Cloning and application of chrysanthemum xiangjun grass glycoside synthesis gene cmmyb78

Abstract

This invention discloses a gene for synthesizing luteolin in chrysanthemum. CmMYB78 And its application in increasing the content of luteolin in chrysanthemum. Isolated from chrysanthemum. CmMYB78 The gene, whose nucleotide sequence is shown in SEQ ID NO:1 in the sequence listing, was constructed... CmMYB78 The overexpression vector was used to genetically transform chamomile using Agrobacterium-mediated transformation to obtain... CmMYB78 Overexpression lines. Measurement. CmMYB78 Overexpression lines showed significantly higher luteolin content than wild-type lines at all four flowering stages: budding, whitening, early flowering, and full bloom. This demonstrates... CmMYB78 CmMYB78 CmMYB78 CmMYB78 CmMYB78 CmMYB78 CmMYB78 Cm The gene has the ability to increase the luteolin content of chrysanthemum, providing a new research approach for luteolin synthesis. This has important theoretical and practical significance for revealing the molecular mechanism of luteolin content in chrysanthemum and for cultivating high-quality chrysanthemum varieties.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to... CmMYB78 Gene cloning and its application in increasing luteolin content. Background Technology

[0002] Gongju ( Chrysanthemum × morifolium *Ramat. Tzvel. cv. Gongju* is a perennial herbaceous plant belonging to the genus *Ramat* in the family Asteraceae. Gongju prefers sunlight and is a short-day plant, intolerant of waterlogging and saline-alkali soil, and thrives in well-drained sandy loam. The capitulum of Gongju is used medicinally, and it is one of the four major medicinal chrysanthemums in China. It has a sweet and bitter taste, is slightly cold in nature, and possesses the effects of dispelling wind and clearing heat, calming the liver and improving eyesight, and detoxifying and reducing swelling. Gongju was first introduced from white chrysanthemum in Deqing, Zhejiang Province, to Shexian County, Anhui Province. Gongju has a neat flower shape, with pure white and tightly clustered petals, making it highly ornamental and medicinal, thus a superior variety for both ornamental and medicinal use. Gongju mainly contains flavonoids, volatile oils, and other effective components. Flavonoids play a key role in the pharmacological activity of Gongju. Luteolin is one of the medicinal components of Gongju, possessing various pharmacological activities such as anti-inflammatory, anti-allergic, and anti-tumor effects; its content is one of the indicators for evaluating the medicinal quality of chrysanthemum. The 2020 Pharmacopoeia of the People's Republic of China (Part I) stipulates that chlorogenic acid (C4) in medicinal chrysanthemum is present in a certain amount of chlorogenic acid. 16 H 18 The content of O9) should not be less than 0.20%, and the content of luteolin (C) should not be less than 0.20%. 21 H 20 O 11 The content of ) should not be less than 0.080%, and 3,5-O-dicaffeoylquinic acid (C 25 H 24 O 12 The content of luteolin and other active ingredients in functional chrysanthemums should not be lower than 0.70%. In recent years, against the backdrop of the deepening construction of beautiful new rural areas, multifunctional chrysanthemums such as chrysanthemum 'Gongju' have become a type of specialty crop with great potential, receiving increasing attention and being widely cultivated and utilized. Functional chrysanthemums such as 'Gongju' are mainly propagated by cuttings and division. Long-term use of these propagation methods has led to a series of genetic degradation problems, manifested as decreased product quality, reduced yield, and weakened resistance. The content of effective components such as luteolin in functional chrysanthemums is generally low. This situation has not only become a key factor restricting its quality improvement but also seriously hindered the further development of the functional chrysanthemum industry. Therefore, exploring and analyzing the genes that enhance luteolin content in 'Gongju' is of great significance for increasing the luteolin content of 'Gongju' and promoting the sustainable development of the functional chrysanthemum industry. Summary of the Invention

[0003] The technical problem this invention aims to solve is to provide a gene for regulating luteolin content in chrysanthemum. CmMYB78 Cloning and its applications.

[0004] This invention relates to the luteolin synthesis gene of chrysanthemum.CmMYB78 The nucleotide sequence is shown in SEQ ID NO:1 in the sequence listing.

[0005] Used for cloning the gene regulating the synthesis of osmanthus glycosides in chrysanthemum. CmMYB78 The primer pair, wherein the primer pair is CmMYB78 -qF and CmMYB78 -qR, whose nucleotide sequences are shown in SEQ ID NO:2 and SEQ ID NO:3 in the sequence listing.

[0006] A gene regulating the synthesis of osmanthus glycosides in chrysanthemum CmMYB78 The nucleotide sequences of the quantitative fluorescent primer pairs are shown in SEQ ID NO:4 and SEQ ID NO:5 in the sequence listing.

[0007] A method for building pGBKT7- CmMYB78 The primer pairs for the plasmid have nucleotide sequences as shown in SEQ ID NO:6 and SEQ ID NO:7 in the sequence listing.

[0008] An overexpression vector, by inserting the luteolin synthesis gene into the empty pORE-R4-35SAA vector. CmMYB78 The specific primer HYG-F / R sequence was obtained and is shown in SEQ ID NO:8 and SEQ ID NO:9 in the sequence listing.

[0009] One of the genes regulating the synthesis of osmanthus glycosides in chrysanthemum. CmMYB78 The encoded amino acid, the sequence of which is shown in SEQ ID NO:10 in the sequence listing.

[0010] Chrysanthemum osmanthus glycoside synthesis gene CmMYB78 Application in increasing the content of osmanthus glycosides in plants.

[0011] In the application described in this invention, the chrysanthemum gene... CmMYB78 It can increase the content of osmanthus glycosides in plants.

[0012] In the application described in this invention, the plant is Chrysanthemum indicum.

[0013] The present invention differs from the prior art in that:

[0014] This invention uses chrysanthemum as material to clone... CmMYB78 Genes are clearly identified through their expression patterns and transcriptional activation activities. CmMYB78 Gene expression and other characteristics; constructing overexpression vectors, using Agrobacterium-mediated genetic transformation of chamomile, and analyzing... CmMYB78 The effect of osmanthus glycosides on the content of osmanthus in chrysanthemum provides a theoretical basis for breeding effective substances in functional chrysanthemums.

[0015] The following description, in conjunction with the accompanying drawings, illustrates a gene regulating the synthesis of osmanthus glycosides in chrysanthemum according to the present invention. CmMYB78 The cloning and its applications will be further explained. Attached Figure Description

[0016] Figure 1 In this invention CmMYB78 Cloning correlation diagram of transcription factors; where A is the total RNA extraction diagram of Chrysanthemum morifolium; B is... CmMYB78 Amplification band diagram;

[0017] Figure 2 In this invention CmMYB78 The expression level analysis of different parts of chrysanthemum is shown in the figure; where the error bar represents the standard error, and different letters indicate that there is a significant difference, P < 0.05;

[0018] Figure 3 In this invention CmMYB78 Graph validating transcription factor self-activation activity; where A represents... CmMYB78 Sequence cut-off site; B is CmMYB78 Analysis of the self-activation activity of transcription factors (pCL1 positive control, pGBKT7 negative control).

[0019] Figure 4 The chrysanthemum in this invention ​ A diagram illustrating the growth and development process of overexpressing plants; where A: chrysanthemum leaf disc; B: callus tissue; C and D: rooting culture;

[0020] ​ In this invention ​ Overexpression line identification diagram; where A: detection of overexpression lines at the DNA level; B: overexpression lines in... ​ Expression level; M: DNA Marker DL2000; WT: Wild type; OX5, OX8, OX19, OX21: Overexpression lines; Error bars represent standard error, different letters indicate significant differences. P <0.05;

[0021] ​ In this invention ​ Analysis of luteolin content at different flowering stages in overexpression lines; where A: four flowering stages of wild-type chrysanthemum; B: four flowering stages of overexpression chrysanthemum (from left to right: bud stage, white bud stage, initial flowering stage, and full bloom stage, scale bar = 1 cm); C: luteolin content at different flowering stages of overexpression plants (error bars represent standard error, different letters indicate significant differences). P <0.05); Detailed Implementation

[0022] 1. Materials and Methods

[0023] 1.1 Experimental Materials

[0024] 1.1.1 Strains

[0025] Agrobacterium strain: EHA105; Escherichia coli strain: DH5α; Yeast strain: Y1H.

[0026] 1.1.2 Plant materials

[0027] After sterilization, the explants of chamomile and chrysanthemum seedlings were inoculated into MS medium containing 30 g / L sucrose and 8 g / L agar powder and cultured under the following conditions: 14 h photoperiod and 25 ± 2 ℃ day and night. The resulting sterile seedlings were used as transgenic materials.

[0028] The seedlings of chamomile and chrysanthemum, as well as the overexpression plants obtained later, were cultivated in the teaching base of the College of Agriculture. The cultivation substrate was humus and perlite in a ratio of 1:1. The cultivation conditions were a temperature of 25±2 ℃, a light duration of 16 h, and a humidity of 70%. The seedlings were used to determine the luteolin content after flowering.

[0029] 1.1.3 Experimental primer sequences

[0030] 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 table below:

[0031] Table 1. Primer names and sequences used in this experiment.

[0032] ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0033] 1.2 Cloning of CmMYB78 transcription factor

[0034] 1.2.1 RNA Extraction

[0035] (1) Material preparation: Soak the pipette tips, centrifuge tubes and mortars used in the experiment in distilled water with proteinase K added for 12 h, sterilize them at high temperature and high pressure and then dry them in an oven.

[0036] (2) Collect leaves of chrysanthemum in a rapid growth state and immediately place them in a mortar pre-cooled at -20 ℃. Add liquid nitrogen to quickly freeze the leaves and grind them in the mortar until they become white powder. The grinding process is carried out on ice to ensure that the sample is kept frozen at all times.

[0037] (3) Add Trizol reagent to completely cover the ground powder, let it stand at room temperature, and then continue grinding until it becomes oily. Usually, 1 mL of Trizol reagent is used for 0.2 g of tissue.

[0038] (4) Add the ground solution to a centrifuge tube, let it stand at room temperature for 5 min, and then put it into a refrigerated high-speed centrifuge at 4 ℃ and 12000 rpm for 6 min.

[0039] (5) Take the supernatant and calculate its volume. Add chloroform at one-fifth of the supernatant volume, shake to extract evenly, and then let stand at room temperature for 5 min. Centrifuge at 12,000 rpm for 5 min at 4 ℃. Repeat this step twice.

[0040] (6) Transfer the supernatant of the centrifuged RNA to a new centrifuge tube, record the volume, then add the same volume of isopropanol, gently and evenly invert the tube, and then place it in a -20°C refrigerator to precipitate the RNA.

[0041] (7) Centrifuge at 4℃, 12000 rpm for 10 min, and discard the supernatant.

[0042] (8) Add 1 mL of 75% ethanol solution prepared in advance with DEPC water, gently invert the container to wash the precipitate thoroughly, centrifuge at 12000 rpm for 10 min at 4℃, and discard the supernatant. Then air dry the RNA precipitate.

[0043] (9) Dissolve the RNA completely in an appropriate amount of RNase-free water or DEPC-treated water.

[0044] (10) Measure the concentration of the dissolved RNA sample and store it in a freezer at –80°C for use in subsequent experiments.

[0045] 1.2.2 Reverse transcription to synthesize cDNA

[0046] cDNA was synthesized by reverse transcription using the FastKing One-Step Genomic cDNA First-Strand Synthesis Kit (TIANGEN). Reaction system: 4 μL 5 × FastKing-RT SuperMix, 1 μL RNA, 15 μL RNase-Free ddH2O, total volume 20 μL. Reaction conditions: 42℃ for 15 min, 95℃ for 3 min; store at –20℃ for later use.

[0047] 1.2.3 Obtaining and Amplifying the CmMYB78 Gene Sequence

[0048] (1) PCR amplification of the target gene

[0049] The CDS sequence of the CmMYB78 gene was searched in the Chrysanthemum genome database, and specific gene amplification primers were designed using Primer 5.0 software. ​ -qF / qR (Table 1), using chrysanthemum cDNA as a template for...​ Gene amplification. The PCR reaction system was as follows: 10 × PCR Buffer: 2.5 μL, MgCl2 (25 mM): 1.5 μL, dNTP (2.5 mM): 2 μL, Primer F (10 μM): 1.0 μL, Primer R (10 μM): 1.0 μL, rTaq (2.5 U / μL): 0.2 μL, cDNA: 1.0 μL, ddH2O: 15.8 μL. Reaction conditions: 95℃ for 5 min, 95℃ for 30 s, 58℃ for 30 s, 72℃ for 60 s, for 35 cycles, 72℃ for 7 min, and stored at 4℃.

[0050] (2) Recovery of PCR products

[0051] Weigh an appropriate amount of agarose and add it to 0.5 × TBE electrophoresis buffer. Heat the mixture, rinse with cold water to cool it down, add EB substitute, and then cool it to a suitable temperature to solidify it into a gel. After spotting the sample, perform gel electrophoresis. After color development, cut the specific fragment with a blade and recover the target fragment using an agarose gel recovery kit. First, under gel imaging, cut the fluorescent target fragment with a blade, trying to avoid including excess agar. Place it in a 1.5 mL centrifuge tube, add 3 gel volumes (0.1 g = 100 μL) of Buffer DE-A solution, mix by inverting, and then heat in a 65 ℃ metal bath (inverting every 2 min). After complete melting, add 0.5 volumes of Buffer DE-A solution of Buffer DE-B solution and mix by inverting. Add the mixture to a 2 mL centrifuge tube containing the matching preparation tube, centrifuge at 12000 rpm for 1 min, and discard the filtrate. Add 500 μL of Buffer W1 solution, centrifuge at 12000 rpm for 1 min, and discard the filtrate. Add 700 μL of Buffer W2 solution, centrifuge at 12000 rpm for 1 min, and discard the filtrate. Repeat this step once. After discarding the filtrate, centrifuge again at 12000 rpm for 2 min. Transfer the centrifuged preparation tube to a new 1.5 mL centrifuge tube, and add 25-30 drops of agar solution to the center of the preparation tube. Add μL of Eluent H2O preheated to 65 ℃, let stand at room temperature for 5 min, centrifuge at 12000 rpm for 1 min, elute once more to increase the concentration of the gel recovery product, and store at -20 ℃ for later use.

[0052] (3) Connection transformation

[0053] The target fragment obtained from the gel extraction was ligated into the pMD19-T vector. The reaction mixture consisted of: Solution I 2.5 μL, pMD19-T 0.5 μL, and gel extraction product 2 μL (1000 ng). The reaction program was: 16 ℃ for 2 h, followed by storage at 4 ℃. The ligated product was added to 100 mL of E. coli DH5α competent cells and completely immersed in an ice bath for 30 min. After the ice bath, the cells were immediately removed and heat-shocked in a 42 ℃ water bath for 90 s. After the heat shock, the cells were ice-shocked for 5 min, ensuring complete immersion in the ice bath. Finally, 700 μL of LB medium was added, and the mixture was incubated at 37 ℃ with shaking for 1.5 h, followed by centrifugation at 12000 rpm for 1 min. A small amount of supernatant was reserved to resuspend the precipitate, which was then spread onto LB agar plates containing ampicillin (Amp) and incubated at 37 ℃.

[0054] (4) Detection

[0055] After the bacteria have grown on the plating, select single colonies, add 700 mL of LB + Amp liquid medium, and incubate at 37℃ with shaking for 3 h. Perform PCR detection, and send a portion of the bacterial solution that shows the corresponding target fragment to the company for testing.

[0056] 1.3 ​ Spatial expression patterns of genes

[0057] RNA was extracted from the roots, stems, leaves, and flowers of *Chrysanthemum indicum* at the same stage of growth, reverse transcribed into cDNA, and stored at -20°C for later use. According to... ​ Sequence-designed quantitative PCR primers qRT-MYB78-F / qRT-MYB78-R (Table 1) were used to detect expression levels in different tissues, with EF1α as the internal reference gene (Table 1).

[0058] The qPCR reaction system is as follows:

[0059] ​ 10.0 ​ 1.6 ​ 1.0 ​ 1.0 ​ 5.0 <![CDATA[ddH2O]]> 1.4 ​ 20.0

[0060] The reaction program was set as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 4℃ cycling; melting curves were selected for 60-95℃; data analysis method was 2 -△△CT Methodological analysis. Experimental data are expressed as mean ± standard error (Mean ± SE). ΔCT (target gene) = CT (target gene) - CT (EF1α), ΔΔCT = ΔCT - ΔCT (maximum).

[0061] 1.4 ​ Assay of transcription factor self-activation activity

[0062] (1) Use BioXM 2.6 software to find enzyme cleavage sites. ​ I and ​ I. Design specific primers CmMYB78NdeI-F and CmMYB78BamHI-R (Table 1) to perform PCR amplification and introduce the restriction sites. ​ Gene fragments were extracted and recovered by agarose gel electrophoresis, and specific fragments were collected.

[0063] (2) Double enzyme digestion, ligation transformation and verification are the same as in 1.2.3, except that the ligation vector is pGBKT7.

[0064] (3) The successfully constructed pGBKT7-CmMYB78 plasmid, pGBKT7 plasmid (negative control) and PCL1 plasmid (positive control) were transformed into competent cells of yeast strain Y187. The yeast culture containing pGBKT7-CmMYB78 plasmid and pGBKT7 plasmid were spread on SD / Trp- (Trp: tryptophan) solid medium, and the yeast culture containing PCL1 plasmid was spread on SD / Leu- (Leu: leucine) solid medium. The cultures were incubated at 30 ℃ for 2-3 days.

[0065] (4) Pick a single clone of bacterial culture, shake it to culture, spot it on SD / Ade- / His- (Ade: adenine, His: histidine) medium containing X-α-gal (X-α-galactosidase), and incubate at 30 ℃ for 3-4 days. Observe the growth status of the colony.

[0066] 1.5 ​ Screening of non-self-activating active fragments

[0067] Using the MEME (http: / / meme-suite.org / ) online gene motif analysis website, input... ​ The gene sequence is used to locate specific motif positions on gene fragments, thereby identifying cutoff sites. Based on the motif positions, three cutoff sites were selected at 561 bp, 693 bp, and 825 bp, and named as follows: ​ 1-561 , ​ 1-693 and ​ 1 -825 Specific primers were designed and ligated into the pGBKT7 vector after enzyme digestion to verify transcriptional activation activity, using the same method as in 1.4. Finally, fragments without transcriptional activation activity were screened by observing colony growth for subsequent experiments.

[0068] 1.6 Genetic transformation and overexpression of chamomile ​ Identification of strains.

[0069] (1) Use BioXM 2.6 software to find enzyme cleavage sites. ​ I and ​ HI, design specific primers (Appendix 1), and perform PCR amplification to introduce the restriction enzyme sites. ​ Gene fragments were extracted and recovered by agarose gel electrophoresis, and specific fragments were collected.

[0070] (2) Double digestion of the pORE-R4-35SAA empty vector and the introduction of restriction enzyme sites ​ Gene fragment. The double enzyme digestion reaction system is as follows:

[0071] ​ 10 ​ 5 ​ 2.5 ​ 2.5 <![CDATA[ddH2O]]> 30.0 ​ 50.0

[0072] The reaction program was set as follows: 37℃ for 3 hours; 65℃ for 20 minutes. The target fragment was collected by agarose gel electrophoresis and stored at -20℃.

[0073] The digested vector and target fragment were ligated using T4 DNA ligase. Ligation system: 1 μL digested gene fragment, 2.5 μL T4 DNA ligase, 1 μL digested pORE-R4-35SAA empty vector, and 0.5 μL ddH2O to a final volume of 5 μL. Reaction conditions: 16℃ for 13 h, then stored at 4℃. Transformation was performed as described in 1.2.3.

[0074] Plasmids were extracted from the successfully sequenced bacterial culture and subjected to double enzyme digestion for verification. The reaction system and procedure were the same as those for the double enzyme digestion system. Agarose gel electrophoresis was used to observe the band size to determine if ligation was successful.

[0075] 1.6.1 Identification of overexpression lines at the DNA level

[0076] (1) First, prepare the extraction solutions: Extraction solution I (50 mL) is prepared by taking 2.685 g of sorbitol, 5 mL of Tris-HCl (1 mol / L), 1 mL of EDTA (0.25 M), and 35 μL of β-mercaptoethanol, and adding distilled water to make up to 50 mL; Extraction solution II (50 mL) is prepared by taking 10 mL of Tris-HCl (1 mol / L), 10 mL of EDTA (0.25 M), 5.85 g of NaCl, and 1 g of CTAB, and adding distilled water to make up to 50 mL. The prepared extraction solutions are sterilized by high temperature and high pressure for 20 min and stored at room temperature for later use.

[0077] (2) DNA was extracted using the CTAB method. 0.3 g of leaves from wild-type and overexpressing plants were placed in a mortar and ground with liquid nitrogen to break the cells and release DNA. 1.5 mL of extraction solution I and 20 μL of β-mercaptoethanol were added and mixed thoroughly. The mixture was then centrifuged at 12,000 rpm and 4 °C for 6 min and the supernatant was discarded.

[0078] (3) Add 400 μL of extraction solution I and 200 μL of extraction solution II (preheated to 65℃) to a centrifuge tube, mix well and resuspend the precipitate. Add 120 μL of 5% sodium dodecyl sarcosinate, place in a constant temperature water bath and adjust the temperature to 65℃, invert once every 10 min, and heat for 30 min.

[0079] (4) After heating, add 600 μL of chloroform-isoamyl alcohol mixture (mixing volume ratio of 24:1), shake for 15 min, centrifuge at 12000 rpm and 4 ℃ for 6 min, and transfer the supernatant to a new centrifuge tube. Repeat the extraction 3 times, add 600 μL of isopropanol (pre-cooled at –20℃), gently invert, stand at –20℃ for 20 min, and centrifuge at 12000 rpm and 4 ℃ for 6 min.

[0080] (5) Remove the supernatant, rinse with 70% ethanol by inversion, centrifuge at 12000 rpm, 4℃ for 6 min, and wash twice. Discard the supernatant, air dry at room temperature, dissolve in RNase-free water, and store at –20℃.

[0081] (6) Design specific primers HYG-F / R (Table 1) containing the overexpression vector fragment. Use the DNA of the overexpression line as a template for PCR amplification, with wild-type DNA as a control. The system and procedure are the same as in 1.2.3. After amplification, perform gel electrophoresis to detect the presence of bands.

[0082] 1.6.2 Identification of overexpression lines at the RNA level

[0083] (1) Select the overexpression line cultured for 30 days and the tissue culture seedlings of wild chamomile. Cut 0.3 g of leaves with scissors to extract their RNA. The extraction method is the same as in 1.2.1. Reverse transcribe to obtain the corresponding cDNA and store at -20℃ for later use.

[0084] (2) Using the extracted cDNA as a template, perform real-time PCR detection, following the same method as 1.3, 2. -△△CT Data were analyzed to identify overexpressing plants.

[0085] 1.7 ​ Determination of osmanthus glycoside content in overexpression lines

[0086] (1) Sample collection: Wild-type plants with the same growth status were selected and ​ After the plants were overexpressed, samples were collected after flowering. Flowers were collected at four different stages: bud stage, white bud stage, early flowering stage, and full bloom stage. After drying in an oven, the flowers were ground in a mortar and pestle and stored at -20℃ for later use.

[0087] (2) Preparation of luteolin standard curve: Weigh 6.0 mg of luteolin standard (Shanghai Guchen), add 4.0 mL of 70% chromatographic methanol solution, and sonicate at 60℃ (40 kHz 120 W) for 40 min. After complete dissolution, dilute to 4 mL to obtain a standard stock solution concentration of 1.5 mg / mL. Take 2 mL, 1.6 mL, 1.2 mL, 0.8 mL, and 0.4 mL of the standard stock solution sequentially and dilute to 2 mL to obtain a standard concentration gradient of 1.5 mg / mL, 1.2 mg / mL, 0.9 mg / mL, 0.6 mg / mL, and 0.3 mg / mL. Filter through a syringe filter (13 mm, 0.22 μm) and add to the sample vial.

[0088] (3) Sample solution preparation: Weigh 0.1 g of sample powder from the wild type and the overexpression line at four flowering stages, add 10 mL of 70% chromatographic methanol solution, and sonicate at 60℃ (40 kHz 120 W) for 40 min. After complete dissolution, bring the volume to 10 mL. Filter through a syringe filter (13 mm, 0.22 μm) and add to the sample vial.

[0089] (4) Analysis of luteolin content: Five standard reference solutions and the sample solution were analyzed using a high-performance liquid chromatograph (Agilent 1260). Chromatographic conditions followed the method for determining luteolin content in chrysanthemum according to the Pharmacopoeia of the People's Republic of China (Part I): detection wavelength 350 nm, column temperature 40 ℃, and injection volume 5 μL. Peak areas were recorded, and a standard curve was plotted with the concentration of each standard reference solution as the ordinate and the peak area as the abscissa. The linear relationship between luteolin content and peak area was obtained as: y = 5 × 10⁻⁶. -5 X + 0.023 (R) 2 =0.987), and this was used to calculate the luteolin content in the sample to be tested.

[0090] 1.8 Data Analysis

[0091] All data results in this experiment were processed, calculated, and plotted using GraPhad Prism 8.3.0 software. SPSS 17.0 was used for data processing and analysis of variance. All experiments were repeated at least three times. The results are expressed as mean ± standard error (mean ± SEM). p < 0.05 indicates a significant difference between the data results.

[0092] 2 Results and Analysis

[0093] 2.1 ​ Cloning of genes

[0094] Total RNA was extracted from Chrysanthemum morifolium using the Trizol method, and RNA quality was assessed by gel electrophoresis imaging. Two bands were observed in the image (…). ​ A) The first band corresponds to the large 28S ribosomal RNA, and the second band corresponds to the small 18S ribosomal RNA. The clear bands without tailing indicate high RNA quality suitable for subsequent experiments. According to... ​ Primers designed for sequence analysis were used to perform PCR amplification with chrysanthemum cDNA as a template. After agarose gel electrophoresis, a clear band was observed between 750 bp and 1000 bp using a gel imaging system. ​ B), after gel extraction and recovery, the bacteria were ligated into the pMD19-T vector using Solution I and then transformed into E. coli. After colony identification, the bacteria were sent to a sequencing company for sequencing. ​ The largest open reading frame is 951 bp, encoding 317 amino acids.

[0095] ​ gene sequence

[0096] GTGCATGGAACATAAGATGACCATTAGATGTATTGACATGATCAGTAGTGGTGGTGAAGAAGATGATCAAATGAATATGATGATAGATGATCTTC

[0097] GAAGAGGTCCATGGACTGTTGAAGAAGACTTCGCTCTTATGAACTACATCGCTCATCATGGCGAAGGCCGATGGAACTCTCTAGCTCGTTGTG

[0098] CCGGGCTCAAGAGAACCGGAAAGAGCTGCAGATTGCGGTGGCTAAATTATCTTCGCCCTGATGTTCGTCGTGGAAATATTACTCTTGAAGAAC

[0099] AACTCTTGATTCTTGAACTTCATTCTCGCTGGGGCAATCGTTGGTCAAAAATAGCACAACACTTGCCAGGAAGAACTGACAACGAGATAAAAA

[0100] ATTATTGGAGAACACGAGTTCAAAAGCACGCTAAACAACTCAAATGCGACGTTAATAGCAAGCAATTTAAAGACACGATGCGTTATCTTTGGAT

[0101] GCCACGACTTGCTGAGCGTATTCAAGCAGCTAATTCCACCACAACCGTAGGTTCGTTTTCCATATCATCGACAGACATCAAGACCACCACTAC

[0102] AACGTATCCATTTGATCAAAATAATATACAAAATGTTGGTACAACCCAATCGGTTATTCCCAACACTTATTCCAGTGATCATGCGAATAACCATAAT

[0103] TACACGCGGGATACTTCAAGGCACACTGCGGTTTCACCGGTGTCTGACTTGACTGATTGCTACTATCCTATCAGTGAGAGCCAAAACCAAAAT

[0104] TTCTTACAAATTAACAACACAGTTGGTGACGAGTTCGATTTTCCAATAATCAGCCCCTCGGCTTACTTCAACCGAGAGATGGATTTTGAAGGAC

[0105] AGATGGAGGAAAACAACCACTGGTCGGGCGAAAATAATGGAGATTTTTCGGACAGTTTGTGGAATGTTGAGGACATATTATATCTTGAACAACA

[0106] TTTTAACAGCATGTAATGATCG

[0107] CmMYB78 protein sequence

[0108] MEHKMTIRCIDMISSGGEEDDQMNMMIDDLRRGPWTVEEDFALMNYIAHHGEGRWNSLARCAGLKRTGKSCRLRWLNYLRPDVRRGNITLEEQL

[0109] LILELHSRWGNRWSKIAQHLPGRTDNEIKNYWRTRVQKHAKQLKCDVNSKQFKDTMRYLWMPRLAERIQAANSTTTVGSFSISSTDIKTTTTTYPF

[0110] DQNNIQNVGTTQSVIPNTYSSDHANNHNYTRDTSRHTAVSPVSDLTDCYYPISESQNQNFLQINNTVGDEFDFPIISPSAYFNREMDFEGQMEENN

[0111] HWSGENNGDFSDSLWNVEDILYLEQHFNSM

[0112] 2.2 ​ Transcription factor expression pattern analysis

[0113] RNA was extracted from the roots, stems, leaves, and flowers of *Chrysanthemum indicum* and reverse transcribed into cDNA. The cDNA was then detected in different tissues of *Chrysanthemum indicum* using qRT-PCR. ​ Gene expression levels were discovered. ​ Gene expression levels varied significantly across different parts of the chrysanthemum plant, with the highest expression in the flower, followed by the leaf, and the lowest expression in the stem and root. The expression level in the flower was 4.1 times that in the stem. ​ ),illustrate ​ Gene expression is tissue-specific.

[0114] 2.3 ​ Validation of transcription factor self-activation activity

[0115] Use the MEME online gene motif analysis website to search ​ Gene motifs, based on their distribution in ​ Three fragments were extracted from the gene sequence for self-activation activity verification. The extraction sites are shown below. ​ A. The three segments are as follows: ​ 1 -561 , ​ 1-693 and ​ 1-825 Combine the above gene fragments with ​ The gene was constructed in the pGBKT7 vector and successfully transferred into pGBKT7- ​ Yeast cultures containing plasmids, the empty pGBKT7 vector, and the PCL1 plasmid were spotted onto SD / Ade- / His- medium containing X-α-gal. After culturing for 3-4 days, it was found that the cultures transformed into pGBKT7-... ​ Yeast colonies can grow normally on SD / Ade- / His-deficient media, and turn blue on X-α-gal-deficient media, showing the same growth pattern as PCL1 (positive control). However, yeast colonies transformed with the pGBKT7 empty vector plasmid (negative control) do not grow or turn blue on X-α-gal-deficient media, indicating that... CmMYB78 Transcription factors have self-activating activity ( Figure 3B).

[0116] Experiments were conducted to screen for non-self-activating fragments. pGBKT7- was extracted. CmMYB78 1-561 pGBKT7- CmMYB78 1 -693 and pGBKT7- CmMYB78 1-825 The plasmid was transformed into competent yeast Y187 cells. After spotting and culturing for 3-4 days, the colony growth was observed. It was found that the plasmid transformed into pGBKT7-... CmMYB78 1-561 and pGBKT7- CmMYB78 1-693 The plasmid colonies failed to grow normally on SD / Ade- / His- medium and did not turn blue on X-α-gal-deficient medium, consistent with the growth status of pGBKT7 (negative control), indicating that these two sequences lacked self-activation activity. Transfection with pGBKT7-... CmMYB78 1-825 The plasmid colonies grew normally on SD / Ade- / His- medium, and turned blue on X-α-gal-deficient medium, consistent with the growth status of PCL1 (positive control), indicating that the site with self-activation activity is between 693 bp and 825 bp. Figure 3 B).

[0117] 2.4 CmMYB78 Obtaining and validating overexpression lines

[0118] 2.4.1 CmMYB78 overexpression lines obtained

[0119] Chamomile was genetically transformed using Agrobacterium infection, and pORE-R4- was introduced. CmMYB78 Agrobacterium bacterial suspension was used to infect chamomile leaves. After infection, the leaves were co-cultured for one week and then transferred to a selection medium. Figure 4 A), after one month, adventitious buds differentiated ( Figure 4 B), after another 2 weeks of cultivation, rooting culture can be carried out ( Figure 4 (C and D) The transformed plants were cultivated and propagated by cuttings to initially obtain 22 strains.

[0120] 2.4.2 Overexpression CmMYB78 Strain verification

[0121] Using Agrobacterium infection method CmMYB78After being transferred into chamomile, 22 overexpression lines were obtained through co-culture, selection culture, and rooting culture. DNA was extracted from the leaves of the overexpressing plants, and PCR amplification was performed using HYG-F / R primers as templates. Ultimately, only 4 overexpression lines showed corresponding specific bands, which were preliminarily identified as OX5, OX8, OX19, and OX21. Figure 5 A). To further investigate the overexpression lines... CmMYB78 Expression levels were determined by reverse transcription of RNA from four overexpression lines, followed by detection of cDNA levels using qRT-PCR. CmMYB78 The expression level was found in overexpression lines. CmMYB78 The expression levels of all strains were higher than those of the wild type. Among them, the highest expression level in the OX5 strain was 6 times that of the wild type, followed by OX21 and OX19, which were 5.5 times and 5.4 times that of the wild type, respectively. The lowest expression level in OX8 was 1.6 times that of the wild type. Figure 5 B).

[0122] 2.5 CmMYB78 Analysis of luteolin content in overexpression lines

[0123] Flowers were collected from wild-type and overexpression plants at four flowering stages: bud stage, white bud stage, initial flowering stage, and full bloom stage. Figure 6 A represents the wild type and 6B represents the overexpression lines. HPLC analysis of luteolin content at different flowering stages in OX5, OX19, and OX21 overexpression plants revealed that the luteolin content in the overexpression lines was significantly higher than that in the wild type at all four flowering stages. The highest luteolin content was observed at the initial flowering stage, followed by the full bloom and budding stages, with the lowest content at the bud stage. The luteolin content in the OX5 line was higher than that in other lines at all flowering stages, reaching a maximum of 2.3 mg / g at the initial flowering stage, which was 2.3 times that of the wild type. Figure 6 C). CmMYB78 Gene overexpression significantly increased the content of luteolin in flowers, indicating that... CmMYB78 It participates in the synthesis of luteolin in chamomile.

[0124] This invention clones from chrysanthemum. CmMYB78 Expression characteristics and transcriptional activation activity were analyzed, and a [system / mechanism] was constructed. CmMYB78 An overexpression vector was used to genetically transform chamomile. The overexpressing plants were identified, and their function was verified by measuring the luteolin content. The following conclusions were drawn:

[0125] 1. Obtained by cloning from chrysanthemum. CmMYB78 Sequence, bioinformatics analysis revealed CmMYB78 The largest open reading frame is 951 bp, encoding 317 amino acids.

[0126] 2. Detection of different organs of Chrysanthemum morifolium using real-time quantitative PCR. CmMYB78 Expression levels were found in all organs, with the highest expression level observed in flowers; yeast one-hybrid assays showed... CmMYB78 It exhibits self-activating activity, and the self-activating active fragment was found to be located at... CmMYB78 On the 693 bp-825 bp of the gene sequence.

[0127] 3. Through Agrobacterium-mediated genetic transformation of chamomile, four strains, OX5, OX8, OX19, and OX21, were obtained after screening and identification. CmMYB78 Among the overexpression lines, the OX5 line showed the highest expression level, being 6 times that of the wild type, followed by OX21 and OX19, which were 5.5 and 5.4 times higher than the wild type, respectively. HPLC analysis of luteolin content at different flowering stages in OX5, OX19, and OX21 overexpressing plants revealed that the luteolin content in the overexpressing lines was significantly higher than that in the wild type at all four flowering stages. The highest luteolin content was observed at the initial flowering stage, followed by the full bloom and budding stages, with the lowest content at the bud stage. The luteolin content in the OX5 line was higher than that in other lines at all flowering stages, reaching a maximum of 2.3 mg / g at the initial flowering stage, which was 2.3 times that of the wild type. This also indicates... CmMYB78 It participates in the regulation of luteolin synthesis.

[0128] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A gene for synthesizing luteolin in chrysanthemum. CmMYB78 Its features are: Chrysanthemum luteolin synthesis gene CmMYB78 The nucleotide sequence is shown in SEQ ID NO:1 in the sequence listing.

2. A gene for synthesizing luteolin as described in claim 1. CmMYB78 Application in increasing luteolin content.

3. The application according to claim 2, characterized in that, The gene CmMYB78 It can increase the content of luteolin in chrysanthemum.

4. A gene for synthesizing luteolin as described in claim 1. CmMYB78 Encoded protein ， The characteristic feature is that the encoded protein has an amino acid sequence as shown in SEQ ID NO:10 in the sequence listing.

5. Amplifying the luteolin-synthetic gene of chrysanthemum as described in claim 1 CmMYB78 primers ， Its characteristic is that its nucleotide sequence is shown in SEQ ID NO:2 and SEQ ID NO:3 in the sequence listing.

6. An overexpression vector, characterized in that, The vector is formed by inserting the chrysanthemum glycoside synthesis gene as described in claim 1 into an empty pORE-R4-35SAA vector. CmMYB78 get.

7. Constructing the overexpression vector as described in claim 6, characterized in that... The specific primer HYG-F / R sequences are shown in SEQ ID NO:8 and SEQ ID NO:9 in the sequence listing.

8. A method for increasing the content of luteolin in chrysanthemum, characterized in that, Improved CmMYB78 Gene expression levels.

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