Application of CmNAC2 in regulating drought tolerance in chrysanthemum

Abstract

This invention belongs to the field of chrysanthemum new variety breeding technology, specifically involving a novel application of the CmNAC2 gene. Overexpression CmNAC2 Genes are later used to enhance plant drought resistance; silencing is also possible. CmNAC2 Genes are used to reduce drought tolerance in plants. The CmMYB15-like gene regulates drought tolerance in chrysanthemums through a dependence on... CmNAC2 Gene, CmNAC2 The gene activates the CmMYB15-like gene by binding to the promoter of the CmMYB15-like gene. CmNAC2 Genes jointly regulate drought resistance in chrysanthemums. Based on these research results, a certain technical foundation can be laid for the regulation mechanism of drought resistance in chrysanthemums and the breeding of new chrysanthemum varieties. At the same time, it can also provide a good reference for the discovery of drought resistance genes in other crops and the breeding of new drought-resistant varieties.

Description

Technical Field

[0001] This invention belongs to the field of chrysanthemum new variety breeding technology, specifically involving a new application of the CmNAC2 gene. Background Technology

[0002] chrysanthemum( Chrysanthemum morifolium Chrysanthemum (Chrysanthemum morifolium) is a perennial herbaceous plant belonging to the genus Chrysanthemum in the Asteraceae family. It is one of China's ten traditional famous flowers, one of the "Four Gentlemen of Flowers," and one of the world's four major cut flowers, enjoying huge market demand. However, in actual cultivation and production, chrysanthemum growth and development are frequently affected by various biotic and abiotic stresses due to factors such as climate and geographical environment. Drought stress is the most common abiotic stress and one of the abiotic stresses that most easily affects chrysanthemum growth, development, and quality. Studies have shown that insufficient water supply during chrysanthemum growth and development directly affects the overall growth and development of the plant, leading to slow growth, decreased quality, and in severe cases, plant death. Typical growth and physiological symptoms of water shortage include, but are not limited to, wilting, chlorosis, and even leaf drop, decreased root vitality, and weakened ability to absorb water and nutrients. Therefore, the cultivation of various resistant chrysanthemum varieties is of great technical significance for the stability of chrysanthemum quality and for meeting market demands.

[0003] Due to the relatively poor targeting and long breeding cycle of conventional hybridization breeding, emerging breeding technologies such as molecular breeding and transgenic breeding, which combine molecular biology techniques, have been widely used in plant breeding. Regarding the cultivation of new drought-resistant chrysanthemum varieties, the full exploration of drought-resistant genes and the regulatory mechanisms of existing chrysanthemum genes in the drought resistance process are important prerequisites for carrying out this work. Summary of the Invention

[0004] The purpose of this application is to provide a new application of the chrysanthemum CmNAC2 gene in plant drought resistance, especially in chrysanthemum, thereby laying a certain technical foundation for the breeding of new drought-resistant chrysanthemum varieties.

[0005] The specific technical solution adopted in this application is as follows.

[0006] chrysanthemum CmNAC2 The application of this gene in regulating drought resistance in chrysanthemums: silencing this gene can significantly reduce the drought resistance of chrysanthemums; the chrysanthemums mentioned CmNAC2 The gene, whose nucleotide sequence is shown in SEQ ID No. 3;

[0007] silence CmNAC2 After gene modification, the overall water retention capacity of chrysanthemum plants can be reduced;

[0008] silence CmNAC2After gene therapy, the degree of damage to the cell membrane and photosystem can be increased, with specific physiological indicators including: increased relative conductivity and MDA content, and decreased FV / FM.

[0009] silence CmNAC2 After gene modification, the antioxidant capacity of chrysanthemum plants can be reduced and the osmotic regulation capacity weakened; specific physiological indicators show that SOD activity, POD activity, and proline content are significantly reduced.

[0010] Application of CmMYB15-like in the regulation of drought resistance in chrysanthemum: Overexpression of the CmMYB15-like gene can improve the drought resistance of chrysanthemum;

[0011] Overexpression of the CmMYB15-like gene can improve the overall water retention capacity of chrysanthemum plants;

[0012] Overexpression of the CmMYB15-like gene can reduce the degree of damage to the cell membrane and photosystem. Specifically, the physiological indicators are: lower relative conductivity and MDA content, and higher FV / FM.

[0013] Overexpression of the CmMYB15-like gene can improve the antioxidant capacity and enhance the osmotic regulation capacity of chrysanthemum plants; specific physiological indicators show that SOD activity, POD activity and proline content are significantly increased.

[0014] CmMYB15-like- CmNAC2 Application of molecular templates in the regulation of drought tolerance in chrysanthemum; the CmMYB15-like gene's regulation of drought tolerance in chrysanthemum depends on... CmNAC2 Genes, together, regulate the drought resistance of chrysanthemums.

[0015] Previous studies have shown that various MYB-type transcription factors are involved in the drought stress response process in plants. For example, in chrysanthemums, research has shown that the interaction between CmSCL4 and CmR1MYB1 jointly regulates the dehydration tolerance of chrysanthemums. However, due to the complexity of crop drought resistance regulation, the discovery of more drought-resistant genes is of great technical significance for improving crop drought resistance.

[0016] In preliminary research, the inventors discovered that the CmMYB15-like gene is involved in the biotic stress response of chrysanthemums (enhancing their aphid resistance). However, further in-depth research on this gene revealed that overexpression of this gene can further enhance the drought resistance of chrysanthemums, and this drought resistance works in conjunction with the CmNAC2 gene. Based on these research results, a certain technical foundation can be laid for understanding the drought resistance regulation mechanism of chrysanthemums and for breeding new chrysanthemum varieties. It also provides valuable reference for the discovery of drought resistance genes in other crops and the breeding of new drought-resistant varieties. Attached Figure Description

[0017] Figure 1 The expression levels of the CmMYB15-like gene at different time points under drought stress;

[0018] Figure 2 Comparison of typical phenotypes of wild-type and CmMYB15-like overexpressing lines after drought stress treatment;

[0019] Figure 3 The results show the survival rate and relative water content of wild-type and CmMYB15-like overexpression lines after drought stress treatment; where: A is the survival rate statistics after 15 days of drought stress treatment; B is the relative water content measurement results of different experimental groups;

[0020] Figure 4 The results show the relative conductivity measurements for different treatment groups.

[0021] Figure 5 FV / FM measurement results for different treatment groups;

[0022] Figure 6 The results of SOD and POD activity assays for different treatment groups are shown; where A represents the SOD activity assay results and B represents the POD activity assay results.

[0023] Figure 7 Results of MDA content determination in different treatment groups;

[0024] Figure 8 Results of proline content determination in different treatment groups;

[0025] Figure 9 The results of bioinformatics analysis of the CmNAC2 gene are shown below; A is a schematic diagram of the conserved domains of the chrysanthemum CmNAC2 protein; B is the secondary structure of the chrysanthemum CmNAC2 protein, with blue representing α-helices, red representing extended strands, green representing β-turns, and purple representing random coils; C is the tertiary structure of the chrysanthemum CmNAC2 protein; D is the hydrophilicity / hydrophobicity analysis of the chrysanthemum CmNAC2 protein; and E is the predicted transmembrane structure of the chrysanthemum CmNAC2 protein.

[0026] Figure 10 For subcellular localization analysis of the CmNAC2 gene; scale bar = 100 μm;

[0027] Figure 11 To validate the transcriptional activation activity of the CmNAC2 gene;

[0028] Figure 12 To verify and analyze the binding of CmNAC2 and CmMYB15-like using yeast single-hybrid experiments;

[0029] Figure 13 To verify the detection of the binding of CmNAC2 and CmMYB15-like using dual-luciferase assay; where: A is a schematic diagram of the connection between CmNAC2 and proCmMYB15-like vectors; B is a fluorescence imaging image of CmNAC2 binding to proCmMYB15-like; C is the relative luciferase activity;

[0030] Figure 14 The expression level of CmNAC2 changed after drought treatment;

[0031] Figure 15 The expression patterns of the CmNAC2 gene in different tissues of chrysanthemum;

[0032] Figure 16 The results show the identification of gene-silenced plants; where: A represents the identification results of different strains in the CmNAC2 silencing experimental group; B represents the identification results of different plant seedlings in the CaLCuV empty vector group; M in the figure is the DL5000 DNA marker, and 1~16 are the sample numbers of different plants.

[0033] Figure 17 The results of qRT-PCR detection of CmNAC2 gene expression levels in gene-silenced plants;

[0034] Figure 18 This serves as a drought resistance phenotype control between plants with the CmNAC2 gene silenced and control plants.

[0035] Figure 19 The results are as follows: A represents the survival rate statistics and relative water content measurements of each strain after 14 days of natural water loss; B represents the relative water content measurements of each strain.

[0036] Figure 20 These are the results of relative conductivity measurements;

[0037] Figure 21 The results are for FV / FM measurements.

[0038] Figure 22 The results are for SOD and POD activity assays; where: A represents the SOD activity assay result; B represents the POD activity assay result.

[0039] Figure 23 The results are for the determination of MDA content;

[0040] Figure 24 These are the results of proline content determination;

[0041] In the accompanying figures, CmNAC (or NAC) refers to the CmNAC2 gene; different lowercase letters indicate significant differences (P<0.05); **p<0.01, *P<0.05. Detailed Implementation

[0042] The present application will be further explained below with reference to the embodiments. Before introducing the specific embodiments, the experimental background of some embodiments is briefly described below.

[0043] Biomaterials:

[0044] chrysanthemum( Chrysanthemum morifolium The 'Shenma' wild-type (WT) and the chrysanthemum CmMYB15-like overexpression lines OE-1 and OE-3 are materials that were used in the inventor's early experimental work and have been disclosed. For example, see "A plant expression vector for the gene CmMYB15-like that improves the aphid resistance of chrysanthemum and its application" (application number 2022105391453).

[0045] tobacco( Nicotiana benthamiana ), yeast one-hybrid strain Y187, yeast one-hybrid strain Y1H, Escherichia coli ( Escherichia coli ) strain DH5α, Agrobacterium tumefaciens ( Agrobacterium tumefaciens strains such as EHA105 and Agrobacterium tumefaciens strain GV3101 are common and frequently used materials in existing biological research and can be obtained through public channels.

[0046] Related vectors such as pGADT7, pGBKT7, pHIS2, pAbAi, plant expression vector pORE-R4, and dual-luciferase (LUC) vector pGreenⅡ-0800-LUC are all common and frequently used plasmids in existing molecular biology research and can be obtained from public channels.

[0047] Since the applicant is a professional scientific research and teaching institution and the inventor is a professional scientific researcher, they have preserved relevant experimental materials for a long time.

[0048] The synthesis and sequencing of the relevant primers were provided by Sangon Biotech (Shanghai) Co., Ltd.

[0049] Main experimental reagents:

[0050] Various rTaq and LATaq DNA polymerases, ApexHF HS DNA high-fidelity polymerase, restriction endonucleases, Solution I ligase, OK CIon DNA homologous recombination ligase, etc., are products of TaKaRa or AG Acrel Company;

[0051] Plasmid DNA extraction and gel recovery kit, Omega Corporation product;

[0052] Plant DNA extraction kit, Dual-Luciferase Reporter Assay Kit for dual-luciferase experiments, etc., products of Nanjing Novizan Co., Ltd.

[0053] Plant RNA extraction kit, product of Beijing Huayueyang Company;

[0054] Yeast experimental culture medium, X-α-gal, 3-AT, AbA, etc., products of Weidi Bio or Zhuangmeng Bio;

[0055] D-Luciferin potassium salt, a product of Digibio Biotechnology Co., Ltd.

[0056] Methods for measuring relevant physiological and biochemical indicators:

[0057] (1) Relative water content

[0058] Take 0.4 g of fresh seedling leaf sample (fresh weight of leaf WF), immerse it in distilled water for several hours until the leaf is saturated with water; take it out and use absorbent paper to remove surface moisture, weigh it immediately (weight of leaf when saturated with water WT); then place it in an oven (80℃) to dry, and weigh it (dry weight of leaf WD).

[0059] Relative moisture content RWC = [(WF-WD) / (WT-WD)] × 100%;

[0060] (2) Relative conductivity

[0061] Take 0.1 g of fresh seedling leaf sample and cut it into small segments with a length of 0.5 cm; rinse it with deionized water and dry the surface moisture with absorbent paper, then place it in a test tube (20 ml specification); add 10 ml of deionized water, shake well, seal, and place at room temperature for 24 h, then measure the conductivity E1 using a digital conductivity meter.

[0062] Then, place it in a boiling water bath and boil for 30 minutes. After cooling, measure the conductivity E2; at the same time, measure its background conductivity E0 (background conductivity (deionized water conductivity) E0=0).

[0063] Electrical conductivity P = [(E1-E0) / (E2-E0)] × 100%;

[0064] (3) Chlorophyll fluorescence parameters: PSⅡ maximum photochemical efficiency Fv / Fm

[0065] The FV / FM was measured using a multifunctional plant efficiency meter (M-PEA, Hansatech Instruments Ltd, UK) in accordance with its instruction manual.

[0066] (4) Content of SOD, POD, MDA, and proline (Pro)

[0067] Superoxide dismutase (SOD) activity was determined using the nitroblue tetrazolium (NBT) method;

[0068] Peroxidase (POD) activity was determined using the guaiacol colorimetric method;

[0069] The content of malondialdehyde (MDA) was determined by the colorimetric method of thiobarbituric acid (TBA).

[0070] The proline (Pro) content was determined by the acidic ninhydrin colorimetric method;

[0071] For specific procedures, refer to existing technologies and routine measurements; further details are unnecessary.

[0072] Example 1

[0073] Based on the previous research on the aphid resistance function of CmMYB15-like response, combined with further genomic bioinformatics analysis and other research experience, the inventors initially speculated that this gene may be related to the drought resistance function of chrysanthemum. Therefore, the inventors conducted further in-depth analysis and verification of the drought resistance function of the gene. The specific experimental results are briefly described below.

[0074] (a) Drought stress treatment

[0075] Select potted chrysanthemum seedlings with good growth and uniform size, which have grown to 6-8 true leaves (using wild-type WT as the control and CmMYB15-like overexpression lines OE-1 and OE-3 as experimental subjects), and cultivate them with normal watering for 7 days (growth conditions: 16 h / 23℃ during the day, 8 h / 22℃ at night, and 5000 Lx of light).

[0076] After normal cultivation, watering was stopped for 15 days as a drought stress treatment.

[0077] During the experiment, samples were taken at different time points according to the experimental design (the 3rd and 4th fully expanded leaves were used as samples) and processed (the samples were flash-frozen in liquid nitrogen and stored at -80℃ or directly used in subsequent experiments).

[0078] (II) Response of CmMYB15-like to drought stress

[0079] During the drought stress treatment, chrysanthemum seedlings from each treatment group were taken, and the substrate around the plant roots was washed with clean water and placed on filter paper for natural dehydration treatment at room temperature (around 25℃). Then, samples were taken at 0, 1, 3, 6, 9, 12, and 24 h after dehydration for RT-qPCR detection to evaluate the response of CmMYB15-like to drought stress.

[0080] For RT-qPCR detection, leaf RNA is first extracted and reverse transcribed into cDNA, which is then used as a template for qPCR. The specific primer design for qRT is as follows:

[0081] qRT-CmMYB15-like-F:5'-GAACCAATAGTTTCGCAATG-3',

[0082] qRT-CmMYB15-like-R: 5'-GACTCGCTAGACGATGGTTG-3'.

[0083] Test results as follows Figure 1 As shown in the figure, the analysis reveals that during leaf water loss, the expression level of CmMYB15-like genes significantly and continuously increased over time, reaching 5.9 times the level at 0 h in 24 h. Therefore, based on these results, it can be preliminarily determined that the CmMYB15-like gene responds to drought stress; or in other words, drought stress can induce a sustained increase in the expression level of the CmMYB15-like gene.

[0084] (III) Overall drought resistance phenotype

[0085] The phenotypic comparison of chrysanthemum seedlings in different treatment groups after 15 days of drought stress is shown in the figure below. Figure 2 As shown, under normal culture conditions, there was no significant difference in growth between the wild-type and overexpression lines. However, after drought stress, compared to the overexpression lines, the wild-type chrysanthemum seedlings exhibited significant water loss and leaf wrinkling, indicating more severe drought damage. Further statistical analysis and measurement of plant survival rate and relative water content were conducted (e.g., ...). Figure 3 As shown in the figure, the survival rates of the overexpression lines OE-1 and OE-3 were 63% and 67%, respectively, while the wild type was only 37%. The relative water content of the overexpression lines OE-1 and OE-3 were 88.76% and 85.98%, respectively, while the wild type was only 58.48%. In other words, the survival rate and relative water content of the wild type were significantly lower than those of the overexpression lines. In other words, CmMYB15-like overexpression can improve the drought resistance of chrysanthemums by reducing water loss.

[0086] (iv) Results of relevant physiological and biochemical indicators

[0087] Having confirmed that CmMYB15-like overexpression can indeed improve the drought resistance of chrysanthemums, the inventors conducted further testing and analysis on the specific effects on the relevant physiological and biochemical indicators of chrysanthemums under drought stress. The details are briefly described below.

[0088] (2) Changes in relative conductivity

[0089] The relative conductivity measurements of different treatment groups are as follows: Figure 4 As shown in the figure. Analysis reveals that under normal culture conditions, there was no significant difference in the relative electrical conductivity of chrysanthemum leaves between wild-type and overexpression lines OE-1 and OE-3, which were 15.71%, 19.26%, and 17.64%, respectively. However, under drought stress conditions, the relative electrical conductivity of leaves between wild-type and overexpression lines OE-1 and OE-3 were 90%, 50.61%, and 41.27%, respectively, with the overexpression lines showing a reduction of 39.43% and 48.78% compared to the wild-type.

[0090] The above results indicate that overexpression of the CmMYB15-like gene can reduce the degree of damage to the cell membrane of chrysanthemum under drought stress, reduce electrolyte leakage, and thus improve the drought resistance of chrysanthemum.

[0091] (2) FV / FM variation

[0092] The results of the measurement of the maximum photochemical efficiency (FV / FM) of the photosystem II (PSII) of leaves in different treatment groups are as follows: Figure 5 As shown in the figure. Analysis shows that under normal culture conditions, there was no significant difference in FV / FM between wild-type and overexpression lines OE-1 and OE-3 chrysanthemum leaves, which were 0.845, 0.845, and 0.846, respectively. However, under drought stress conditions, the FV / FM of wild-type and overexpression lines OE-1 and OE-3 leaves were 0.63, 0.756, and 0.736, respectively, with the wild-type decreasing by 0.128 and 0.108 compared to the overexpression lines.

[0093] The above results indicate that drought stress reduces the light energy conversion efficiency within the PSII reaction center of each line. However, overexpression of CmMYB15-like can improve the light energy conversion efficiency of chrysanthemum leaves to some extent, thereby enhancing the plant's photosynthetic capacity and providing stress resistance.

[0094] (3) SOD and POD activity

[0095] The SOD and POD measurement results of different treatment groups are as follows: Figure 6As shown in the figure. Analysis shows that under normal culture conditions, there were no significant differences in SOD and POD activities between wild-type and overexpression lines OE-1 and OE-3 chrysanthemum leaves; the SOD activities were 28.17, 25.61, and 27.03 U·g-1, respectively, and the POD activities were 1321.6, 1598.8, and 1589.2 U·g-1·min-1, respectively. However, under drought stress, the SOD and POD activities of wild-type and overexpression lines OE-1 and OE-3 were significantly increased. Specifically, the SOD activities of leaves of wild-type and overexpression lines OE-1 and OE-3 were 141.59, 166.02, and 173.93 U·g-1, respectively, which were 24.44 and 32.34 U·g-1 higher than those of wild-type. The POD activities of leaves of wild-type and overexpression lines OE-1 and OE-3 were 2260.4, 3235.2, and 3396.4 U·g-1·min-1, respectively, which were 974.8 and 1136 U·g-1·min-1 higher than those of wild-type.

[0096] The above results indicate that drought stress increases the activities of SOD and POD in crops, but overexpression of CmMYB15-like enhances these activities to an even greater extent. In other words, overexpression of CmMYB15-like strengthens drought resistance by enhancing reactive oxygen species scavenging capabilities.

[0097] (4) Changes in MDA content

[0098] The changes in MDA content in different treatment groups are as follows: Figure 7 As shown in the figure. Analysis reveals that under normal culture conditions, there was no significant difference in MDA content between wild-type and overexpression lines OE-1 and OE-3 chrysanthemum leaves, which were 29.59, 27.05, and 31.21 μmol·g⁻¹, respectively. However, under drought stress, the MDA content in the leaves of wild-type and overexpression lines OE-1 and OE-3 were 47.15, 32.12, and 37.62 μmol·g⁻¹, respectively, representing a decrease of 15.03 and 9.53 μmol·g⁻¹ compared to the wild-type. This result indicates that CmMYB15-like overexpression can enhance the antioxidant capacity of chrysanthemum cell membranes.

[0099] (5) Changes in proline content

[0100] The changes in proline content in different treatment groups are as follows: Figure 8As shown in the figure. The analysis shows that even under normal culture conditions, overexpression of CmMYB15-like increases proline content. The proline content in the leaves of wild-type and overexpression lines OE-1 and OE-3 chrysanthemums was 284.98, 428.28, and 399.93 μg·g-1, respectively, which were 143.3 and 114.95 μg·g-1 higher than that of wild-type.

[0101] After drought stress treatment, the proline content in the leaves of wild type and overexpression lines OE-1 and OE-3 was 376.68, 606.58 and 595.28 μg·g-1, respectively, which were 229.90 and 218.60 μg·g-1 higher than that of wild type.

[0102] This result indicates that overexpression of CmMYB15-like significantly increases the proline content in chrysanthemums, and under drought stress, it further enhances the drought resistance of chrysanthemums by significantly increasing proline content (the increase in proline content can improve the stability of protoplasmic colloids and intra-tissue metabolism in chrysanthemum cells, slow down cell dehydration, and thus improve its drought resistance).

[0103] Example 2

[0104] Based on the fact that CmMYB15-like can be used to enhance the drought resistance of chrysanthemums as clearly demonstrated in Example 1, the inventors conducted further experimental explorations in order to further investigate the upstream regulatory pathways of CmMYB15-like. The relevant experimental details are briefly described below.

[0105] (I) Screening for upstream transcription factors of CmMYB15-like genes based on yeast one-hybrid technology

[0106] First, bioinformatics analysis was performed on the cis-acting elements in the CmMYB15-like promoter sequence region;

[0107] Subsequently, based on the above bioinformatics analysis results, the CmMYB15-like promoter sequences (FR, F1R, FR1) were cloned in segments, and the cloned sequence fragments were ligated and recombinated with the pHIS2 vector, respectively.

[0108] Next, the correctly ligated recombinant plasmid was transformed into Y187 yeast competent cells, plated on SD / -Trp-Leu medium, and incubated upside down at 30°C for 3 days. After amplification and centrifugation, the bacterial cell mass was resuspended in 50 μL of TE buffer, and 5 μL of bacterial culture was aspirated and serially diluted before being spotted onto SD / -Trp-Leu medium containing different concentrations of 3-AT (3-amino-1,2,4-triazole). After incubation at 30°C for 3 days, the appropriate 3-AT screening concentration was determined.

[0109] Finally, the pHIS2-bait plasmid and the chrysanthemum 'Shenma' yeast single-hybrid library were co-transformed into Y187 yeast competent cells and plated on the SD / -Trp-Leu-His medium containing a suitable 3-AT concentration as selected above. After culturing for 3 days, positive single colonies were picked for PCR detection and sequencing identification (the pGADT7 universal primer was used for sequencing identification).

[0110] Analysis of upstream regulatory transcription factors of the CmMYB15-like gene revealed that its promoter region contains multiple cis-regulatory elements, including potential binding elements for NAC-type transcription factors: ABRE, G-box, NACRS (CGTG), MYC, mybst1 (TATCC), NRS1 (TAGTT), and NRS2 (GAATC). This suggests that CmMYB15-like transcription may be regulated by NAC-type transcription factors.

[0111] Based on the above results, recombinant vectors were constructed using the full-length 964 bp CmMYB15-like promoter sequence, as well as truncated sequences of 178 bp and 447 bp, respectively. These vectors were then used as "bait" to screen for *Saccharomyces cerevisiae* libraries. Combined with relevant sequencing and protein annotation results, the NAC2 transcription factor was ultimately selected for further research.

[0112] It should be noted that the CmMYB15-like promoter sequence (964 bp) is shown in SEQ ID No. 1, and is as follows:

[0113] 。

[0114] (II) Cloning and Sequence Analysis of Chrysanthemum CmNAC2 Gene

[0115] Based on the aforementioned identification of the NAC2 transcription factor as the research object, the inventors further cloned and sequenced the gene, and the relevant experimental details are briefly described below.

[0116] First, referring to the instructions of the plant RNA extraction kit, RNA was extracted from the leaves of chrysanthemum 'Shenma' as a sample, and then cDNA was prepared by reverse transcription using the AG reverse transcription kit (follow the instructions).

[0117] Subsequently, the primer pairs for PCR amplification were designed as follows:

[0118] CmNAC2-ORF-F: 5'-ATGACTTTGGAGTTGCCTCC-3',

[0119] CmNAC2-ORF-R: 5'- TTACATTTGGTATGATTTTGG-3';

[0120] Using the prepared cDNA as a template, PCR amplification was performed using the primers mentioned above. The reference design for a 50 μl amplification system is as follows:

[0121] rTaq, 25 μL;

[0122] CmNAC2-ORF-F primers, 2 μL (10 μM);

[0123] CmNAC2-ORF-R primers, 2 μL (10 μM);

[0124] cDNA template, 2 μL;

[0125] Add ddH2O to a final volume of 50.0 μL;

[0126] The PCR reaction program was as follows: 94℃ for 2 min; 94℃ for 30 s, 55℃ for 15 s, 72℃ for 1 kb / 1 min, 35 cycles; extension at 72℃ for 5 min.

[0127] The amplification products were analyzed by 1% agarose gel electrophoresis, and the target band was recovered using a gel recovery kit.

[0128] Next, the recovered target band was ligated to the pMD18-T vector. The 10 μl ligation system was designed as follows:

[0129] PCR amplification product (i.e. the recovered target band gene fragment), 1.0 μL (100 ng).

[0130] pMD18-T, 1.0 μL (50 ng);

[0131] Solution I, 5.0 μL;

[0132] ddH2O, 3.0 μL;

[0133] Connect at 16℃ for 30 minutes;

[0134] After ligation, 5 μL of the ligation product was added to the DH5α competent cells that had been slowly thawed on ice beforehand. The cells were gently aspirated and mixed, then placed on ice for 30 min, followed by heat shock at 42°C for 60 s. After heat shock, the cells were quickly transferred to an ice bath and placed on ice for 2 min.

[0135] After standing, add 700 μL of antibiotic-free LB liquid medium and incubate at 37°C and 200 rpm for 60 min.

[0136] After the resuscitation culture was completed, the cells were centrifuged at 400 rpm for 5 min, and 200 μL of supernatant was retained to resuspend the bacterial cells. The cells were then spread on LB solid medium containing ampicillin and incubated upside down at 37°C overnight. After the culture was completed, positive single colonies were picked and transferred to LB liquid medium containing ampicillin for culture. Samples were taken for bacterial PCR identification and sequencing analysis.

[0137] The final sequencing results showed that the obtained CmNAC2 protein sequence is shown in SEQ ID No. 2 (284AA), as follows:

[0138] MTLELPPGFRFHPNDEELVMHYLIRKCASQSISVPIIADIDLYKFDPWQLPGMAVYGEKEWYFFSPRDRKYPNGSRPNRAAGTGYWKATGADKPIGKPKAVGIKKALVFYAGKAPRGVKTNWIMHEYRLANVDRSAGKRSNN LRLDDWVLCRIYNKKGVLEKHINSEVKSTQFSEMEIETKPKITPYAPMSNMSSLHPPPSIPHHVMDDGINFESSESVPTLQTDSSSEHEREVQSEVKKDDFQFNYMDSFADDAFTPQNQYYNDFQLSPLQDIFMFMPKSYQM;

[0139] Correspondingly, the specific CmNAC2 coding sequence (855bp) is shown in SEQ ID No. 3, as follows:

[0140] ATGACTTTGGAGTTGCCTCCTGGATTTAGATTTCACCCGAATGATGAAGAGCTGGTTATGCATTATCTTATTAGGAAATGTGCTTCTCAATCGATTTCGGTCCCTATTATTGCTGATATTGATTTGTATAAATTCGATCCTTGGCAGCTTCCTGGTATGGCTGTGTATGGAGAAAAGGAGTGGTACTTTTTTTCGCCTAGGGACAGAAAGTATCCTAATGGGTCCCGGCCTAACAGAGCGGCCGGGACAGGATACTGGAAGGCTACCGGGGCAGATAAGCCAATTGGGAAGCCGAAAGCGGTTGGGATAAAGAAGGCGTTGGTGTTTTACGCCGGTAAAGCACCAAGAGGGGTGAAAACAAATTGGATAATGCACGAGTATCGTTTAGCTAATGTTGATAGATCTGCTGGCAAACGGAGCAACAATCTTAGGTTAGATGATTGGGTATTATGTCGAATATACAACAAGAAAGGTGTTTTGGAGAAACACATAAACTCGGAAGTAAAATCAACACAATTCTCAGAGATGGAAATCGAAACAAAGCCAAAAATCACACCATATGCTCCCATGAGTAATATGTCCTCGTTACATCCGCCACCATCAATACCACATCATGTCATGGACGATGGAATTAATTTCGAGTCATCCGAATCAGTGCCTACTTTGCAAACAGATTCAAGTTCAGAACACGAACGAGAAGTCCAAAGCGAGGTTAAAAAGGACGATTTTCAGTTCAATTACATGGATTCTTTCGCAGATGACGCATTTACACCTCAAAACCAATATTACAATGACTTTCAACTCTCTCCATTACAAGACATATTCATGTTTATGCCAAAATCATACCAAATGTAA。

[0141] For the obtained CmNAC2 gene, using existing bioinformatics software, the preliminary analysis results show that (the relevant results are as Figure 9As shown in the image, CmNAC2 has a highly conserved DNA-binding domain at its N-terminus, composed of five substructures (A, B, C, D, and E), belonging to the NAC family. The total molecular weight of CmNAC2 is 32.72 KD, its theoretical isoelectric point is 6.72, its protein instability coefficient is 48.60, indicating structural instability. Its average hydrophilicity is -0.665, classifying it as a hydrophilic protein. This protein contains a conserved NAC-DNA-binding domain. Prediction of the secondary and tertiary structures of CmNAC2 reveals that it contains 51.3% α-helices, 18.3% extended strands, 20.5% β-turns, and 9.9% random coils. Analysis of the protein's hydrophilicity / hydrophobicity shows a minimum value of -2.800 and a maximum value of 2.011. Prediction of the protein's transmembrane structure shows that all amino acids from position 1 to 284 are extracellular domains.

[0142] (III) Subcellular localization of the chrysanthemum CmNAC2 gene

[0143] Based on the aforementioned cloning of the CmNAC2 gene, the inventors conducted further subcellular localization studies on the expression sites of this gene in cells. The specific experimental details are briefly described below.

[0144] Based on the sequences obtained from the aforementioned cloning and sequencing, and using the pMD18-T plasmid containing the CmNAC2 gene as a template, primers containing BamHI and EcoRI restriction sites were designed as follows:

[0145] pORE-R4-CmNAC2-F: 5'-ctagaaggccttggatccATGACTTTGGAGTTGCCTCCTG-3',

[0146] pORE-R4-CmNAC2-R:5'-aaagtcgacgaattcCATTTGGTATGATTTTGGCATAAAC-3';

[0147] Referring to the aforementioned procedures or existing routine procedures, PCR amplification was performed to obtain the target fragment containing the restriction enzyme sites of the vector for subsequent ligation. Electrophoresis was used to detect and the amplified product was recovered and purified.

[0148] Subsequently, the purified product and the pORE-R4 plasmid (overexpression vector) were subjected to double enzyme digestion. The 50 μl enzyme digestion system was designed as follows:

[0149] PCR amplification product, 2.0 μL (2.0 μg).

[0150] Quick BamHI, 2.0 μL;

[0151] Quick EcoRI, 2.0 μL;

[0152] 10X QuickCut Green Buffer, 5.0 μL;

[0153] ddH2O, 39.0 μL;

[0154] Enzyme digestion was performed at 37℃ for 2 hours. After enzyme digestion, the target fragment was detected by electrophoresis and recovered.

[0155] The CmNAC2 recovered from the above enzyme digestion was ligated with the pORE-R4 vector product; the 10 μl ligation system was designed as follows:

[0156] Target gene fragment, 1.0 μL (100 ng);

[0157] pORE-R4, 2.0 μL (approximately 50 ng);

[0158] Solution I, 5.0 μL;

[0159] ddH2O, 2.0 μL;

[0160] Connect at 16℃ for 30 minutes;

[0161] After ligation, the ligation product was transformed into DH5α Escherichia coli competent cells using the heat shock method. After transformation, the cells were plated on Kan-resistant LB medium and incubated overnight at 37°C. Positive transformed colonies were then picked for bacterial PCR detection and sequencing identification to ensure that the plasmid ligation and transformation were correct.

[0162] The recombinant plasmid pORE-R4-CmNAC2, which was correctly identified and sequenced, was extracted and further transformed into EHA105 and GV3101 (psoup) Agrobacterium competent cells, respectively. Then, the selected and identified positive Agrobacterium containing the recombinant plasmid pORE-R4-CmNAC2 (with pORE-R4 empty vector as a control) were inoculated into YEB liquid medium containing 50 mg / L Kan and 50 mg / L Rif and cultured at 28°C with shaking until the OD 600 reached about 1.5.

[0163] After the culture was completed, the cells were collected by centrifugation at 4000 rpm for 5 min. The cells were then resuspended in infection buffer (10 mM MES, 10 mM MgCl2, 200 μM AS, pH=5.7) and the OD600 was adjusted to about 1.0. The cells were then incubated at room temperature (about 25℃) in the dark for about 4 h (this was used as the infection solution).

[0164] Select tobacco leaves that are about 5-6 weeks old, and inject the prepared infection solution into the leaves from the underside of the leaves using a 1 mL syringe. After injection, incubate in the dark for 1 day, and then continue to incubate under normal light conditions for another day. Observe the fluorescence signal and take pictures under a laser confocal microscope (NiKon, A1HD25).

[0165] (For any experimental procedures not described in detail above, please refer to the aforementioned procedures or standard procedures in this field; they will not be repeated here.)

[0166] Experimental results are as follows Figure 10 As shown in the figure. Analysis shows that the GFP fluorescence signal in tobacco from the pORE-R4 empty control group is distributed throughout the entire cell, while the GFP fluorescence signal in tobacco transformed with pORE-R4-CmNAC2 is specifically expressed in the cell nucleus and co-localizes with the nuclear localization marker, indicating that the CmNAC2 gene localization acts in the cell nucleus.

[0167] (iv) Analysis of transcriptional activation activity of the CmNAC2 gene

[0168] As described in the previous screening process, the CmNAC2 gene obtained by screening belongs to the upstream gene of the CmMYB15-like gene. Therefore, it is necessary to further analyze and clarify the transcriptional activation activity of this gene. The specific experimental process is briefly described below.

[0169] Referring to the foregoing description and existing technical procedures, using the pMD18-T plasmid containing the CmNAC2 gene as a template, primers containing BamHI and EcoRI restriction sites were designed; the target fragment containing the restriction sites of the vector for subsequent ligation was obtained by PCR amplification, and the amplified product was obtained by electrophoresis detection and purification.

[0170] Subsequently, the purified product was ligated with the pGBKT7 vector to construct the recombinant plasmid pGBKT7-CmNAC2;

[0171] Next, the recombinant plasmid pGBKT7-CmNAC2 was transformed into Y2H yeast competent cells (pCL1 was transformed into a positive control, and pGBKT7 was transformed into a negative control). After transformation, the cells were plated (positive control pCL1 was plated on SD / -Leu medium, and negative controls pGBKT7 and pGBKT7-CmNAC2 were plated on SD / -Trp medium) and incubated upside down at 30°C for 3 days.

[0172] After culturing, single colonies were resuspended in 0.9% NaCl solution and spotted onto SD / -His-Ade medium with and without X-α-gal, respectively. After incubation overnight, colony growth was observed and photographed for identification. For identification and analysis:

[0173] Blue color on SD / -His-Ade medium supplemented with X-α-gal indicates transcriptional activation activity; otherwise, no transcriptional activation activity is observed (pCL1 is a positive control, indicating transcriptional activation activity, while pGBKT7 is a negative control, indicating no transcriptional activation activity).

[0174] Experimental results are as follows Figure 11 As shown in the figure, yeast transformed with the pGBKT7 vector cannot grow normally, while yeast transformed with the pCL1 and pGBKT7-CmNAC2 vectors can grow normally and can activate the activity of α-galactosidase, showing blue on SD / -Ade-His medium containing X-α-gal, indicating that CmNAC2 has transcriptional activation activity.

[0175] (v) Yeast monohybrid verification of whether CmNAC2 binds to the CmMYB15-like promoter.

[0176] Based on the above experimental results, in order to further clarify whether CmNAC2 can bind explicitly to the CmMYB15-like promoter, the inventors conducted further experimental verification using yeast single-hybrid experiments. The specific experimental process is briefly described below.

[0177] First, referring to the foregoing description and existing technical procedures, based on the Kpn I and SaII restriction sites, the CmMYB15-like promoter segments (FR, F1R, FR1) were ligated into the pAbAi vector, and the recombinant plasmid was further transformed into yeast Y1H competent cells to obtain a Y1H yeast strain containing the above three promoter fragment sequences.

[0178] Simultaneously, referring to the above, the CmNAC2 gene was ligated and recombined with the pGADT7 vector, and the correctly identified recombinant plasmid pGADT7-CmNAC2 was extracted and prepared for use.

[0179] Subsequently, pGADT7 (empty control) and recombinant plasmid pGADT7-CmNAC2 were transformed into the aforementioned Y1H yeast competent cells containing the CmMYB15-like promoter fragment, respectively.

[0180] After transformation, the negative control (pGADT7+pAbAi-FR / F1R / FR1) and the experimental group (pGADT7-CmNAC2+pAbAi-FR / F1R / FR1) were respectively plated on SD / -Leu-Ura medium and incubated upside down at 30℃ for 3 days.

[0181] After culturing, select single clones, suspend them in 0.9% NaCl solution, and adjust the OD. 600=Approximately 0.2, take 5 μL and serially dilute, then spot onto SD / -Leu medium with no AbA and with AbA concentration gradients of 100, 200, 500, and 1000 ng / mL, respectively. After incubating upside down at 30℃ for 3 days, observe the colony growth and take pictures.

[0182] Experimental results are as follows Figure 12 As shown in the figure. Analysis shows that both the negative control and the experimental group showed colony growth on SD / -Leu medium without AbA. However, on SD / -Leu medium containing 200 ng / mL AbA, the negative control did not show colony growth, while the experimental group still showed colony growth. The full-length FR segment showed the strongest binding, the FR1 segment showed a slightly weaker binding, and the F1R segment showed the weakest binding. This result preliminarily indicates that CmNAC2 can indeed specifically bind to the CmMYB15-like promoter.

[0183] (vi) Verification of the binding of CmNAC2 to the CmMYB15-like promoter by dual-luciferase assay

[0184] Based on the preliminary verification results above, the inventors further verified and analyzed the binding of CmNAC2 to the CmMYB15-like promoter using a dual-luciferase assay. The relevant experimental results are briefly described below.

[0185] First, referring to the foregoing description and existing technical procedures, the CmMYB15-like promoter sequence was ligated and recombined with the pGreenⅡ-0800-LUC plasmid to construct the recombinant plasmid: pGreenⅡ-0800-LUC-pro-CmMYB15-like; and then further transformed into GV3101 (psoup) Agrobacterium competent cells;

[0186] Subsequently, positive Agrobacterium tumefaciens containing recombinant plasmids pORE-R4-CmNAC2 and pGreenⅡ-0800-LUC-pro-CmMYB15-like, as well as the pORE-R4 empty vector, were inoculated into YEB liquid medium (50 mg / L Kan, 50 mg / L Rif) and cultured at 28℃ with shaking at 200 rpm until OD600=1.0.

[0187] After the culture was completed, the bacterial cells were collected by centrifugation at 4000 rpm for 5 min. The bacterial cells were resuspended in infection buffer (10 mM MES, 10 mM MgCl2, 150 μM AS, pH=5.7) and the OD600 was adjusted to about 0.8 as the infection solution.

[0188] During infection, tobacco leaves aged 5-6 weeks were used as the target. The control group (pORE-R4 empty + pGreenⅡ-0800-LUC-pro-CmMYB15-like) and the experimental group (pORE-R4-CmNAC2 + pGreenⅡ-0800-LUC-pro-CmMYB15-like) were mixed evenly at a ratio of 1:1 (volume ratio). The infection solution was then injected into the leaves from the underside of the leaves using a 1mL syringe. After injection, the leaves were cultured in the dark for 1 day and then placed under normal light conditions for another day.

[0189] Before testing, D-fluorescein potassium salt was sprayed on the underside of the leaves. After spraying, the leaves were left to stand in the dark for 5 minutes before the fluorescence signal was observed and photographed using a fully automated chemiluminescence image analysis system (Tamon 5200).

[0190] In addition, the activities of firefly luciferase and sea cucumber luciferase were detected using the Dual Luciferase Reporter Assay Kit (Novizan, Nanjing) (follow the instructions for operation).

[0191] Experimental results are as follows Figure 13 As shown in the figure, the analysis reveals that CmNAC2 significantly activates the expression of the luciferase gene driven by the CmMYB15-like promoter. Furthermore, fluorescence detection results (with René luciferase RLuc fluorescence as an internal control, co-transformed pORE-R4+35S-RLuc-proCmMYB15-like-Luc as the control group, and co-transformed pORE-R4-CmNAC2+35S-RLuc-proCmMYB15-like-Luc as the experimental group) show a significant increase in LUC fluorescence value in the experimental group. These results clearly indicate that CmNAC2 activates the expression of the CmMYB15-like gene in vivo.

[0192] Example 3

[0193] Building upon Example 2's identification of CmNAC2 as an upstream regulator of the CmMYB15-like gene, the inventors further analyzed the function of CmNAC2 in drought resistance in chrysanthemum. A brief summary of the specific experimental findings is as follows.

[0194] (I) Response of CmNAC2 under drought stress

[0195] Based on the relevant analytical materials in Example 1, the inventors further analyzed the response of CmNAC2 under drought stress (the specific experimental process and operation can be referred to the above and conventional operation of existing technology).

[0196] The results of CmNAC2 expression level detection showed that ( Figure 14 As drought duration increases, CmNAC2 Gene expression levels were also significantly upregulated, reaching a peak at 12 h, which was 7.27 times that of the control group. This indicates that... CmNAC2 It can also respond to drought stress in chrysanthemums.

[0197] Further analysis of CmNAC2 expression patterns under normal growth conditions showed that ( Figure 15 (Samples were taken from the above-ground apical buds, leaves, stems, and roots of wild-type chrysanthemum 'Shenma' during vegetative growth). CmNAC2 was expressed in apical buds, leaves, stems, and roots, but the expression level was lowest in apical buds and highest in stem tissue, which was 12.9 times that of apical buds. Based on this, it can be preliminarily inferred that CmNAC2 mainly plays a role in stems.

[0198] (II) Construction of transiently silenced CmNAC2 gene chrysanthemum

[0199] To further verify and evaluate the function of the CmNAC2 gene in drought resistance of chrysanthemum, the inventors constructed a vector for transient silencing of the CmNAC2 gene based on virus-induced gene silencing (VIGS) technology and created chrysanthemum germplasm with the CmNAC2 gene silenced. The specific experimental details are briefly described below.

[0200] First, an artificial interference small RNA sequence (amiRNA) was selected based on the CmNAC2 gene sequence, and interference primers (Oligo primers) were designed accordingly as follows:

[0201] CmNAC2-I miR-s: 5'-gaTTTGAGGTGTAAATGCGTCCTtctctcttttgtattcc-3',

[0202] CmNAC2-II miR-a: 5'-gaAGGACGCATTTACACCTCAAAtcaaagagaatcaatga-3',

[0203] CmNAC2-III miR*s: 5'-gaAGAACGCATTTACTCCTCAATtcacaggtcgtgatatg-3',

[0204] CmNAC2-IVmiR*a: 5'-gaATTGAGGAGTAAATGCGTTCTtctacatatattcct-3',

[0205] Primer A: 5'-CTGCAAGGCGATTAAGTTGGGTAAC-3',

[0206] Primer B: 5'-GCGGATAACAATTTCACACAGGAAACAG-3'.

[0207] Subsequently, based on the primer design described above, overlapping PCR technology was used for amplification:

[0208] In the first round of PCR amplification, PCR amplification was performed using primer sets of "primer A + CmNAC2-IV miR*a", "CmNAC2-III miR*s + CmNAC2-II miR-a", and "CmNAC2-I miR-s + primer B", respectively.

[0209] The 50μl amplification system was designed as follows:

[0210] 2X ApexHF FS PCR Master Mix, 25.0 μL;

[0211] Forward oligo, 2.0 μL;

[0212] Reverse oligo, 2.0 μL;

[0213] Plasmid DNA, 2.0 μL;

[0214] ddH2O, 19.0 μL;

[0215] The PCR reaction program was as follows: 94℃ for 2 min; 98℃ for 10 s, 60℃ for 30 s, 72℃ for 5 s, 35 cycles; extension at 72℃ for 5 min; the PCR amplification products were detected by 1% agarose gel electrophoresis and purified by gel extraction.

[0216] Using the recovered products from the first round of PCR as templates, a second round of PCR amplification was performed. The amplification system of 50 μl was designed as follows:

[0217] 2X ApexHF FS PCR Master Mix, 25.0 μL;

[0218] Oligo A, 2.0 μL;

[0219] Oligo B, 2.0 μL;

[0220] The first round of PCR amplification yielded 3.0 μL of recovered product.

[0221] ddH2O, 18.0 μL;

[0222] After the PCR amplification product was recovered and purified, it was double-digested with Kpn I and Xba I, ligated into the PCVA vector for recombination, and transformed into DH5α competent E. coli cells to ensure the correct construction of the recombinant plasmid.

[0223] Next, the correctly constructed recombinant plasmid vector pCVA-amiRCmNAC2 and the pCVA and pCVB vector plasmids were transformed into Agrobacterium tumefaciens strain EHA105. The correctly transformed recombinant strains were screened and identified and inoculated into YEB (50 mg / L Kan + 50 mg / L Rif) liquid medium and cultured overnight. The cells were collected by centrifugation, resuspended in resuspension (10 mM MES, 200 μM AS, 10 mM MgCl2) and the OD600 was adjusted to about 1.5.

[0224] Equal volumes of CaLCuV-amiRCmNAC2 (pCVA-amiRCmNAC2+ pCVB) resuspension from the treatment group and CaLCuV (pCVA + pCVB) from the control group were mixed and allowed to stand at room temperature in the dark for 4 h to serve as the infection solution. Vacuum infection was used to infect the leaves of chrysanthemum tissue culture seedlings with a growth of about 6-8 cm. During infection, the time was 5-8 min / time, and the process was repeated 3-4 times.

[0225] After infection, the plants were rinsed under running water to remove the bacterial solution on the plant surface. They were then cultured at a low temperature of 8-10℃, in a humid and dark environment for 3 days, and then transplanted into seedling trays and placed in a culture environment of 22-25℃.

[0226] Samples were taken for PCR identification after 2 weeks of culture; samples were taken for detection of changes in the expression level of the target gene after 4 weeks of culture to assess whether the gene silencing line was successfully constructed.

[0227] Some experimental results are as follows Figure 16 , Figure 17 As shown in the figure, the analysis shows that a CmNAC2 gene silencing line was successfully obtained. Furthermore, the gene expression level detection results indicate that the expression level of CmNAC2 in the gene silencing line is only 0.46 times that of the empty vector control group, which indicates that the CmNAC2 gene silencing effect is good.

[0228] (III) Drought resistance phenotype of CmNAC2 silent plants

[0229] Following the experimental procedure described in Example 1 above, CmNAC2 silent plants (with no load as the control) were subjected to 14 days of drought stress treatment. Plant phenotypes were observed, samples were collected, and photographs were taken for analysis.

[0230] Relevant experimental results are as follows Figure 18 , Figure 19As shown in the figure, under normal culture conditions, both the CaLCuV empty control plants and the CmNAC2 silent plants grew well; however, after drought stress treatment, the leaves of the CmNAC2 gene-silenced plants showed more severe water loss and wrinkling, indicating a deeper degree of stress damage. Further statistical analysis revealed that after drought stress treatment, the survival rate of gene-silenced plants was 23%, compared to 83% for the empty control plants; additionally, the relative water content of gene-silenced plants was 35%, while that of the empty control plants was 86%. These results indicate that silencing the CmNAC2 gene can accelerate water loss to some extent, thereby reducing the drought resistance of chrysanthemums.

[0231] (iv) Changes in relevant physiological and biochemical indicators

[0232] Referring to Example 1, changes in relevant physiological indicators were measured. A brief summary of the specific changes is as follows.

[0233] (1) Changes in relative conductivity

[0234] Comparison of relative conductivity measurements results as follows Figure 20 As shown in the figure, the analysis reveals that the relative electrical conductivity of the leaves of the silenced plant was 64%, which is 4.59 times that of the uncontrolled plant (14%). This result indicates that silencing the CmNAC2 gene leads to increased damage to the cell membrane and more severe electrolyte leakage.

[0235] (2) FV / FM variation

[0236] Comparison results of the maximum photochemical efficiency (FV / FM) measurement of leaf photosystem II (PSII) are as follows: Figure 21 As shown in the figure, the analysis reveals that after drought stress treatment, the FV / FM ratio of the unloaded control plants was 0.76, which is 1.4 times that of the silenced plants (0.54). This result indicates that silencing the CmNAC2 gene reduces the light energy conversion efficiency within the PSII reaction center of chrysanthemum, thereby reducing the plant's photosynthetic capacity and stress resistance.

[0237] (3) Changes in SOD and POD activity

[0238] The results of SOD and POD activity assays are as follows: Figure 22As shown in the figure. Analysis reveals that after drought stress treatment, the SOD and POD activities of gene-silenced plants were significantly lower than those of the control plants. Specifically, the SOD activity of the empty vector control plants was 222.96 U·g⁻¹, an increase of 106.51 U·g⁻¹ compared to the 116.45 U·g⁻¹ of the silenced plants; the POD activity of the empty vector control plants was 3363.6 U·g⁻¹·min⁻¹, an increase of 1523 U·g⁻¹·min⁻¹ compared to the 1837.6 U·g⁻¹·min⁻¹ of the silenced plants. These results indicate that silencing the CmNAC2 gene reduces the SOD and POD activities of plants, thereby reducing reactive oxygen species scavenging capacity and weakening drought resistance.

[0239] (4) Changes in MDA content

[0240] MDA content test results are as follows Figure 23 As shown in the figure, the MDA content of the empty control plant was 26.69 μmol·g⁻¹, which was 29.86 μmol·g⁻¹ lower than that of the silenced plant (56.55 μmol·g⁻¹). This result indicates that silencing the CmNAC2 gene deepens the degree of oxidative damage to the cell membrane, thereby weakening its antioxidant capacity.

[0241] (5) Changes in proline content

[0242] The results of the proline content test are as follows: Figure 24 As shown, the proline content of the unsupported control plants was 378.23 μg·g⁻¹, an increase of 150.61 μg·g⁻¹ compared to the 227.62 μg·g⁻¹ of the silenced plants. This result indicates that silencing the CmNAC2 gene reduces cellular osmoregulation by decreasing proline content, thereby increasing the instability of protoplasmic colloids and intratissue metabolism, ultimately leading to accelerated cellular dehydration and weakened drought resistance.

Claims

1. CmNAC2 The application of genes in regulating drought tolerance in chrysanthemums is characterized by, silence CmNAC2 The gene is used to reduce the drought tolerance of chrysanthemums; CmNAC2 The gene, whose nucleotide sequence is shown in SEQ ID No.

3.

2. Utilize CmNAC2 The method for breeding chrysanthemum varieties based on genes is characterized by, Through silence CmNAC2 Genes were used to breed low-water-consumption chrysanthemum varieties; The CmNAC2 The gene, whose nucleotide sequence is shown in SEQ ID No. 3.

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