. Figure 5 B), seedlings were obtained through screening. Figure 5 C), then transfer it to a rooting medium for further culture. The differentiated buds gradually begin to root, grow into complete plants, and eventually obtain hygromycin-resistant seedlings. Figure 5 D). This experiment ultimately yielded 9 overexpression lines.

[0145] 2.4.2 Validation of CmMYB106 overexpression in groundcover chrysanthemum

[0146] DNA was extracted from groundcover chrysanthemum and the CmMYB106 overexpressing line, respectively. Specific primers HYG-F / R (Table 1) were designed for PCR amplification. Of the nine initially obtained overexpressing lines, only four showed specific bands, and the band positions matched the expected fragment sizes, preliminarily confirming that OX1, OX4, OX5, and OX9 plants were CmMYB106 overexpressing lines. Figure 6 A). To further investigate the transcriptional level of CmMYB106 in the overexpression lines, three well-growing overexpression lines were selected from the four overexpression lines. RNA was extracted from these lines and reverse transcribed to obtain cDNA. Using the cDNA as templates, qRT-PCR was used to detect the expression level of CmMYB106 in these four lines. Data analysis revealed that the expression levels of CmMYB106 in the overexpression lines were all higher than those in the wild-type groundcover chrysanthemum, at 27.97, 33.47, and 44.83, respectively. Among them, OX4 had the highest relative expression level, which was 4.5 times that of the wild-type groundcover chrysanthemum. Figure 6 B).

[0147] 2.5 Cold resistance analysis of CmMYB106 overexpression lines

[0148] 2.5.1 Phenotypic changes in CmMYB106 overexpression lines under low temperature conditions

[0149] To investigate the function of the CmMYB106 gene in groundcover chrysanthemum under low-temperature stress, wild-type groundcover chrysanthemum (WT) with the same growth potential and the screened CmMYB106 overexpressing lines were subjected to low-temperature treatment at four temperatures: 25℃, 10℃, 5℃, and -5℃. Figure 7As shown, with the decrease of temperature, the growth of both wild type and CmMYB106 overexpression lines were inhibited, and the leaves became obviously soft and drooping, and the angle of leaf axil was larger. However, there were differences between the phenotypes of WT and CmMYB106 overexpression lines. It was found that the growth of CmMYB106 overexpression lines was better than that of WT, and the angle of leaf axil of CmMYB106 overexpression lines was smaller than that of WT, and the leaves of CmMYB106 overexpression lines were more straight than those of WT. The wilting degree of WT was more serious.

[0150] 2.5.2 Changes of antioxidant enzymes in CmMYB106 overexpression lines under low temperature

[0151] It was found that the SOD activity of CmMYB106 overexpression lines was significantly higher than that of WT at 25℃. With the decrease of temperature, the SOD activities of both lines increased, but the increase of CmMYB106 overexpression lines was greater. At 5℃, the SOD activity of CmMYB106 overexpression lines reached 386.58 μg·L -1 min -1 , which was increased by 173.53 μg·L -1 min -1 . Although the activity decreased at-5℃, CmMYB106 overexpression lines still maintained a high level. It was found that the change trend of POD activity was similar to that of SOD. With the decrease of temperature, the POD activities of both lines increased significantly, and the POD activity of CmMYB106 overexpression lines was higher than that of WT under all low temperature conditions. Especially at 5℃, the POD activity of CmMYB106 overexpression lines reached 19.25 μg·L -1 min -1 , which was 1.44 times of that of WT. It was found that the CAT activity also showed an increasing trend with the decrease of temperature, and the activity slightly decreased at-5℃ and reached the highest value of 4.23 μg·L -1 min -1 at 5℃. Although the CAT activity of CmMYB106 overexpression lines was slightly higher than that of WT under different temperatures, the overall difference was not as significant as that of SOD and POD, which might be because the CAT activity was complexly regulated by other factors ( Figure 8 ).

[0152] As Figure 9As shown, under low-temperature stress, the MDA content in both WT and CmMYB106 overexpressing plants increased continuously with decreasing temperature. The increase was rapid between 25℃ and 5℃, but slowed down after 5℃. The largest difference in MDA content was observed between the two plants at -5℃, with the WT plant containing 1.29 times more MDA than the CmMYB106 overexpressing plant. The proline content in both WT and CmMYB106 overexpressing plants increased with decreasing temperature between 25℃ and 5℃, then decreased at -5℃. The proline content in CmMYB106 overexpressing plants was higher than that in WT plants under multiple temperature treatments, most significantly at 5℃, where the proline content in CmMYB106 overexpressing plants was 2.84 times that in WT plants. The results showed that the CmMYB106 overexpressing lines were less susceptible to ROS damage than WT lines, further indicating that the CmMYB106 overexpressing plants were more resistant to stress than WT lines, and that the increase of the CmMYB106 gene had a resistance effect to low temperature stress.

[0153] 2.5.3 Under low temperature conditions, overexpression of CmMYB106 strain H2O2 and O2 - Changes in accumulation

[0154] Wild-type groundcover chrysanthemum and CmMYB106 overexpression lines were treated at 4℃. After 3 hours, leaves of similar size, intact and undamaged, were collected for DAB and NBT staining and photographed. At 25℃, both WT and CmMYB106 overexpression lines accumulated small amounts of O2. - ( Figure 10 A) and H2O2 ( Figure 10 B), while O2 under low temperature conditions - The accumulation of O2 and H2O2 increased rapidly compared to the 25℃ treatment condition. However, the O2 accumulation in WT... - The accumulation of H2O2 in the OX4 overexpression line was significantly higher than that in the CmMYB106 overexpression line. - The CmMYB106 overexpression line showed the lowest accumulation of H2O2 and the smallest staining area. The results indicate that the CmMYB106 overexpression line exhibits the lowest H2O2 and O2 accumulation. - The accumulation of the gene is less than that of the wild type. Overexpression of the CmMYB106 gene in ground cover chrysanthemum can enhance the plant's cold resistance.

[0155] 2.5.4 Changes in cold-resistance-related genes in the CmMYB106 strain under low-temperature conditions

[0156] To further investigate the mechanism of potential cold tolerance through CmMYB106 overexpression, we measured the gene expression levels of cold-related genes CmCBF1 and CmCOR, and antioxidant genes CmPOD, CmCAT, and CmSOD. Figure 11) under low temperature stress, the relative expression levels of the antioxidant enzyme-related genes CmCAT, CmPOD and CmSOD of the overexpression plants were increased under low temperature conditions, which were 2.12, 4.75 and 3.85 times of the wild type, respectively. At the same time, the expression levels of the CBF-dependent regulation pathway genes CmCBF1 and CmCOR were also rapidly increased, which were 4.42 and 3.84 times of the wild type, respectively; under normal temperature conditions, the expression levels of the five genes between the WT plants and the CmMYB106 overexpression strain had no significant difference. It is shown that CmMYB106 is jointly involved in the process of responding to low temperature stress by multiple genes.

[0157] The CmMYB106 is cloned from the ground cover chrysanthemum 'Yingjie Hongfei', the expression characteristics, transcription activation activity are analyzed, the CmMYB106 overexpression vector is constructed, the ground cover chrysanthemum is genetically transformed, and the overexpression plants are identified, and the function is verified by low temperature treatment. The following conclusions are drawn:

[0158] 1. The CmMYB106 sequence is cloned from the ground cover chrysanthemum, and bioinformatics analysis finds that the full length of CmMYB106 gene is 948bp, the maximum open reading frame is 945bp, and it encodes 315 amino acids.

[0159] 2. The CmMYB106 expression amount in different organs of the ground cover chrysanthemum is detected by real-time fluorescent quantitative PCR, and it is found that the expression amount in each organ is the highest in the root, which is 3.39; through yeast single hybridization, it is shown that CmMYB106 has self-activation activity, and it is found that the self-activation activity fragment is located on 549-647bp of the CmMYB106 gene sequence.

[0160] 3. The ground cover chrysanthemum is genetically transformed by Agrobacterium mediation, and after screening and identification, four CmMYB106 overexpression strain lines OX1, OX4, OX5 and OX9 are obtained, wherein the CmMYB106 relative expression amount of the OX4 plant is the highest, which is 3.34 times of the wild type ground cover chrysanthemum. Under low temperature treatment (25℃; 10℃; 5℃; -5℃), the CmMYB106 overexpression strain line leaves are emerald green, and the leaf axil angle does not change obviously, while the wild type leaves appear wilting, and the leaf axil angle increases. Through the physiological index data, it is found that the antioxidant enzyme activities (SOD, CAT, POD) of the CmMYB106 overexpression strain line are higher than those of the wild type ground cover chrysanthemum, and the MDA content is lower than that of the wild type. Through DAB and NBT staining, it is found that the H2O2 and O2 -The accumulation amount of anthocyanin in the CmMYB106 overexpression lines was less than that in the wild type plants. Under low temperature conditions, the cold tolerance related genes also changed. The expression amount of CmCBF1 and CmCOR, which were CBF-dependent regulation pathway genes, in the CBF-dependent regulation pathway of the CmMYB106 overexpression line CBF increased by 4.42 and 3.84 times compared with the wild type, which further indicated that CmMYB106 was involved in the regulation of cold tolerance of C. morifolium.

[0161] The above-described embodiments are merely preferred embodiments of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A cold tolerance gene in ground cover chrysanthemum CmMYB106 In the use of enhancing the cold tolerance of ground cover chrysanthemum, wherein, The cold-resistant gene of the ground cover chrysanthemum CmMYB106 The nucleotide sequence of the cold-resistant gene is shown as SEQ ID NO: 1 in the sequence listing.

Images