Application of NO-mediated CqDOF3 gene in regulating plant drought resistance

Abstract

This invention discloses the application of the CqDOF3 gene in regulating plant drought resistance under NO-mediated regulation. The CqDOF3 gene's CDS sequence is shown in SEQ ID No. 1, and its protein sequence is shown in SEQ ID No. 2. This invention investigated the mechanism by which NO regulates the quinoa DOF3 gene in response to drought stress, finding that NO upregulates CqDOF3 gene expression, enhances antioxidant enzyme activity, and reduces reactive oxygen species accumulation, thereby significantly improving the drought resistance of transgenic plants, promoting plant physiological adaptation, enriching the theoretical basis of plant stress resistance, and contributing to the cultivation of high-quality quinoa varieties adapted to drought environments.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the CqDOF3 gene mediated by NO in regulating plant drought resistance. Background Technology

[0002] Quinoa (Chenopodium quinoa Willd.) is a traditional Andean crop, widely valued for its seeds' high protein content and favorable amino acid composition. However, drought resistance varies among species, and varieties that are both drought-resistant and high-yielding are lacking in practical production. Furthermore, limited understanding of the genetic background of numerous domestic and international germplasm resources hinders in-depth and systematic research at the molecular level, limiting the application and contribution of molecular breeding techniques. The publication of the quinoa reference genome in 2017 spurred quinoa breeding and cultivation research, while also providing new research opportunities for exploring the drought resistance mechanisms and metabolic regulation of quinoa at the molecular level. However, to date, research on the molecular mechanisms of quinoa drought resistance remains relatively limited and urgently requires further exploration and strengthening.

[0003] DOF (DNA binding with one finger) is a plant-specific family of transcription factors, first discovered in maize. It is plant-specific and has not been reported in other eukaryotes (such as yeast and animals). A typical DOF protein consists of 200–400 amino acids. Its N-terminus contains a highly conserved DOF domain of 50–52 amino acids, while its C-terminus contains nuclear localization signaling and transcriptional regulatory domains. Its structure is a Cys2 / Cys2Zn2+ finger structure, regulating DNA-protein and protein-protein interactions and recognizing specific cis-elements (AT) / AAAG in the promoter regions of its target genes. It has been shown to participate in abiotic stress in crops such as Arabidopsis thaliana, rice, and maize, but no studies have been reported in quinoa.

[0004] The regulatory role of gaseous signaling molecules in plant stress responses has attracted increasing attention. Nitric oxide (NO) is a gaseous signaling molecule widely present in plants, and its synthesis includes both enzymatic and non-enzymatic pathways. It plays a crucial role in various physiological processes, including plant growth and development, signal transduction, and responses to biotic and abiotic stresses. Exogenous application of NO donors (such as SNPs) has been shown to significantly improve the drought tolerance of various plant species. However, the molecular-level regulation of DOF family members, particularly in quinoa, and the mechanisms by which they exert their drought-resistance effects remain unclear. Therefore, a systematic study of the drought resistance mechanism of the quinoa DOF3 gene and the role of exogenous NO in regulating the drought resistance of transgenic plants is of great significance, aiming to provide a scientific basis and new strategies for drought-resistant breeding of quinoa. Summary of the Invention

[0005] To address the aforementioned technical limitations, this application proposes the application of the CqDOF3 gene mediated by NO in regulating plant drought resistance; it overcomes the deficiencies and defects mentioned in the background art.

[0006] The purpose of this invention is to provide an application of the CqDOF3 gene in regulating plant drought resistance under NO-mediated conditions, characterized in that the CDS sequence of the CqDOF3 gene is shown in SEQ ID No. 1, and the protein sequence encoded by the CqDOF3 gene is shown in SEQ ID No. 2.

[0007] Furthermore, the primer pair for amplifying or detecting the CqDOF3 gene according to claim 1 includes CqDOF3-F and CqDOF3-R; wherein the nucleotide sequence of CqDOF3-F is shown in SEQ ID No. 3, and the nucleotide sequence of CqDOF3-R is shown in SEQ ID No. 4.

[0008] Furthermore, the CqDOF3 gene positively regulates plant drought resistance by modulating the activity of plant-related antioxidant enzymes.

[0009] Furthermore, overexpression of the CqDOF3 gene as described in claim 1 includes the following steps:

[0010] S1: Construction of recombinant expression vector: Using an expression vector with SacⅠ and XbaⅠ restriction sites, CqDOF3 is linked between the SacⅠ and XbaⅠ restriction sites of the expression vector to form a recombinant vector overexpressing CqDOF3;

[0011] S2: The constructed pCAM-CqDOF3 overexpression vector was transformed into Agrobacterium GV3101, and then the vector was transformed into quinoa callus and Arabidopsis thaliana by Agrobacterium-mediated transformation to achieve the expression of the CqDOF3 gene, thereby obtaining transgenic plants overexpressing CqDOF3.

[0012] Furthermore, primer pairs used to detect CqDOF3 gene expression in transgenic plants include:

[0013] (a): The primer pair used to detect transgenic quinoa callus includes 35S-F and 35S-R; wherein the nucleotide sequence of 35S-F is shown in SEQ ID No. 5 and the nucleotide sequence of 35S-R is shown in SEQ ID No. 6;

[0014] (b): The primer pairs used to detect transgenic Arabidopsis include F3-F and F3-R; wherein the nucleotide sequence of F3-F is shown in SEQ ID No.7 and the nucleotide sequence of F3-R is shown in SEQ ID No.8.

[0015] Furthermore, the plant in question is quinoa.

[0016] Furthermore, the CqDOF3 gene enhances plant drought resistance by regulating the activity of plant-related antioxidant enzymes.

[0017] The application of the CqDOF3 gene mediated by NO in regulating plant drought resistance provided by this invention has the following beneficial effects:

[0018] This invention has achieved significant beneficial effects by studying the application of the CqDOF3 gene in regulating plant drought resistance under NO-mediated regulation. The study found that NO can significantly upregulate the expression of CqDOF3, enhance the activity of antioxidant enzymes, and reduce the accumulation of reactive oxygen species under drought stress, thereby effectively improving the drought resistance of quinoa. This research not only provides new theoretical support for understanding plant stress resistance, but also provides potential genetic engineering methods for breeding drought-resistant plants, which will help to cultivate high-quality quinoa varieties adapted to drought environments. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This invention relates to the conserved domains, secondary structures, and tertiary structures of the CqDOF3 protein. Figure 1 A is a conservative structural domain. Figure 1 B is a two-level structure. Figure 1 C represents a three-level structure;

[0021] Figure 2 This invention relates to the construction of a phylogenetic tree; Figure 2 A is the sequence alignment diagram; Figure 2 B represents the phylogenetic tree;

[0022] Figure 3 This invention is for predicting CqDOF3 interacting proteins;

[0023] Figure 4 This refers to the relative expression level of CqDOF3 in this invention; Figure 4 A represents the expression level of the CqDOF3 gene in the roots and leaves of quinoa under different drought treatments. Figure 4 B represents the expression level of CqDOF3 under NO+ drought treatment; Figure 4 C represents the expression level of CqDOF3 under PTIO+ drought treatment; Figure 4 D represents the organizational expression mode of CqDOF3;

[0024] Figure 5 This invention relates to the construction of a vector that overexpresses the CqDOF3 gene; Figure 5 A is a schematic diagram of CqDOF3 gene overexpression; Figure 5 B represents the amplification of the CqDOF3 gene; Figure 5 C represents the ligation verification of the cloning vector; Figure 5 Verification using double enzyme digestion (D); Figure 5 E represents the detection of Agrobacterium tumefaciens liquid PCR using the ligation vector pCAM-CqDOF3-GFP; Figure 5 B. Figure 5 In C, M represents DNA Marker DL 5000; Figure 5 D and Figure 5 In E, M represents DNA Marker DL 5000 and DNA Marker DL 2000; the number indicates the target band.

[0025] Figure 6 This is the subcellular localization of the CqDOF3 protein of this invention; from left to right, they are green fluorescent protein, chloroplast autofluorescence, bright field, and superimposed field.

[0026] Figure 7 This is a positive verification of the genetic transformation of quinoa and Arabidopsis thaliana in this invention; Figure 7 A is a positive PCR identification of quinoa callus overexpressing; Figure 7 B represents the relative expression level of the CqDOF3 gene in callus tissue; Figure 7 C represents the result of overexpressing Arabidopsis thaliana; Figure 7 D represents positive PCR identification of Arabidopsis thaliana overexpressing plants. Figure 7 A and Figure 7 In D, M represents DNA marker DL 2000, N represents negative control (wild type), P represents positive control (plasmid), and the number represents the target band; Figure 7 E represents the qRT-PCR validation of 16 Arabidopsis thaliana lines overexpressing the CqDOF3 gene;

[0027] Figure 8 This invention relates to the phenotype and biomass of CqDOF3 transgenic quinoa callus under drought stress mediated by exogenous NO. Figure 8 A represents the phenotype of quinoa callus tissue after 30 days of different treatments; Figure 8 B represents the fresh weight of quinoa callus tissue. Figure 8 C represents the dry weight of quinoa callus tissue;

[0028] Figure 9 This invention relates to the antioxidant enzyme activity, osmotic regulators, and malondialdehyde content of CqDOF3 transgenic callus under drought stress mediated by exogenous NO. Figure 9 A represents SOD activity; Figure 9B represents POD activity; Figure 9 C represents CAT activity; Figure 9 D represents the proline content; Figure 9 E represents the soluble protein content; Figure 9 F represents the malondialdehyde content;

[0029] Figure 10 This invention describes the phenotype, fresh weight, and dry weight of CqDOF3 transgenic Arabidopsis thaliana lines under drought stress mediated by exogenous NO. Figure 10 A represents the Arabidopsis thaliana drought phenotype; Figure 10 B represents fresh weight; Figure 10 C represents dry weight;

[0030] Figure 11 This invention describes the water loss phenotype of detached leaves of the CqDOF3 transgenic Arabidopsis thaliana strain, the water loss rate of detached leaves, and the relative water content. Figure 11 A represents the dehydration phenotype of detached leaves from transgenic Arabidopsis thaliana lines; Figure 11 B represents the water loss rate of the detached leaf; Figure 11 C represents the relative water content under drought stress mediated by exogenous NO.

[0031] Figure 12 This invention relates to the seed germination rate and root length of CqDOF3 transgenic Arabidopsis thaliana lines under drought stress mediated by exogenous NO. Figure 12 AB represents the germination rate of transgenic Arabidopsis seeds treated with 0 and 200 mM mannitol; Figure 12 C represents the seed germination rate of CqDOF33 transgenic Arabidopsis thaliana lines treated with exogenous NO mannitol; Figure 12 D represents the root length of seeds from CqDOF3 transgenic Arabidopsis thaliana lines under exogenous NO-mediated drought stress; Figure 12 E represents the root length phenotype;

[0032] Figure 13 This invention relates to photosynthetic pigments in transgenic Arabidopsis thaliana lines with CqDOF3 under drought stress mediated by exogenous NO3. Figure 13 A represents chlorophyll content; Figure 13 B represents the chlorophyll a content; Figure 13 C represents the chlorophyll b content; Figure 13 D represents the carotenoid content;

[0033] Figure 14 This invention relates to the antioxidant enzyme activity, osmotic regulators, and MDA content of CqDOF3 transgenic Arabidopsis thaliana lines under drought stress mediated by exogenous NO. Figure 14 A represents SOD activity; Figure 14 B represents POD activity; Figure 14 C represents CAT activity; Figure 14 D represents the soluble protein content; Figure 14 E represents the proline content; Figure 14 F is the relative conductivity; Figure 14 G represents the malondialdehyde content;

[0034] Figure 15 This invention relates to the level of membrane lipid peroxidation in transgenic Arabidopsis thaliana lines with CqDOF3 under drought stress mediated by exogenous NO. Figure 15 A shows NBT staining of leaves from Arabidopsis thaliana overexpressing the gene; Figure 15 B represents DAB staining of Arabidopsis thaliana leaves overexpressing DAB. Figure 15 C represents the rate of superoxide anion production; Figure 15 D represents the hydrogen peroxide content. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1

[0037] See Figures 1 to 15 As shown, this application provides the application of the CqDOF3 gene in regulating plant drought resistance under NO-mediated conditions. The nucleotide sequence of the CqDOF3 gene is shown in SEQ ID No. 1, and the amino acid sequence of the protein expressed by the CqDOF3 gene is shown in SEQ ID No. 2.

[0038] SEQ ID No. 1:

[0039]

[0040] SEQ ID No. 2:

[0041] MMDPAIKLFGKTIPVIGDNESPRYDSNESSNSDLHDVDDKESTEDAMEIEQSDDPAPPPEEDSTNPPIMSDTNDNEKAPDSEKESEEMDEKESDDQKSSQQKTLKKPDKILP CPRCNSMDKFCYYNNYNVNQPRHFCKSCQRYWTAGGTMRNVPVGAGRRKNKSSASRYCHITVSEALQAARLDVPNGVHHPGLKTNGTVLTFGPDNPLCESMLNIADKKVLNG TTATPRNGFHNMDQVVCRSKENGDDRSSGSTVTTTSSMDEKATAGQQGQIKPNLNGFTNQMPCYQWPYMWNPAVPVPPFFPSAFPMSFYPPPPYWNGAMPWNFPWVSPCPPGQ KSPESNPNSPTLGKHSRDGEMLNQSENEKSIGTPTVLVPKTLRIDDPDEAANSSIWSTLGIKKGDSVSKGGGFFNTLQSKKKDDDLHNIDVPPALQANPAALCRSMNFQESA.

[0042] The primer pairs for amplifying the CqDOF3 gene include CqDOF3-F and CqDOF3-R; wherein the nucleotide sequence of CqDOF3-F is shown in SEQ ID No. 3; and the nucleotide sequence of CqDOF3-R is shown in SEQ ID No. 4.

[0043] SEQ ID No. 3:

[0044] AGAACACGGGGGACGAGCTCATGATGGATCCAGCGATTAAATTGTTTG;

[0045] SEQ ID No.4:

[0046] ACCATGGTGTCGACTCTAGAAGCACTCTCTTGGAAGTTCATTGAC;

[0047] Primer pairs for amplifying the CqDOF3 gene in transgenic quinoa callus include 35S-F and 35S-R; the nucleotide sequence of 35S-F is shown in SEQ ID No. 5; and the nucleotide sequence of 35S-R is shown in SEQ ID No. 6. Primer pairs for amplifying the CqDOF3 gene in transgenic Arabidopsis thaliana include F3-F and F3-R; the nucleotide sequence of F3-F is shown in SEQ ID No. 7; and the nucleotide sequence of F3-R is shown in SEQ ID No. 8.

[0048] SEQ ID No. 5:

[0049] GATGGATCCAGCGATTAAA;

[0050] SEQ ID No. 6:

[0051] AGAAGCACTCTCTTGGAAGT;

[0052] SEQ ID No. 7:

[0053] GATGGATCCAGCGATTAAAT

[0054] SEQ ID No. 8:

[0055] AGAAGCACTCTCTTGGAAGT

[0056] Overexpression of the CqDOF3 gene can improve the drought resistance of plants.

[0057] A recombinant expression vector was constructed using the CqDOF3 gene, and the recombinant expression vector was transferred into plants to improve the drought resistance of the plants.

[0058] The expression vector for overexpressing CqDOF3 has SacⅠ and XbaⅠ restriction sites. CqDOF3 is linked between the SacⅠ and XbaⅠ restriction sites of the expression vector to form a recombinant vector for overexpressing CqDOF3.

[0059] The overexpression vector was pCAMBIA2300-35S-EGFP.

[0060] The plant selected was Arabidopsis thaliana or quinoa callus.

[0061] The CqDOF3 gene enhances plant drought resistance by increasing antioxidant capacity.

[0062] Example 2

[0063] An application of the CqDOF3 gene mediated by NO in regulating plant drought resistance, characterized by the following steps:

[0064] (1) Bioinformatics of the quinoa CqDOF3 gene;

[0065] (2) Expression pattern and subcellular localization of CqDOF3 in quinoa;

[0066] (3) The CqDOF3 gene was overexpressed in quinoa callus and Arabidopsis thaliana, and their drought resistance under exogenous NO regulation was analyzed.

[0067] I. Bioinformatics Analysis of Quinoa CqDOF3 Gene

[0068] 1. Method

[0069] 1.1 Bioinformatics analysis of the CqDOF3 gene:

[0070] Bioinformatics analysis of the CqDOF3 gene was performed using the software shown in Table 1.

[0071]

[0072] Table 1

[0073] 2. Results

[0074] 2.1 Bioinformatics analysis of the CqDOF3 gene

[0075] 2.1.1 Analysis of the basic physicochemical properties of the protein encoded by the CqDOF3 gene:

[0076] The full-length CDS of the quinoa DOF3 gene is 1352 bp. The protein encoded by the CDS contains 450 amino acids, has a molecular weight of 49.6 kDa, a theoretical isoelectric point of 5.13 (classifying it as an acidic protein), an instability index of 55.6 (classifying it as an unstable protein), and an average hydrophilicity of -0.901, which is unevenly distributed throughout the peptide chain (classifying it as a hydrophilic protein). The CqDOF3 protein contains a typical DOF domain between amino acids 108 and 166, belonging to the DOF transcription factor family (…). Figure 1 A); Secondary structure prediction showed that the protein is mainly composed of three components: α-helix, extended strand, and random coil, accounting for 6.44% (29), 8.00% (36), and 85.56% (385), respectively, with no β-sheet. Figure 1 B), the predicted secondary structure of the tertiary domain of this protein is consistent with the results. Figure 1 C).

[0077] 2.1.2 Phylogenetic analysis of quinoa DOF3 protein

[0078] Homologous sequence alignment analysis of quinoa DOF3 protein with 14 other species revealed a highly conserved sequence LKKFDKIIPCPRONSMDTKFCYYNNYNVYQPRHFCKSCQRYWTAGGTMRNPVGAGRRK in almost all protein sequences. Figure 2 A). Phylogenetic analysis showed that the 15 species could be divided into four groups, with quinoa DOF3 being most closely related to spinach DOF3, and most distantly related to Arabidopsis thaliana and rice. Figure 2 B).

[0079] 2.1.3 Predictive Analysis of Quinoa DOF3 Protein Interaction

[0080] Protein interaction prediction revealed that CqDOF3 protein may interact with ADO3 (Adagio protein), COL5 (zinc finger protein CONSSTANS-LIKE 5), APRR9, F12L6.19 (CASP-like protein 4D1), APRR7, NAC020, CCA1 (CIRCADIANCLOCK-ASSOCIATED 1), T3A4.5 (CCT motif family protein), HAM1 (MYST family histone acetyltransferase 1), and MEE13.8 (hydrolase superfamily protein).

[0081] 2.1.4 Analysis of cis-regulatory elements in the promoter of the quinoa DOF3 gene

[0082] Analysis of the cis-regulatory elements of the quinoa DOF3 gene promoter revealed (Table 2) that the promoter region of this gene contains 9 hormone-responsive elements (auxin, gibberellin, jasmonic acid, zeatin, and salicylic acid), 1 defense and stress-responsiveness element (TC-rich repeats), 4 anaerobic-inducible elements (ARE), 2 low-temperature-responsive elements (I-box, LTR), 1 defense and stress-responsiveness element (TC-rich repeats), and 12 light-responsive elements.

[0083]

[0084] Table 2

[0085] II. Subcellular localization and tissue-specific expression analysis of CqDOF3 in quinoa

[0086] 1. Method:

[0087] 1.1 Amplification of the target fragment:

[0088] (1) RNA extraction and cDNA synthesis

[0089] The experimental material was quinoa variety HZ-2-13. Quinoa seeds were grown in an environment of 24℃ / 22℃ (day / night), 16 h light / 8 h darkness, and relative humidity of 75±5%. When quinoa seedlings reached 60 days old, quinoa leaves were collected, placed in cryovials, and then flash-frozen in liquid nitrogen and stored at -80℃ for subsequent experiments. RNA was extracted from quinoa leaves using the Trizol method with the AGRNAex Pro RNA extraction kit. cDNA synthesis was performed using the Evo M-MLV reverse transcription premixed kit; detailed instructions for both methods can be found in their respective product manuals.

[0090] (2) Design of primers for the CqDOF3 gene and gene cloning

[0091] The overexpression and subcellular localization vector was pCAMBIA2300-35S-EGFP. Specific primers were designed using homologous recombination. Primers F (SEQ ID No. 3) and R (SEQ ID No. 4) were designed based on the CDS sequence of CqDOF3. The amplified sequence was inserted into the multiple cloning site downstream of the CaMV35S promoter of the vector and fused with the GFP expression protein. The primers were synthesized by Shanghai Sangon Biotech, and the restriction enzyme sites were SacⅠ and XbaⅠ.

[0092] The CqDOF3 gene amplification reaction system (PCR) is shown in Table 3.

[0093]

[0094] Table 3

[0095] The conditions for CqDOF3 gene amplification reaction are shown in Table 4.

[0096]

[0097] Table 4

[0098] (3) Purification and recovery of PCR products:

[0099] The PCR products were purified and recovered using the Steady Pure DNA Gel Extraction Kit. For detailed steps, please refer to the instruction manual.

[0100] 1.2 Linearization of expression vector plasmids:

[0101] In this study, the pCAMBIA2300-GFP empty vector plasmid was linearized using SacⅠ and XbaⅠ. The enzyme digestion system is shown in Table 3, and the enzyme digestion reaction procedure is shown in Table 5. After digestion, the target vector fragment was detected by 1% agarose gel electrophoresis and recovered from the gel. The fragment was then stored at -20℃.

[0102]

[0103] Table 5

[0104]

[0105] Table 6

[0106] 1.3 Homologous recombination reaction:

[0107] The CqDOF3 gene amplification product was ligated with the enzyme-digested pCAMBIA2300-GFP vector via homologous recombination. The reaction conditions were set at 50℃ for 15 min, followed by an ice bath for 5 min. The resulting recombinant product was stored at -20℃ for later use.

[0108] 1.4 Transformation of E. coli with recombinant expression vector pEGFP-CqDOF3:

[0109] Place E. coli competent cells DH5α on ice. When they become a mixed ice-water state, take 10 μL of the homologous recombination reaction product and add it to a test tube containing DH5α. Incubate on ice for 30 min, then incubate in water at 42℃ for 50 s, and react on ice for 2 min. Then add 700 μL of LB medium and incubate at 220 r / min and 37℃ for about 45-75 min. Spread evenly on LB solid medium (containing 50 μg / mL Kan) and incubate overnight at 37℃ inverted.

[0110] 1.5 Restriction digestion identification of recombinant plasmid pCAM-CqDOF3-EGFP:

[0111] When a single colony grows on an overnight culture plate, select a single colony and incubate it in 10 mL of LB medium containing 50 μg / mL Kan for 6-8 h. Using a suitable bacterial culture as a template, perform PCR identification. Send the identified positive bacterial cultures to Shanghai Sangon Biotech Co., Ltd. for sequencing, extract plasmids for enzyme digestion verification, and the suitable plasmid is identified as pCAM-CqDOF3-EGFP.

[0112] 1.6 Transformation of Agrobacterium with recombinant expression vector pCAM-CqDOF3-EGFP:

[0113] Agrobacterium competent cells GV3101 were placed on ice, and 10 μL of plasmid was added to a test tube containing GV3101. The cells were then incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. LB liquid medium (antibiotic-free) was added, and the cells were cultured at 28°C and 230 r / min for 2-3 h. The culture was then spread onto LB solid medium containing antibiotics (50 μg / mL Rif + 50 μg / mL Kan), and incubated upside down in the dark at 28°C for 48 h. A single colony was picked and cultured in 10 mL of LB liquid medium (containing Kan and Rif) at 28°C and 230 r / min for 12-18 h. The cultured bacterial solution was identified by PCR and then frozen at -80°C.

[0114] 1.7 Genetic transformation of tobacco

[0115] Infection solutions were prepared using *Agrobacterium* with the recombinant expression vector pCAM-CqDOF3-EGFP. *Agrobacterium* culture with the empty vector pCAMBIA2300-35S-EGFP was used as a control (CK). The bacterial cells were resuspended in an equal volume of MS liquid medium (containing 1% sucrose, 10 mM MES, 150 μM AS, 10 mM MgCl2, pH 5.8), and the OD600 was adjusted to 0.5. After incubation on ice for 2–3 h, the culture was used for infection. *Nicotiana benthamiana* plants that had grown for 30 days and were in good condition were selected. The bacterial solution was injected into the mesophyll tissue from the lower epidermis of the leaf abaxial surface using a sterile syringe, and the injection area was marked. Infected plants were cultured in the dark at 25°C for 12 h, then transferred to light for another 48 h. Transformation of *Nicotiana benthamiana* was then performed.

[0116] 1.8 Subcellular localization observation

[0117] Take a clean glass slide and drop an appropriate amount of sterile 10% glycerol in its center as a moisturizing and light-transmitting medium for observing the sample. Carefully cut the marked area of ​​the tobacco leaf injected with the recombinant vector using sterile scissors, avoiding the veins. Place the cut leaf tissue on the glycerol drop in the center of the glass slide. Then cover it with a coverslip, taking care to avoid air bubbles that may affect the observation. Place the coverslip face down on the stage of a laser confocal scanning microscope. Set the excitation wavelength to 488 nm and start the microscope for scanning observation. Capture and record the distribution of the green fluorescence signal of GFP (green fluorescent protein) through the imaging system to determine the subcellular localization of the fusion protein.

[0118] 1.9 Analysis of CqDOF3 gene expression patterns under drought stress

[0119] This study selected plump, disease- and pest-free quinoa seeds, sowing 20 seeds evenly in each pot. After sowing, the pots were thoroughly irrigated and then placed in a plant growth chamber (culture conditions: 25℃, 16 h / 8 h light-dark cycle, 70% humidity, 2000 Lx light intensity) for routine cultivation. When the seedlings reached 50 days of age, plants with uniform growth were selected for experimental treatment, with two groups: a control group (CK) and a drought stress group (20% PEG 6000). Based on the research group's previous study on the gene expression patterns of quinoa in response to drought stress, leaf and root samples were collected from both groups at four time points: 0 h, 6 h, 12 h, and 24 h after treatment. Each treatment was performed in triplicate. After all samples were collected, they were immediately rapidly frozen in liquid nitrogen and then transferred to a -80 ℃ freezer for storage for subsequent molecular experiments.

[0120] 1.10 Analysis of CqDOF3 gene expression pattern mediated by NO

[0121] Quinoa seedlings were cultured as described in section 1.9. Three treatments were set up: PEG (20% PEG), SNP + PEG (100 μmol L⁻¹ SNP + 20% PEG), and PTIO + PEG (200 μmol L⁻¹ PTIO + 20% PEG). Treatment times were 0 h, 6 h, 12 h, and 24 h. Leaf samples were taken from quinoa seedlings. Each treatment was performed in triplicate. After all samples were collected, they were rapidly frozen in liquid nitrogen and stored at -80°C for subsequent quantitative experiments.

[0122] 1.11 Extraction of total RNA from plant tissues and synthesis of the first strand of cDNA

[0123] Take out the quinoa sample stored at -80℃. RNA was extracted using the Trizol method with the AG RNAex Pro RNA extraction kit. cDNA synthesis was performed using the Evo M-MLV reverse transcription premix kit. For detailed steps of both, please refer to their respective instructions.

[0124] 1.12 Real-time quantitative PCR (qRT-PCR) analysis

[0125] Using cDNA from different samples obtained above as templates, real-time quantitative PCR was performed for analysis, with each treatment stage repeated in triplicate. The specific expression pattern of the CqDOF3 gene was analyzed using the 2−ΔΔCT method. Finally, analysis of variance was performed using IBM SPSS Statistics 27 software, and the Duncan method was used to compare the significance of differences between treatments at the 0.05 level. Plotting was performed using Origin 2021.

[0126] 2. Results

[0127] 2.1 Analysis of CqDOF3 gene expression patterns under drought stress

[0128] In leaf tissue ( Figure 4 A) With increasing treatment time, the expression level of CqDOF3 showed a trend of first decreasing and then increasing. It reached a peak at 6 h of drought treatment, which was 175.78 times that of the control. In the roots, it also reached the highest level at 6 h of treatment, which was 22.75 times that of the control. This indicates that drought strongly induces the expression of the CqDOF3 gene in leaves and roots.

[0129] 2.2 Analysis of CqDOF3 gene expression pattern mediated by NO

[0130] To understand whether exogenous NO induces CqDOF3 gene expression, we analyzed the expression patterns of CqDOF3 gene under PEG+SNP and PEG+PTIO treatments. The results showed that under exogenous NO treatment, the expression level of CqDOF3 gene exhibited a trend of first increasing and then decreasing. Figure 4 B), reaching its peak at 6 h, was 32.74 times that of the control. CqDOF3 gene expression level significantly decreased at 6 h after PEG+PTIO treatment. Figure 4 The expression of CqDOF3 decreased by 38% compared to 6h. This indicates that exogenous NO significantly induces the expression of the CqDOF3 gene.

[0131] 2.3 Tissue-specific analysis of the CqDOF3 gene

[0132] The expression pattern of the CqDOF3 gene in various tissues of quinoa was analyzed using qRT-PCR. The results showed that ( Figure 4 (D) The expression of this gene exhibits significant tissue specificity, with the highest expression level in leaves, followed by roots, stems, and flowers, and the lowest expression level in seeds; there is no significant difference in expression levels between stems and flowers. These results indicate that CqDOF3 expression varies significantly among different quinoa tissues, with dominant expression in leaves.

[0133] 2.4 Cloning of the CqDOF3 gene and construction of the vector

[0134] This study used quinoa leaf cDNA as a template and employed homologous recombination-specific primers for PCR amplification. The amplified product was detected by agarose gel electrophoresis, showing a target fragment size of approximately 1350 bp, consistent with the expected fragment size. The target fragment was then recovered, purified, and sequenced to verify the absence of base mutations. Figure 5 B), by using homologous recombination technology, a large fragment of the vector was ligated to the target gene, and the resulting cells were transformed into competent E. coli cells. Colony PCR and double enzyme digestion amplification both yielded a specific band of approximately 1350 bp, consistent with the size of the target fragment, indicating that homologous recombination was successful. Figure 5 CD), the recombinant vector construction was completed. After transforming the recombinant vector pCAMBIA2300-CqDOF3 into Agrobacterium GV3101 competent cells, bacterial culture PCR was performed for identification. Figure 5 E), all amplification products showed specific bands matching the size of the target fragment, proving that the quinoa CqDOF3 gene overexpression vector pCAMBIA2300-CqDOF3 had been successfully constructed. Figure 5 A).

[0135] 2.5 Fluorescence detection of pCAMBIA2300-CqDOF3-EGFP fusion protein

[0136] To clarify the subcellular localization characteristics of the CqDOF3 protein, an empty GFP vector and a recombinant CqDOF-EGFP fusion protein vector were introduced into tobacco leaf epidermal cells via Agrobacterium-mediated transient transformation. The results were observed using laser confocal microscopy. Figure 6 The empty GFP vector showed obvious green fluorescence signals in both the nucleus and cell membrane regions, while the CqDOF3-EGFP fusion protein showed fluorescence signals that were specifically concentrated in the nucleus, indicating that the CqDOF3 protein is subcellularly located in the nucleus.

[0137] III. Analysis of drought resistance of quinoa CqDOF3 mediated by exogenous NO

[0138] 1. Method:

[0139] 1.1 Agrobacterium-mediated transformation of quinoa callus

[0140] 1.1.1 Quinoa aseptic seedling cultivation

[0141] Select plump, uniformly sized quinoa seeds, rinse them under running water for 30 minutes, and then blot dry with sterile filter paper. The seeds are then sterilized. The sterilization process is as follows: first, soak the seeds in 75% ethanol for 45 seconds, then rinse them six times with sterile water; next, soak them in 0.1% HgCl2 solution for 5 minutes, then rinse repeatedly with sterile water until no HgCl2 residue remains. Finally, blot dry with sterile filter paper, and evenly sow the sterilized seeds onto MS solid medium. Transfer the seeds to a light incubator and culture for 7 days. The culture conditions are set as follows: constant temperature of 25℃, photoperiod of 16 h / 8 h, and light intensity of 4000 Lx. After the sterile seedlings have grown, the hypocotyls of the sterile seedlings are harvested in a clean bench as explants for quinoa callus induction.

[0142] 1.1.2 Agrobacterium-mediated genetic transformation of quinoa callus

[0143] (1) Preparation of bacterial culture: The bacterial culture containing the target band stored at -80℃ was added to 50 mL of LB liquid medium (containing 50 μg / mL Kan and 50 μg / mL Rif), placed in a shaker at 28℃ and 210 rpm, and shaken until OD600=0.8. Then, it was dispensed into 50 mL centrifuge tubes, placed in a centrifuge at 4℃ and 4000 r / min for 10 min, the precipitate was collected, and the bacterial culture was resuspended in MS liquid medium containing 2% sucrose (containing 0.1 mmol / L AS). The bacterial culture OD600=0.6 was ready for use.

[0144] (2) Cut off the hypocotyl: Cut off the hypocotyl (about 1 cm) of the sterile seedling that has grown for 7 days in the ultra-clean workbench and place it in sterile water.

[0145] (3) Infecting hypocotyl: Carefully pour out sterile water, pour in the resuspended bacterial solution to cover the hypocotyl, infect for 10 min, then pour out the bacterial solution and use sterile filter paper to absorb the residual bacterial solution on the surface of the hypocotyl.

[0146] (4) Inoculation: Inoculate with MS medium containing 2.5 mg / L NAA, 2.0 mg / L 6BA, 50 mg / L AS and 20 mg / L Pro, and incubate in the dark at 25°C for 72 h.

[0147] (5) When mycelial zones are about to appear under the explants, wash the seedlings for 10 min in 2% sucrose MS liquid medium containing 300 mg / L Carb, blot dry the surface of the explants with sterile filter paper, and inoculate them into the MS medium containing 300 mg / L Carb from step 4. Incubate in the dark at 25°C, subculture once every 10 days, halving the Carb content each time, for a total of 3 subcultures to obtain transgenic callus. After the callus has been cultured for 30 days, measure the relevant indicators.

[0148] 1.1.3 PCR and qRT-PCR detection of CqDOF3 gene-transformed quinoa callus:

[0149] Callus tissues from wild-type quinoa and transgenic quinoa were harvested, and DNA was extracted and used as templates for PCR amplification using Taq DNA polymerase. A forward primer (SEQ ID No. 5, 35S-F: GATGGATCCAGCGATTAAA) was designed at the 35S promoter, and a reverse primer (SEQ ID No. 6, 35S-R: AGAGCACTCTCTTGGAAGT) was designed at the target gene, producing a 1300 bp fragment. Furthermore, RNA was extracted from wild-type and transgenic quinoa callus tissues and qRT-PCR was performed using Supereal Pre Mix Plus enzyme to detect the expression level of the CqDOF3 gene in transgenic quinoa callus tissues.

[0150] 1.1.3 Analysis of drought resistance in transgenic quinoa callus

[0151] The obtained callus tissues were transferred to MS solid medium supplemented with 0 mM, 300 mM mannitol and 300 mM + 15 μmol SNP respectively after co-culture and cultured for 30 days. Subculture was performed every 15 days. Samples were taken after the culture was completed for subsequent experiments.

[0152] 1.2 Arabidopsis genetic transformation:

[0153] 1.2.1 Obtaining transgenic Arabidopsis thaliana:

[0154] Transgenic Arabidopsis plants were obtained by referring to the flower-dipping method.

[0155] 1.2.2 Screening and purification of positive plants:

[0156] The pCAMBIA1302-35S-EGFP vector is hygromycin resistant. Transformed Arabidopsis seeds were screened on 1 / 2 MS solid medium containing hygromycin until purified to the T3 generation.

[0157] 1.2.3 PCR identification and qRT-PCR analysis of positive plants:

[0158] After culturing at 22℃ and a photoperiod of 16 h / 8 h for 14 days, resistant seedlings with good growth were selected and transplanted to soil. When the plants had 7-8 true leaves, DNA was extracted from basal leaves and identified by PCR using vector-specific primers for the target fragment (F3-F: 5′-GATGGATCCAGCGATTAAAT-3′; F3-R: 5′-AGAAGCACTCTCTTGGAAGT-3′, expected amplification fragment 1300 bp). To screen transgenic Arabidopsis lines with high CqDOF3 gene expression to meet the needs of subsequent functional verification experiments, this study selected genetically stable homozygous T3 generation transgenic Arabidopsis lines as materials, with Actin1 as an internal control. The expression level of CqDOF3 in T3 generation transgenic Arabidopsis was detected by qRT-PCR. The line with the lowest expression level was selected as a control, and two lines with the highest relative expression levels were selected for propagation and used in subsequent experiments.

[0159] 1.2.4 Drought Resistance Analysis of Transgenic Arabidopsis

[0160] The overexpressing Arabidopsis plants were transplanted and grown in soil for 15 days. The control group received normal watering, while the treatment groups were subjected to either natural drought for 7 days or natural drought for 7 days after spraying with 0.05 mmol / L SNP. SNP treatment required 3 days of pretreatment, with uniform spraying on both sides of the leaves, repeated every 24 hours. The SNPs used had to be freshly prepared and used immediately. Samples were collected 7 days after treatment, with three biological replicates for each group. Samples were stored at -80℃ for later use.

[0161] 2. Results

[0162] 2.1 Positive identification of transgenic callus tissue:

[0163] like Figure 7 A. The PCR reaction successfully amplified a 1300 bp band, and qRT-PCR confirmed that the relative expression level of the CqDOF3 gene in the overexpressed callus tissue was significantly higher than that in the wild type. Figure 7 B) indicates that CqDOF3 gene overexpression quinoa callus tissue was successfully constructed.

[0164] 2.2 Analysis of physiological parameters of transgenic quinoa callus under NO-mediated drought stress:

[0165] 2.2.1 Phenotypic, fresh weight, and dry weight analysis of transgenic quinoa callus tissue under NO-mediated drought stress:

[0166] like Figure 8 As shown in Figure A, under normal conditions, the overexpressing callus tissue grew better than the wild-type. Under drought stress, cell division in both the WT and OE lines was significantly inhibited, and their growth status was significantly affected. However, the addition of SNPs significantly alleviated the stress on the callus tissue, and the overexpressing callus tissue showed a better growth trend than the wild-type. Furthermore, as... Figure 8 In both B and C cases, under normal growth conditions and simulated drought stress conditions, the fresh and dry weight of quinoa callus overexpressing CqDOF3 was higher than that of wild-type callus. Furthermore, the fresh and dry weight of quinoa callus overexpressing CqDOF3 after SNP addition was significantly higher than that under drought stress. These results indicate that CqDOF3 overexpression positively regulates the drought stress tolerance of callus, and the addition of SNP can alleviate the inhibitory effect of drought stress on quinoa callus growth.

[0167] 2.2.2 Antioxidant System Analysis of Quinoa Callus

[0168] This study analyzed the effect of CqDOF3 overexpression on the antioxidant enzyme activity of quinoa callus. The results are as follows: Figure 9As shown in AC, under normal conditions, there were no significant differences in the activities of SOD, POD, and CAT in WT and OE callus tissues. After drought stress, the activities of POD and CAT in the overexpressing lines were significantly higher than those in WT, and the SOD activity also increased slightly. After the addition of SNP, the activities of all three antioxidant enzymes were further increased. These results indicate that CqDOF3 overexpression can enhance the antioxidant enzyme activity of quinoa callus tissue under drought stress, and exogenous SNP treatment can further alleviate drought-induced oxidative damage.

[0169] 2.2.3 Osmotic Regulation System and MDA Analysis of Quinoa Callus

[0170] like Figure 9 As shown in the diagram, under normal conditions, there was no significant difference in proline (Pro) and soluble protein content between WT and OE calli, while MDA content was slightly higher in the overexpression lines. After drought stress treatment, the soluble protein and Pro content in the overexpression calli significantly increased. After exogenous SNP treatment, the soluble protein and Pro content in the overexpression lines further increased, while the MDA content significantly decreased. These results indicate that CqDOF3 overexpression may enhance the drought resistance of calli by promoting the accumulation of osmotic regulatory substances and inhibiting membrane lipid peroxidation; SNP treatment helps maintain the cell membrane stability and physiological function of transgenic calli under drought conditions, thereby alleviating stress damage.

[0171] 2.3 Analysis of Arabidopsis lines overexpressing the CqDOF3 gene:

[0172] 2.3.1 Identification of transgenic Arabidopsis thaliana lines:

[0173] Positive results were verified in genetically transformed Arabidopsis plants, such as... Figure 7 C. Fourteen Arabidopsis thaliana strains were initially screened for positive verification. All Arabidopsis strains amplified the target fragment of 1300 bp (7D), indicating successful acquisition of Arabidopsis thaliana overexpression plants. Then, qRT-PCR analysis was performed on the overexpressing Arabidopsis thaliana plants. Using OE2, with the lowest expression level, as a control, the CqDOF3 gene was significantly upregulated in the overexpressing Arabidopsis thaliana, with OE3 and OE10 showing the highest expression levels among all lines, 20.25 times and 21.42 times that of the control, respectively. Figure 7 Therefore, these two strains were used as experimental materials for subsequent drought resistance analysis.

[0174] 2.3.2 Phenotypic, fresh weight, and dry weight analysis of transgenic Arabidopsis thaliana under NO-mediated drought stress:

[0175] like Figure 10As shown in Figure A, under normal growth conditions, the overexpression line was taller than the wild-type line, with no significant differences in other phenotypic characteristics. However, after 7 days of natural drought, the wild-type line exhibited more severe wilting than the overexpression line. Subsequently, while the plants were subjected to 7 days of natural drought, they were sprayed with SNP. SNP, as a NO donor, significantly reduced the damage caused by drought stress. The results showed that after spraying with SNP, the wilting degree of both the wild-type and overexpression lines was reduced compared to the plants under drought stress, and the leaf damage of the overexpression line was less severe than that of the wild-type plants. The results of plant fresh weight and dry weight showed that (…). Figure 10 (BC) The quality of overexpressing plants was significantly higher than that of wild-type plants. In summary, the overexpressing lines exhibited stronger drought stress tolerance, and exogenous NO significantly reduced the damage of drought stress to plants and improved plant quality.

[0176] 2.3.3 Analysis of Arabidopsis plant phenotype, detached leaf water loss rate, and relative leaf water content:

[0177] As the main organ of a plant, water loss primarily occurs in the leaves. For example... Figure 11 As shown in Figure A, there was no significant difference in leaf water loss among all lines in the first 120 minutes. However, as time increased, WT leaves lost water faster, and this trend was consistent with... Figure 11 The results of the water loss rate determination of the detached leaves in section B are consistent. For example... Figure 11 As shown in Figure C, under normal conditions, the relative water content of leaves in the overexpressing lines was slightly higher than that of the wild type (WT). After drought stress treatment, the relative water content of all lines decreased, but the relative water content of the two OE lines remained significantly higher than that of WT. Exogenous SNP spraying further promoted the recovery of relative water content under drought conditions. These results indicate that CqDOF3 overexpression can effectively alleviate water loss caused by drought stress, maintain the water balance in plants, and thus enhance their drought resistance.

[0178] 2.3.4 Seed germination and root length measurement of transgenic Arabidopsis thaliana under NO-mediated drought stress:

[0179] like Figure 12As shown in AC, under normal conditions, all seeds of WT, OE3, and OE10 germinated on day 5, with a germination rate of 100%. After the addition of 200 mM mannitol, the germination rate decreased in all cases, but the germination rates of OE3 and OE10 were significantly higher than those of WT. However, after the addition of 0.1 mmol / L SNP, the germination rates of WT, OE3, and OE10 all increased significantly, by 58.6%, 62.33%, and 59.33%, respectively, compared to the germination rates under simulated drought stress. The results indicate that the germination rate of the OE lines was significantly higher than that of WT under drought stress, and the addition of SNP significantly improved the seed germination rate. Figure 12 As shown in DE, under 200 mM mannitol treatment, the growth of all plants was inhibited, with the WT plants showing more significant growth inhibition and shorter root lengths. However, the addition of SNPs significantly alleviated the inhibitory effect of drought stress on plant growth, resulting in a marked increase in root length compared to the drought-stressed state. The results indicate that under drought stress, the OE line had longer roots than the WT line, and that SNPs can alleviate the inhibitory effect of drought on plant growth and increase root length.

[0180] 2.3.5 Analysis of photosynthetic pigments:

[0181] Chlorophyll, as the "energy conversion station" of plants, can absorb light energy and convert and transfer energy. Under drought stress, chlorophyll function is severely impacted, and chlorophyll content can reflect the degree of stress on plants. Figure 13 As shown in Figure A, after drought stress, the total chlorophyll content of the OE3 and OE10 lines was 5.28% and 4.42% higher than that of the WT line, respectively. However, after exogenous SNP application under drought stress, the chlorophyll content of all plants increased compared to the drought stress state. This indicates that overexpression of the CqDOF3 gene can increase plant drought resistance, and exogenous SNP application can increase chlorophyll content under drought stress, thus increasing plant drought resistance. (Chlorophyll a content, chlorophyll b content, and carotenoid content are also mentioned.) Figure 13 (BD) The results showed that after direct application of SNP, the chlorophyll content of the OE lines was significantly higher than that of the WT lines. The results indicated that under drought treatment, the chlorophyll content of the OE lines was higher than that of the WT lines, and that exogenous application of SN could increase the chlorophyll content in plants under drought stress.

[0182] 2.3.6 Antioxidant System Analysis:

[0183] like Figure 14As shown in AC, under normal conditions, there was no significant difference in the activities of SOD and POD enzymes related to the antioxidant system between WT and OE lines. CAT enzyme activity in OE lines was significantly higher than that in WT lines. However, after drought stress, the SOD content in OE lines increased slightly; POD content was 16.24% and 42.29% higher than that in WT lines, respectively; CAT content was also significantly higher than that in WT lines, being 2.94 times and 2.59 times higher, respectively. After exogenous application of SNP, the SOD content of all lines increased slightly, and the POD content increased by 49.34%, 10.48%, and 31.18% compared to the drought stress treatment; the CAT content increased by 14.8%, 22.65%, and 19.86% compared to the drought stress treatment. These results indicate that CqDOF3 overexpression lines have stronger antioxidant enzyme activity under drought stress, and exogenous SNP treatment can further enhance their activity, thereby strengthening the drought tolerance of the plants.

[0184] 2.3.7 Osmosis Regulation System and MDA Analysis:

[0185] like Figure 14 As shown in the DG, under normal conditions, there were no significant differences in soluble protein, proline (Pro) content, and relative conductivity among the different strains. After drought stress, the soluble protein, Pro content, and relative conductivity of all strains increased. Specifically, the overexpression strain (OE) had soluble protein content 19.05% and 19.79% higher than the WT strain, respectively; Pro content was significantly higher than the WT strain by 28.42% and 20.04%, respectively; and relative conductivity was significantly lower than the WT strain by 18.62% and 20.60%, respectively. MDA content was also lower than the WT strain. Exogenous application of SNP alleviated the physiological damage caused by drought stress. The soluble protein content of each strain increased by 11.58%, 19.97%, and 1.37% compared to the drought treatment, respectively; Pro content increased by 12.06%, 3.51%, and 30.61%, respectively; and relative conductivity decreased by 8.31%, 10.12%, and 4.41%, respectively. Furthermore, the MDA content in the OE strain decreased further, while it increased significantly in the WT strain. These results indicate that CqDOF3 may enhance plant drought resistance by promoting the accumulation of osmotic regulators and reducing membrane lipid peroxidation; exogenous NO can synergistically enhance osmotic regulation and reduce membrane system damage, thereby improving drought adaptability.

[0186] 2.3.8 Analysis of H2O2 and O2- content

[0187] like Figure 15As shown in Figures AB, under normal conditions, there was no significant difference in DAB and NBT histochemical staining between the WT and OE lines. After drought stress, the degree of membrane lipid peroxidation in WT plants was severe, and the DAB and NBT histochemical staining of leaves was more pronounced than that of Arabidopsis overexpressing lines, indicating that WT plant leaves suffered more severe damage under drought stress and accumulated more O2- and H2O2; under exogenous NO-mediated drought stress, the staining intensity of all lines was reduced. Figure 15 CD further showed that under drought stress, both the O2- production rate and H2O2 content were significantly reduced, and significantly lower than WT; under exogenous NO-mediated drought stress, both the O2- production rate and H2O2 content were reduced in all lines. The results indicate that overexpression of CqDOF3 can enhance the plant's ability to cope with drought stress, reduce the degree of membrane lipid peroxidation, and exogenous NO treatment further enhances the antioxidant defense system.

Claims

1. An application of the CqDOF3 gene mediated by NO in regulating plant drought resistance, characterized in that, The CDS sequence of the CqDOF3 gene is shown in SEQ ID No. 1, and the protein sequence encoded by the CqDOF3 gene is shown in SEQ ID No.

2.

2. The application of the NO-mediated CqDOF3 gene in regulating plant drought resistance according to claim 1, characterized in that, Primer pairs for amplifying or detecting the CqDOF3 gene as described in claim 1 include CqDOF3-F and CqDOF3-R; wherein the nucleotide sequence of CqDOF3-F is shown in SEQ ID No. 3, and the nucleotide sequence of CqDOF3-R is shown in SEQ ID No.

4.

3. The application of the NO-mediated CqDOF3 gene in regulating plant drought resistance according to claim 1, characterized in that, The CqDOF3 gene positively regulates plant drought resistance by modulating the activity of plant-related antioxidant enzymes.

4. The application of the NO-mediated CqDOF3 gene in regulating plant drought resistance according to claim 1, characterized in that, Overexpression of the CqDOF3 gene according to claim 1 includes the following steps: S1: Construction of recombinant expression vector: Using an expression vector with SacⅠ and XbaⅠ restriction sites, CqDOF3 is linked between the SacⅠ and XbaⅠ restriction sites of the expression vector to form a recombinant vector overexpressing CqDOF3; S2: The constructed pCAM-CqDOF3 overexpression vector was transformed into Agrobacterium GV3101, and then the vector was transformed into quinoa callus and Arabidopsis thaliana by Agrobacterium-mediated transformation to achieve the expression of the CqDOF3 gene, thereby obtaining transgenic plants overexpressing CqDOF3.

5. The application of the NO-mediated CqDOF3 gene in regulating plant drought resistance according to claim 1, characterized in that, Primer pairs used to detect CqDOF3 gene expression in transgenic plants include: (a): The primer pair used to detect transgenic quinoa callus includes 35S-F and 35S-R; wherein the nucleotide sequence of 35S-F is shown in SEQ ID No. 5 and the nucleotide sequence of 35S-R is shown in SEQ ID No. 6; (b): The primer pairs used to detect transgenic Arabidopsis include F3-F and F3-R; wherein the nucleotide sequence of F3-F is shown in SEQ ID No. 7 and the nucleotide sequence of F3-R is shown in SEQ ID No.

8.

6. The application of the NO-mediated CqDOF3 gene in regulating plant drought resistance according to claim 1, characterized in that, The plant in question is quinoa.

7. The application of the NO-mediated CqDOF3 gene in regulating plant drought resistance according to claim 4, characterized in that, The CqDOF3 gene enhances plant drought resistance by regulating the activity of plant-related antioxidant enzymes.

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