Chrysanthemum gene cmbhlh104, encoding protein and use in plant drought improvement

Abstract

The application discloses a chrysanthemum gene CmbHLH104, a coding protein and application in plant drought resistance improvement, and belongs to the technical field of genetic engineering.The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the coded protein is shown as SEQ ID NO.2.The application obtains a chrysanthemum 'Fall Color' transgenic strain with higher drought tolerance, and under the drought and water loss stress, the chrysanthemum CmbHLH104 gene can rapidly respond and enhance the drought resistance of the plant, thereby providing a theoretical basis and utilization value for improving the drought resistance and adaptability of other plants and breeding by using the gene.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a chrysanthemum gene CmbHLH104, its encoded protein, and its use in improving plant drought resistance. Background Technology

[0002] Drought, as a significant abiotic stress affecting plant metabolism and limiting plant growth and development, leads to a series of socio-environmental problems, including further deterioration of the ecological environment, hindering industrial, agricultural, and pastoral production, and exacerbating desertification. Simultaneously, drought stress severely impacts plant growth and ornamental value, becoming a limiting factor for the application of many plants. Current plant adaptation mechanisms have evolved to alleviate drought stress. Drought-resistant plants can respond to drought stress through their own resistance mechanisms and complex regulatory networks, enabling them to quickly signal, respond, and appropriately adapt to water deficit problems. Therefore, the breeding and application of drought-resistant and water-saving transgenic plant varieties can effectively improve agricultural production efficiency. Transcription factors (TFs) are a class of trans-acting factors that can directly regulate gene expression. They participate in plant responses to drought stress by regulating plant hormone regulation and synthesis, plant tissue development and growth, and abiotic stress mechanisms. The transcription factor family plays a crucial role in many plant abiotic stresses; among them, NAC, WRKY, bHLH, and MYB have been shown to enhance stress tolerance by stimulating stress response genes.

[0003] bHLH transcription factors are named for their highly conserved structural features. Each bHLH transcription factor possesses a conserved domain containing approximately 50-60 amino acids (aa), consisting of two conserved domains: a basic region and a helix-loop-helix (HLH) region. The basic region is the DNA-binding region, specifically recognizing E-box and G-box motifs; the HLH region, located at the C-terminus, regulates the expression of downstream target genes. bHLH transcription factors are one of the most widespread transcription factor superfamilies in eukaryotes and the second largest transcription factor family in plants, playing crucial roles in plant growth, development, secondary metabolism, and responses to environmental stress. Numerous studies have shown that bHLH transcription factors are involved in key processes in plant responses to drought stress, such as stomatal development, root hair formation, and epidermal trichome formation.

[0004] Chrysanthemums are plants with high ornamental and economic value due to their beauty, color, and fragrance. Growing worldwide, they are ornamental plants with global application value. Their growth and productivity are susceptible to many abiotic and biotic stresses, among which drought is a significant stressor severely affecting chrysanthemum quality, yield, and geographical distribution. 'Fall Color' is a new type of chrysanthemum variety cultivated from wild chrysanthemums through a "wild breeding" method. It is characterized by superior ornamental value, strong stress resistance, and tolerance to extensive management. It possesses characteristics such as drought resistance, cold resistance, rich flower colors, good ornamental value, strong coverage, easy propagation, low maintenance requirements, and strong adaptability, making it an excellent drought-resistant plant material. Currently, the discovery and molecular mechanism research of drought-response genes in the ground cover chrysanthemum 'Fall Color' are relatively scarce. Therefore, discovering drought-resistant genes in chrysanthemums and elucidating the molecular regulatory mechanisms of drought resistance has considerable potential value for developing new water-saving chrysanthemum germplasm resources. Summary of the Invention

[0005] To address the aforementioned shortcomings in the prior art, this invention provides a chrysanthemum gene CmbHLH104, its encoded protein, and its use in improving plant drought resistance. During periods of drought and water loss, this gene can rapidly respond to and adapt to stress, thereby enhancing the drought resistance of plants.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0007] A chrysanthemum gene, CmbHLH104, was cloned from the RNA of chrysanthemum 'Fall Color' leaves by PCR. The core coding region is 675 bp in length, and its sequence is shown in SEQ ID NO.1.

[0008] Furthermore, the gene is a gene sequence that has more than 80% homology with the sequence described in SEQ ID NO.1 and encodes a protein with the same function.

[0009] The protein encoded by the chrysanthemum gene CmbHLH104 has an amino acid sequence as shown in SEQ ID NO.2, consisting of 224 amino acids with a molecular weight of 54.746 kDa and a theoretical isoelectric point (pI) of 5.16. It contains a single nuclear localization signal: “EKECSRKRGR”. Predictive analysis of the protein's amino acid sequence revealed two structural features: a single nuclear localization signal and a typically conserved bHLH domain. Furthermore, the bHLH domain of CmbHLH104 is highly consistent with that of its homologous protein, bHLH104. This characteristic is consistent with the evolutionary characteristics of bHLH proteins, placing CmbHLH104 in the bHLH IVc subfamily.

[0010] A plasmid containing the chrysanthemum CmbHLH104 gene mentioned above.

[0011] A recombinant expression vector containing the aforementioned chrysanthemum CmbHLH104 gene.

[0012] Transgenic cell lines containing the aforementioned chrysanthemum CmbHLH104 gene.

[0013] Engineered bacteria containing the aforementioned chrysanthemum CmbHLH104 gene.

[0014] A gene chip comprising the aforementioned chrysanthemum gene CmbHLH104.

[0015] The above-mentioned chrysanthemum gene CmbHLH104 is used in screening or identifying drought-resistant varieties.

[0016] The above-mentioned chrysanthemum gene CmbHLH104, plasmid, recombinant expression vector, transgenic cell line, engineered bacteria or gene chip are used in the improvement of drought resistance-related qualities or germplasm resources of chrysanthemum, or in the cultivation of transgenic chrysanthemums.

[0017] A formulation for enhancing the drought resistance of chrysanthemum, wherein the active ingredient is a component that promotes the expression of the chrysanthemum gene CmbHLH104.

[0018] Furthermore, the active ingredient is a small molecule compound, shRNA, gRNA, or short peptide.

[0019] The beneficial effects of this invention are:

[0020] This invention has obtained a transgenic chrysanthemum line 'Fall Color' with high drought tolerance. At the same time, under drought and water loss stress, the chrysanthemum CmbHLH104 gene can respond rapidly, enhancing the plant's drought resistance. This provides a theoretical basis and application value for using this gene to improve the drought resistance of other plants and for breeding. Attached Figure Description

[0021] Figure 1 The homologous sequence alignment results of the chrysanthemum CmbHLH104 gene of this invention;

[0022] Figure 2 This is the phylogenetic tree of the chrysanthemum CmbHLH104 gene of the present invention;

[0023] Figure 3 This is the result of the subcellular localization of the chrysanthemum gene CmbHLH104 in this invention;

[0024] Figure 4 Positive identification results for plants transgenic with the CmbHLH104 gene;

[0025] Figure 5 The growth status of wild-type and transgenic 'Fall Color' plants after drought stress treatment;

[0026] Figure 6 Determination of water loss rate in wild-type and transgenic 'Fall Color' plants;

[0027] Figure 7 Determination of proline content in wild-type and transgenic 'Fall Color' plants;

[0028] Figure 8 Stomatal measurements were performed on wild-type and transgenic 'Fall Color' plants; where a is a diagram showing the stomatal shape of wild-type and transgenic 'Fall Color' plants; and b is a diagram showing the number of stomata in wild-type and transgenic 'Fall Color' plants.

[0029] Figure 9 Chlorophyll content was measured in wild-type and transgenic 'Fall Color' plants;

[0030] Figure 10 Determination of MDA content in wild-type and transgenic 'Fall Color' plants under stress;

[0031] Figure 11 Determination of soluble protein content in wild-type and transgenic 'Fall Color' plants under stress;

[0032] Figure 12 For wild-type and transgenic 'Fall Color' plants, superoxide anion (O3) levels under stress 2- The determination of );

[0033] Figure 13 The determination of SOD activity in wild-type and transgenic 'Fall Color' plants under stress;

[0034] Figure 14 To determine the POD activity of wild-type and transgenic 'Fall Color' plants under stress. Detailed Implementation

[0035] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0036] Example 1: Extraction of the CmbHLH104 gene from chrysanthemum

[0037] 1. Take leaves of ground cover chrysanthemum 'Fall Color', flash freeze them in liquid nitrogen, and store them in a -80℃ freezer for total RNA extraction. Total RNA was extracted using the Plant Total RNA Isolation Kit purchased from Chengdu Lanbo Biotechnology Co., Ltd. cDNA synthesis was performed using Reverse Transcriptase M–mLV(RNase H-) from Dalian Takara Biotechnology Co., Ltd., following the product instructions for first-strand synthesis.

[0038] Using the first strand of cDNA synthesized in the above kit as an amplification template, and using F: 5'-ttcggatcttccagaGATATCATGGATCCTTTTGAGAA-3' (SEQ ID NO.3) and R: 5'-caactgccgttcgacGATATCAGCAGCGGGCGGCCTCA-3' (SEQ ID NO.4) as primers, 'Fall Color' cDNA was used as a template and EcoRV was used as the restriction enzyme site for amplification by PCR.

[0039] The amplification system is shown in Table 1. The amplification conditions are: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 35 cycles, and finally 72℃ extension for 5 min.

[0040] Table 1 PCR amplification system

[0041] reagents Dosage DNA 100-150ng Primer F 5pmol Primer R 5pmol Taq Plus polymerase 1U 10×PCR buffer 2.5μL <![CDATA[Mg 2+ ]]> 1.5mM <![CDATA[ddH2O]]> Up to 25μL

[0042] 2. After PCR amplification, electrophoresis analysis was performed. The amplified fragment of approximately 675 bp was recovered using an OMEGA DNA recovery kit. The amplified fragment was inserted into the Peasy-T1 Simple cloning vector and transformed into E. coli DH5α competent cells. Single colonies were picked for colony PCR to identify positive clones. The positive clones were sent to Chengdu Qingke Biotechnology Co., Ltd. to determine the coding sequence of the chrysanthemum 'Fall Color' gene CmbHLH104, as shown in SEQ ID NO.1. The open reading frame of the obtained gene was then translated into an amino acid sequence using ORF Finder software, as shown in SEQ ID NO.2.

[0043] Example 2: Sequence homology and homology analysis of Chrysanthemum 'Fall Color' CmbHLH104

[0044] Sequencing results were imported into DNAMAN and compared with the CmbHLH104 sequence. The results showed that the cloned gene sequence was highly consistent with the bHLH104 transcription factor domain. Psort II was used to predict the subcellular location of the CmbHLH104 gene, showing that it is located in the nucleus. This is consistent with the bioinformatics prediction results and conforms to the basic characteristics of transcription factors. A single nuclear localization signal, EKECSRKRGR, was predicted using NLS-mapper. Comparison of this transcription factor with the protein sequences of other bHLH transcription factor family members, and domain analysis of the CmbHLH104 amino acid sequence using MEGA 7.0, revealed that this gene mainly possesses two structural features: a single nuclear localization signal and a typical conserved bHLH domain (e.g., Figure 1 Further, phylogenetic trees were constructed using multiple sequence alignment of highly homologous bHLH proteins from other plants (e.g., Figure 2 The study found that the chrysanthemum 'Fall Color' CmbHLH104 protein had the highest similarity (94.64%) to pyrethrum TcbHLH104-like (Tanacetumcinerariifolium, GEV16879.1), indicating a close phylogenetic relationship between the two. This demonstrates that the CmbHLH104 protein is highly consistent with bHLH proteins from other plants, further confirming that CmbHLH104 belongs to the bHLHⅣc subfamily.

[0045] Example 3: Construction of the plant expression vector for the chrysanthemum gene CmbHLH104

[0046] Plasmids were extracted from the bacterial culture that had been correctly sequenced. The plasmid containing the CmbHLH104 gene was double-digested at two restriction enzyme sites, HindIII and SacI. The DNA fragment was then recovered using a DNA recovery kit and ligated into the corresponding digested Super1300 expression vector. The resulting vector was named 35S:pCAMBIA1300-CmbHLH104.

[0047] Example 4: Subcellular localization of the chrysanthemum gene CmbHLH104

[0048] Based on the subcellular expression vector structure of pCAMBIA1300-35S-YFP, two restriction enzyme sites, SacI and KpnI, were selected. Primers were designed using the principle of homologous recombination, based on the vector sequence and the CmbHLH104 gene sequence with the stop codon removed. The primers were synthesized by Chengdu Qingke Biotechnology Co., Ltd. The specific primer sequences are as follows:

[0049] SacI-F: 5'-TTCGGATCTTCCAGAGATATCATGGATCCGTTTGAGAA-3' (SEQ ID NO.5)

[0050] KpnI-R: 5'-CAACTGCCGTCGACGATATCAGCAGCGGGCGGCCTCA-3' (SEQ ID NO. 6).

[0051] Using the RETMix homologous recombination kit, the CmbHLH104 gene (with the stop codon removed) was ligated into the linearized pCAMBIA1300-35S-YFP vector, which had been digested with SacI and KpnI, to construct the fusion vector 35S:CmbHLH104-pCAMBIA1300-YFP. Wild-type Nicotiana benthamiana leaves were injected with both the empty pCAMBIA1300-35S-YFP and the fusion vector 35S:CmbHLH104-pCAMBIA1300-YFP for transient expression. After slide preparation, the samples were observed under a laser confocal microscope. Results are shown in the figure below. Figure 3 .

[0052] from Figure 3 As can be seen, fluorescent signals were detected in all tobacco leaves transformed with pCAMBIA1300-35S-YFP, while fluorescent signals were detected only in the cell nucleus in tobacco leaves transformed with 35S:CmbHLH104-pCAMBIA1300-YFP. This indicates that the CmbHLH104 gene is located in the cell nucleus and functions there.

[0053] Example 4: Genetic transformation of the CmbHLH104 gene

[0054] Thaw *Agrobacterium* LBA4404 competent cells on ice. Add 5 μL of recombinant plasmid 35S:pCAMBIA1300-CmbHLH104 to 200 μL of competent cells, gently mix with a pipette tip, incubate on ice for 30 min, flash freeze in liquid nitrogen for 5 min, incubate in a 37°C water bath for 5 min, place on ice for 5 min, and then add LB liquid medium. Incubate at 28°C for 2–3 h. Spread the bacterial culture evenly on LB solid medium containing 50 μg / mL Kan and 20 μg / mL Rif, and incubate at 28°C for 48 h until single colonies appear. Pick a single colony and inoculate it into LB liquid medium, incubate at 28°C with shaking at 250 rpm for 24 h. Transfer 1 mL of the bacterial culture to 50 mL of LB liquid medium containing 50 μg / mL Kan and 20 μg / mL Rif, and incubate at 28°C with shaking at 200 rpm until OD500 is reached. 600≈0.4; Centrifuge the bacterial culture with the above OD value at 4℃, 8000r / min for 10min to collect the bacterial cells, discard the supernatant, resuspend the bacterial cells in 1 / 2MS liquid medium (pH about 5.6-6.0), and dilute with an equal volume of 1 / 2MS solution.

[0055] Select healthy, sterile 'Fall Color' seedlings that are 1-2 months old, and cut 1cm pieces from the upper tender leaves. 2 Square leaf discs, with the top facing up, were pre-cultured on MS medium and then immersed in the above-treated Agrobacterium bacterial solution for infection. During this process, the bacterial solution was constantly agitated to ensure full contact between the leaf discs and the solution. The dried leaf discs were then inoculated onto co-culture medium with the top facing up for dark incubation. After co-culture, the leaf discs were transferred to delayed culture for 10 days and then to meristematic culture medium. When the shoots emerging from the leaf discs reached 2 cm, the shoots were cut off and transferred to rooting selection medium containing 5 μg / mL Hyg to grow into seedlings.

[0056] After the seedlings have grown roots and new leaves for 4 weeks, 0.1g of leaf tissue is cut into a 1.5mL centrifuge tube, the tissue is ground, and DNA is extracted using the CTAB method. The transgenic plants are then subjected to PCR positive identification (e.g., ...). Figure 4 This process was repeated to obtain T1 generation transgenic plants, and the screening steps were repeated until positive overexpression homologous plants were selected. 'Fall Color' itself contains the CmbHLH104 gene, so amplifying the CmbHLH104 gene cannot confirm the positivity of the transgenic plant. Therefore, the Hyg resistance tag on the Super1300-CmbHLH104 overexpression vector was amplified to determine whether the transgenic plant was positive.

[0057] Example 5: Drought stress treatment of transgenic 'Fall Color' CmbHLH104

[0058] 1. Seedling to mature plant cultivation

[0059] Wild-type 'Fall Color' (WT) and three transgenic 'Fall Color' lines (Line-A, Line-B, Line-C) were aseptically propagated to obtain tissue culture aseptic seedlings. After washing the roots, the seedlings were soaked in sterile water for 3 days to allow them to recover. They were then transplanted into 10cm square pots, with an equal amount of nutrient substrate (peat:vermiculite = 1:1) added to each pot and mixed well. After about 30 days of routine care and cultivation, plants with uniform growth were selected for stress resistance analysis.

[0060] 2. Phenotypic identification of transgenic 'Fall Color' CmbHLH104 under drought stress treatment

[0061] The stress treatment involved natural drought dehydration, while the control group received normal irrigation with clean water. Phenotypic characteristics of wild-type and 'Fall Color' overexpressing seedlings and adult plants under drought stress were observed and recorded through photography. Seedling phenotypes under drought stress are shown below. Figure 5 ;

[0062] like Figure 5 As shown, the CmbHLH104 gene overexpression lines Line-A, Line-B, and Line-C all showed stronger drought tolerance than WT, indicating that CmbHLH104 gene overexpression improves the resistance to drought stress in 'Fall Color'.

[0063] Example 6: Determination of relevant physiological indicators under drought stress treatment

[0064] 1. Cultivation of chrysanthemum 'Fall Color' plants

[0065] The cultivation of chrysanthemum 'Fall Color' was the same as in Example 5. Wild-type 'Fall Color' (WT) and overexpression transgenic lines (Line-A, Line-B, Line-C) aseptic tissue culture seedlings were transplanted into 10cm square pots (each pot containing an equal amount of nutrient substrate). Before transplanting, the potting soil was watered to ensure full absorption. Under sufficient light, the plants were cultured in a conventional manner for about 30 days.

[0066] 2. Treatment and determination of leaf water loss rate

[0067] Twenty-four hours before sampling, 'Fall Color' (WT) and transgenic lines (Line A, Line B, Line C) were subjected to a bottom soaking treatment. 0.5g of leaves from both WT and transgenic plants were harvested, and the initial leaf weight was recorded as m0. The leaves were placed in a 25℃ constant temperature and humidity incubator for natural drought treatment. The leaves were weighed and recorded at 1h, 2h, 3h, 4h, 6h, 8h, 10h, and 12h after treatment. These measurements represent the natural water loss rate of detached leaves from wild-type and transgenic 'Fall Color' plants under drought stress. The results are shown in [Figure number missing]. Figure 6 .

[0068] like Figure 6 As shown, over time, the water loss rate of detached leaves in the transgenic lines (Line A, Line B, Line C) tended to be consistent and significantly lower than that of the WT plants. This indicates that under relatively severe environmental stress, the leaves of the transgenic lines have a stronger water retention capacity than those of the WT plants, significantly reducing the water transpiration rate and improving the plants' resistance to drought.

[0069] 3. Determination of stomatal aperture

[0070] The cultivation of 'Fall Color' (WT) and transgenic lines (LineA, LineB, LineC) 'Fall Color' plants was the same as in (1); approximately 18 leaves each from WT and transgenic lines (LineA, LineB, LineC) were placed in 100 mL of MES-KCl buffer and treated with light for 2.5 h to induce stomatal opening; 6 leaves from each of LineA, LineB, and LineC lines that had completed induction were placed in MES-KCl buffer containing 40 μmol / L ABA or 300 mmol / L mannitol. After 2 h of light treatment, the epidermis was peeled off to prepare slides, photographed under a microscope, and the stomatal diameter was counted (50 stomata were selected for each treatment). The results are shown in […]. Figure 7 .

[0071] There is a strong correlation between stomatal movement and leaf water loss, such as Figure 7 As shown, after mannitol and ABA induction treatments, the stomatal closure degree of transgenic lines (LineA, LineB, LineC) was significantly higher than that of WT plants. Under abiotic stress, the stomatal aperture of transgenic lines (LineA, LineB, LineC) was significantly lower than that of WT plants. This indicates that plants overexpressing the CmbHLH104 gene can improve leaf water retention capacity by shrinking stomatal aperture under drought stress, thereby reducing plant transpiration and enhancing the stress tolerance of overexpressing plants.

[0072] 4. Chlorophyll content determination under drought stress

[0073] Prepare 50mL centrifuge tubes and label them. Add 30mL of 95% ethanol solution to each tube. Weigh 0.3g of leaves from drought-stressed and normally cultured plants, completely immerse the leaves in the 95% ethanol solution, and incubate in the dark at 25℃ for 48h. The 95% ethanol extract has maximum absorbance values ​​(A649 and A665) at wavelengths of 649nm and 665nm, respectively. Therefore, measure A649 and A665 using a UV spectrophotometer, using the 95% ethanol extract as a blank control. This step is repeated twice. Measure the absorbance of each sample at wavelengths of 665nm and 649nm using a microplate reader. The results are shown in the figure. Figure 8 .

[0074] like Figure 8As shown, under normal culture conditions, the chlorophyll content of the 'Fall Color' transgenic plants was higher than that of the wild-type WT plants. After drought stress treatment, the chlorophyll content of the 'Fall Color' lines (Line-A, Line-B, Line-C) overexpressing the CmbHLH104 gene was significantly higher than that of the WT plants after stress treatment. This indicates that the drought resistance of the 'Fall Color' plants overexpressing the CmbHLH104 gene is significantly higher than that of the wild-type WT plants.

[0075] 5. Determination of superoxide dismutase (SOD) content under drought stress

[0076] Prepare the SOD reaction solution using 0.05M pH 7.8 phosphate buffer:Met:NBT:EDTA-Na2:FD:distilled water in a ratio of 15:3:3:3:3:2.5. Mix well in a beaker and store in the dark. Pipette 20 μL of enzyme solution and add 3 mL of SOD reaction solution, using phosphate buffer as a blank control. Place the centrifuge tube under 4000 Lux light for 30 min. Perform the reaction in a separate blank tube under the same conditions in the dark. Zero the microscope using the blank tube in the dark and measure the absorbance at 560 nm using a microplate reader. Define 50% inhibition of NBT photoreduction as one unit of enzyme activity (U). Results are shown below. Figure 9 .

[0077] like Figure 9 As shown, under drought stress treatment, the SOD activity of all plant lines was increased, with the transgenic plants (Line-A, Line-B, Line-C) showing a significantly higher increase than the WT plants.

[0078] 6. Determination of peroxidase (POD) content under drought stress

[0079] Take 50 mL of PBS (pH 6.0, 0.2 M) buffer in a beaker, add 28 μL of guaiacol (2-methoxyphenol), and heat and stir on a magnetic stirrer until the guaiacol dissolves. After the solution cools, add 19 μL of 30% H2O2, mix well, and store at 4°C for later use. Take 3 mL of the above reaction solution and add 40 μL of enzyme solution. Use PBS instead of enzyme solution as a control to zero the OD measurement. 470 The value changes over 40 seconds; see the results. Figure 10 .

[0080] like Figure 10 As shown, under normal conditions, the POD activity of transgenic lines (Line-A, Line-B, Line-C) was significantly higher than that of WT; under drought stress, the POD activity of transgenic lines (Line-A, Line-B, Line-C) was increased and significantly different from that of WT.

[0081] 7. Determination of malondialdehyde (MDA) content under drought stress

[0082] Take 0.5g samples (leaves) from each of the normal conditions and drought-treated WT, Line-A, Line-B, and Line-C, cut them into small pieces, place them in a mortar, add 5mL of 5% TCA solution, grind into a homogenate, transfer to a centrifuge tube, and centrifuge at 3000r / min for 20min. Take 2mL of the supernatant in a centrifuge tube, add an equal volume of 0.67% TBA (thiobarbituric acid), mix, boil in a 100℃ water bath for 30min, quickly cool with cold water, and centrifuge at 3000r / min for 10min. Take the supernatant and measure the OD value at 450nm, 532nm, and 600nm (zeroed with deionized water). Perform three replicates. The results are shown in the figure. Figure 11 .

[0083] like Figure 11 As shown, under drought stress, the MDA content of all plants increased significantly; among them, the increase in MDA content of WT plants was significantly greater than that of transgenic lines. The results indicate that plants overexpressing the CmbHLH104 gene play a role in the antioxidant mechanism within the plant, indirectly participating in the regulation of cellular peroxidation under stress. The results are as follows... Figure 11 As shown.

[0084] 8. Determination of proline (Pro) content under drought stress

[0085] Weigh 25 mg of proline and dissolve it in a small amount of distilled water. Dilute to 250 mL with distilled water to obtain the proline stock solution (containing 100 μg of proline per mL). Take six 50 mL volumetric flasks and add 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, and 3 mL of the proline stock solution, respectively. Dilute to volume with distilled water and set aside. Take seven test tubes and pipette (tube 0 is the blank group) with a series of standard concentrations of proline stock solution, dilute to the mark with distilled water, and mix well. The proline concentrations in each flask are 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 6 μg / mL, respectively.

[0086] Take 0.1g of fresh leaf samples under normal conditions and drought treatment, respectively. Grind the samples in a mortar with 1mL (0.5mL, 0.5mL) of 3% sulfosalicylic acid solution in two portions, then bring the volume to 10mL and transfer to a centrifuge tube. Centrifuge at 4500g for 3min. Collect the supernatant and bring the volume to 10mL with 3% sulfosalicylic acid solution. Transfer 2mL of the extract to a clean, dry glass test tube, add 2mL of acidic ninhydrin and 2mL of glacial acetic acid, heat at 100℃ for 1h, terminate the reaction by placing in ice water, add 4mL of toluene, shake thoroughly for 15-20s, and allow to separate into layers. Using toluene as a control, measure the absorbance at 520nm using a spectrophotometer. Repeat this step twice and three times. The results are shown in the figure. Figure 12 .

[0087] A large accumulation of proline can maintain osmotic balance in the cytosol and reduce the damage to cells caused by adverse stresses, such as... Figure 12 As shown, under drought stress, the proline content in both WT and transgenic plants increased significantly, and the transgenic plants showed a highly significant difference compared to WT plants.

[0088] 9. Determination of soluble protein content under drought stress

[0089] Weigh 25 mg of bovine serum albumin, dissolve it in distilled water, and bring the volume to 100 mL. Take 40 mL of this solution and dilute it to 100 mL with distilled water. Add 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1 mL of the protein solution to six test tubes, respectively. Dilute each tube to 1 mL with distilled water and mix well. Add 5 mL of Coomassie Brilliant Blue reagent to each tube, mix well, and let stand. Measure the absorbance at 595 nm. Plot a standard curve with protein content on the x-axis and absorbance on the y-axis.

[0090] Take 0.3g samples (fresh leaves) from both normal and drought-treated conditions, place them in a pre-chilled mortar, add 3mL of pre-chilled distilled water in two portions, and grind on ice to form a homogenate. Transfer the homogenate to a centrifuge tube and centrifuge at 4℃ and 12000g for 20min. The supernatant is the crude protein extract. Add 1mL of the above protein extract to 5mL of Coomassie Brilliant Blue solution, shake well, react for 5min, and measure the absorbance at 595nm. Determine the protein content using a standard curve. The results are shown in [Figure number missing]. Figure 13 .

[0091] Soluble proteins are important osmotic regulators and nutrients; their increase and accumulation can enhance the water-retention capacity of cells and protect cellular life-sustaining substances and biomembranes. For example... Figure 13 As shown, under drought stress, the soluble content in both transgenic and WT plants increased.

[0092] 10. Superoxide anion (O 2- Determination of content

[0093] Take 0.1 g of fresh leaf samples from plants under normal conditions and those under drought treatment. Add 1 mL of extraction buffer and homogenize on ice. Transfer to centrifuge tubes and centrifuge at 10000 g for 20 min at 4 °C. The supernatant is the crude enzyme extract, which should be placed on ice for testing. The specific procedures were performed according to the Suzhou Keming reagent kit, with three replicates. Results are shown below. Figure 14 .

[0094] like Figure 14 As shown, under drought stress, the transgenic lines (Line A, Line B, Line C) produced superoxide anion (O2) 2- The content of ) and superoxide anion (O) in WT plants 2- The content of ) differed by 2 times, approximately twice that under normal conditions, showing a highly significant difference from WT. This indicates that overexpression of the CmbHLH104 gene can reduce oxidative damage to plants under stress.

[0095] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A chrysanthemum gene CmbHLH104 characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. The chrysanthemum gene of claim 1 CmbHLH104 The encoded protein is characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO.

2.

3. A chrysanthemum gene comprising the one described in claim 1 CmbHLH104 The plasmid.

4. A recombinant expression vector comprising the chrysanthemum gene of claim 1. CmbHLH104 4. A recombinant expression vector comprising the chrysanthemum gene of claim 1.

5. An engineered bacterium comprising the chrysanthemum gene of claim 1. CmbHLH104 5. An engineered bacterium comprising the chrysanthemum gene of claim 1.

6. A gene chip, characterized by The chrysanthemum gene of claim 1 CmbHLH104 .

7. The chrysanthemum gene of claim 1 CmbHLH104 The use of the plasmid of claim 3, the recombinant expression vector of claim 4, the engineering bacteria of claim 5 or the gene chip of claim 6 in the improvement of drought-resistant germplasm resources of chrysanthemum.

8. The chrysanthemum gene of claim 1 CmbHLH104 The use of the plasmid of claim 3, the recombinant expression vector of claim 4, the engineering bacteria of claim 5 or the gene chip of claim 6 in cultivating transgenic drought-resistant chrysanthemum varieties.

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