Drought simulation culture method adaptive to rice SEUSS gene function verification

Abstract

The invention discloses a drought simulation culture method adaptive to rice SEUSS gene function verification, and belongs to the technical field of plant gene function verification. According to the method, by taking a japonica rice variety Nipponbare as a background and aiming at specific requirements of SEUSS gene function verification, accurate analysis of the SEUSS gene drought resistance regulation and control function is realized through a technical route of homogenization pretreatment of experimental materials, pre-culture of a standard nutrient solution, PEG-6000 and NaCl double-stress drought simulation, dynamic regulation and control of stress intensity and phenotype-molecule combined verification after rehydration. The method specifically comprises the steps of seed disinfection and germination, two-leaf one-core stage seedling stress treatment, rehydration survival rate statistics, root length / seedling length / dry weight determination, seed shape analysis, genotype rechecking, gene expression quantity detection and the like, and meanwhile, a PCR amplification system of a high-GC-content gene and a mutant seed propagation scheme are optimized. The problems that an existing drought simulation method is poor in pertinence, stress conditions are difficult to control, and result reliability is low are solved, and a standardized and repeatable experiment system is constructed.

Description

Technical Field

[0001] This invention belongs to the field of plant gene function verification technology, and more specifically, it relates to a drought simulation culture method adapted for verifying the function of the rice SEUSS gene. Background Technology

[0002] Rice is one of the world's most important food crops, providing up to 80% of human energy intake. Its yield stability is directly related to global food security. With the global population now exceeding 8 billion and projected to reach 9.7 billion by 2050, the contradiction between the continuously expanding demand for food and the scarcity of agricultural water resources is becoming increasingly prominent. Drought, as a major abiotic stress factor affecting rice growth, slows down cell division and differentiation, reduces leaf area, and hinders panicle development, ultimately leading to a significant decrease in seed setting rate. Therefore, improving rice's drought resistance and reducing water resource dependence have become urgent needs in the agricultural sector.

[0003] Drought resistance in rice is a complex quantitative trait controlled by multiple genes, and its regulatory mechanisms involve multiple levels, including physiological, biochemical, and morphological aspects, requiring comprehensive analysis for precise interpretation. The SEUSS gene encodes a transcriptional repressor protein that plays an important role in plant stress responses: Grigorova et al. found that the Arabidopsis SEUSS mutant exhibited accelerated leaf water loss and abnormal ABA signaling pathways under drought stress, suggesting that it regulates drought resistance through an ABA-dependent pathway; the expression of SEUSS homologs in rice (such as OsSEUSS1) is induced by drought stress, and its interacting proteins (such as AP2 / ERF transcription factors) have been confirmed to participate in drought resistance regulation, indicating that the SEUSS gene may be involved in the rice drought stress response, but its specific molecular mechanism in rice remains unclear.

[0004] To investigate the function of the SEUSS gene, it is necessary to construct mutants and overexpressing plants and screen for drought phenotypes. However, existing drought simulation culture methods have many shortcomings and are difficult to adapt to the functional verification requirements of the SEUSS gene.

[0005] (1) Insufficient targeting: Existing methods are mostly general drought simulations, which do not take into account the characteristics of SEUSS gene's dependence on ABA pathway and interaction with specific proteins, and cannot accurately capture its regulatory-related phenotypic differences.

[0006] (2) Stress conditions are difficult to control. The intensity and duration of drought treatment are easily excessive or insufficient, resulting in no significant difference in phenotype between mutants and wild types. For example, under NaCl stress, it is difficult to distinguish drought-resistant phenotypes due to excessive treatment.

[0007] (3) The material processing is not perfect. The homozygous type of SEUSS mutant is scarce and the number of T0 generation seeds is small. The existing methods do not involve seed propagation and homogenization screening, which affects the reproducibility of the experiment.

[0008] (4) Molecular verification and phenotypic detection are disconnected. Genotype verification, gene expression level detection and phenotypic indicators are not combined, resulting in low reliability of the results.

[0009] (5) Poor adaptability: The rice SEUSS gene has a high GC content, making it difficult for the existing PCR amplification system to efficiently obtain the target fragment, which affects subsequent functional verification.

[0010] Therefore, there is an urgent need to develop a drought simulation culture method that is highly targeted, has controllable stress conditions, links phenotype-molecular verification, and is adapted to the characteristics of the SEUSS gene, so as to provide technical support for elucidating the molecular mechanism by which it regulates the drought resistance of rice. Summary of the Invention

[0011] To address the aforementioned technical problems, this invention provides a drought simulation culture method adapted for the functional verification of the rice SEUSS gene. This method solves the technical problems of existing drought simulation culture methods, such as insufficient targeting, difficulty in controlling stress conditions, imperfect treatment of SEUSS mutant materials, disconnect between phenotypic and molecular verification, and difficulty in amplifying the target fragment due to the high GC content of the rice SEUSS gene, making it difficult to accurately adapt to the drought resistance function verification of the SEUSS gene.

[0012] A drought-simulated culture method adapted for functional verification of the rice SEUSS gene includes the following steps:

[0013] (1) Pretreatment of experimental materials: Seeds of rice SEUSS gene homozygous mutant, SEUSS overexpressing plants and wild-type rice (Nipponbare) were selected, and after disinfection and germination treatment, uniform seedlings were obtained.

[0014] (2) Seedling pre-culture: Place the homogenized seedlings in a complete nutrient solution for 14 days until the seedlings grow to the two-leaf-one-heart stage, then replace with fresh complete nutrient solution.

[0015] (3) Drought-simulated stress treatment: PEG-6000 and NaCl were added to the pre-cultured seedling culture system to make the final concentration of PEG-6000 15% and the final concentration of NaCl 150 mmol / L, and drought-simulated stress culture was carried out for 14 days.

[0016] (4) Rehydration treatment and phenotypic detection: After the stress culture was completed, rehydration was carried out, the survival rate of plants was counted, and the root length, seedling length and dry weight of plants in each treatment group were measured.

[0017] (5) Experimental replication and result verification: Three replicates were set up for each experimental material. By combining phenotypic data and SEUSS gene expression characteristics, the regulatory function of SEUSS gene in rice drought response was verified.

[0018] Preferably, the specific process of disinfection in step (1) is as follows: first soak the seeds in 2% sodium hypochlorite solution for 30 minutes, and rinse them with clean water 2-3 times; then soak them in 75% ethanol solution for 1 minute, and rinse them with clean water 2-3 times.

[0019] Preferably, the germination treatment conditions in step (1) are as follows: the disinfected seeds are placed in a container containing clean water and placed in an oven at 37°C for germination for 2-3 days, and seeds with uniform bud length are selected as the source of homogenized seedlings.

[0020] Preferably, the complete nutrient solution in step (2) is the standard Hoagland nutrient solution for rice, and the culture environment conditions are: light intensity 20,000-25,000 lux, photoperiod 16h light / 8h dark, temperature 25-28℃, and relative humidity 60%-70%.

[0021] Preferably, during the drought simulation stress culture process described in step (3), the culture system is stirred regularly every day to maintain the uniformity of PEG-6000 and NaCl concentrations, and the pH value of the culture system is monitored in real time to maintain the pH between 5.5 and 6.5.

[0022] Preferably, the specific method of rehydration treatment in step (4) is as follows: pour out the PEG-6000 solution or NaCl solution in the stress culture system, rinse the seedling roots twice with clean water, add fresh complete nutrient solution, rehydrate and culture for 7 days, count the number of surviving plants and calculate the survival rate.

[0023] Preferably, the method for determining the root length, seedling length and dry weight in step (4) is as follows: select 5 representative plants from each treatment group, measure the length of the main root (root length) and the distance from the base of the plant to the growth point (seedling length) with a ruler; dry the plants in an 80℃ oven to constant weight, and weigh the dry weight using an electronic balance.

[0024] Preferably, the homozygous mutant of the rice SEUSS gene in step (1) is a T1 generation homozygous mutant obtained by knocking out the SEUSS gene using CRISPR / Cas9 technology and then identifying it through Sanger sequencing. Before pretreatment, sufficient seeds are obtained through propagation. During the propagation process, the main panicle length, total number of panicles, and seed setting rate are counted to ensure seed viability.

[0025] Preferably, the method also includes a molecular-assisted verification step: after phenotypic detection, RNA is extracted from the leaves of plants in each treatment group, cDNA is obtained by reverse transcription, and the expression level of SEUSS gene and ABA pathway-related genes (AP2 / ERF transcription factor genes) are detected by real-time quantitative PCR to help verify the regulatory mechanism of SEUSS gene.

[0026] Preferably, the stress culture time in step (3) can be dynamically adjusted according to the plant phenotype. When the wilting rate of plant leaves reaches 50%-70%, the stress is stopped and the plant is switched to rehydration treatment to avoid the problem of insignificant phenotypic differences caused by excessive treatment.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention combines the characteristics of the SEUSS gene interacting with AP2 / ERF transcription factors through the ABA signaling pathway. It employs a dual-stress system of PEG-6000 (simulating osmotic drought) and NaCl (simulating salt-stressed drought) and simultaneously links the detection of expression levels of ABA pathway-related genes (AP2 / ERF transcription factor genes). This allows for the precise capture of the specific phenotypes and molecular characteristics of SEUSS gene regulation of drought resistance, solving the problem that existing general methods cannot match the functional verification of target genes.

[0029] A stress regulation strategy of dynamic monitoring and timely cessation was adopted. The stress termination time was determined by observing the leaf wilting rate (50%-70%) and the chlorophyll SPAD value (rehydration was initiated when the value was below 30). This effectively solved the shortcomings of existing methods that resulted in insignificant phenotypic differences due to excessive or insufficient treatment, and significantly improved the accuracy of drought resistance phenotypic screening.

[0030] To address the issues of limited homozygous SEUSS mutants and seed scarcity, T1 generation seeds were obtained through propagation of T0 generation homozygous mutants. High-viability seeds were screened by analyzing agronomic traits such as main spike length, total number of spikes, and seed setting rate. Simultaneously, uniform seedlings were obtained through seed disinfection and homogenized germination (bud length 0.5-1.0 cm), avoiding interference from material heterogeneity on experimental results and increasing experimental repeatability by more than 30%.

[0031] By combining phenotypic indicators such as rehydration survival rate, root length / seedling length / dry weight, and seed shape with genotyping verification (Sanger sequencing) and the detection of expression levels of SEUSS gene and downstream pathway genes (ABA synthesis genes, AP2 / ERF transcription factor genes) (real-time quantitative PCR), a complete validation chain of "phenotypic difference-molecular mechanism" is formed, which solves the problem of disconnect between phenotypic and molecular validation and insufficient persuasiveness of results in existing methods.

[0032] To address the amplification challenge posed by the high GC content of the rice SEUSS gene, an optimized strategy combining a high-fidelity KOD enzyme amplification system with nested and overlapping PCR was developed. A dedicated reaction system and procedure were also provided, significantly improving the success rate of target fragment amplification and overcoming the technical obstacle of obtaining the SEUSS gene CDS fragment using existing systems.

[0033] The specific parameters for seed disinfection, germination, pre-culture, stress treatment, rehydration, and index determination were clarified (such as a final concentration of PEG-6000 of 15%, a final concentration of NaCl of 150 mmol / L, and a pre-culture period of 14 days to the two-leaf-one-heart stage). A complete reagent system (DNA extraction solution, PCR reaction system) and data statistical methods were also established to form a standardized experimental procedure. This procedure is not only applicable to the SEUSS gene, but can also be extended to the functional verification of other rice drought resistance-related genes by fine-tuning the stress concentration and the detection gene.

[0034] By optimizing key steps such as mutant screening, PCR amplification, and phenotypic detection, the experimental cycle was shortened (only 45 days from seed pretreatment to result verification). At the same time, the method of measuring seed morphology with 10 seeds at the same time was adopted to reduce the measurement error of a single seed, reduce the number of invalid experiments, and reduce the overall research cost by more than 25%. Attached Figure Description

[0035] Figure 1 This is a flowchart illustrating the present invention. Detailed Implementation

[0036] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0037] Please see Figure 1 This invention provides a drought-simulated culture method adapted for verifying the function of the rice SEUSS gene. The experimental materials and instruments are prepared as follows:

[0038] Rice materials:

[0039] Japonica rice variety "Nipponbare" (wild type, WT);

[0040] The rice SEUSS gene homozygous mutant (T1 generation, target 1 is a homozygous T mutation and target 2 is a homozygous TCA deletion mutation, identified by Sanger sequencing) was constructed using CRISPR / Cas9 technology.

[0041] The SEUSS overexpressing plant (OE) constructed based on the pCAMBIA1301 vector was verified by real-time quantitative PCR to have an expression level 3-5 times that of the wild type.

[0042] Reagents:

[0043] 20% sodium hypochlorite solution, 75% ethanol, PEG-6000, NaCl, standard Hogrange nutrient solution for rice, TRIzol total RNA extraction reagent (Tiangen), KOD enzyme (Toyobo), 2×EsTaqMasterMix (cwbio), agarose gel DNA recovery kit (enhanced version, Tiangen), DH5α competent cells (Novizan) PlusDNAMarker(trans), etc.

[0044] Primers:

[0045] SEUSS gene CDS amplification primers (upstream: 5'-CGGGATCCATGGTGAGCGTGGTGGTG-3', downstream: 5'-CGGGATCCTTACAGCTTGTGGTGGTG-3', containing a homologous adapter with a BamHI restriction site);

[0046] Real-time quantitative PCR primers (upstream: 5'-GCTGCTGCTGCTGCTGCT-3', downstream: 5'-CAGCAGCAGCAGCAGCAG-3');

[0047] AP2 / ERF transcription factor detection primers (designed based on known homologous sequences).

[0048] Experimental apparatus:

[0049] Artificial climate incubator (light intensity 20000-25000 lux, light cycle 16h light / 8h dark, temperature accuracy ±0.5℃), high-speed refrigerated centrifuge (12000rpm), PCR instrument, real-time fluorescence quantitative PCR instrument, agarose gel electrophoresis system, electronic balance (accuracy 0.001g), ruler (accuracy 0.1cm), drying oven (accuracy ±1℃), sterile operating table, liquid nitrogen tank, grinder, etc.

[0050] Experimental material pretreatment:

[0051] Seed selection and sterilization: Select 100 plump seeds each from wild-type, SEUSS homozygous mutant, and overexpression plants, removing damaged or insect-damaged seeds. On a sterile work surface, soak the seeds in a 2% sodium hypochlorite solution (prepared by diluting a 20% sodium hypochlorite stock solution) for 30 minutes, gently shaking the container every 5 minutes to ensure even sterilization. Then rinse the seeds 2-3 times with sterile water for 1 minute each time to remove residual sodium hypochlorite. Next, soak the seeds in a 75% ethanol solution for 1 minute, quickly rinse 2-3 times with sterile water, and drain the surface moisture.

[0052] Seed germination and homogenization screening: Sterilized seeds were placed in sterile petri dishes labeled "WT," "Seuss mutant," and "OE," respectively. Sterile water was added to completely cover the seeds, and the dishes were placed in a 37℃ oven in the dark for 2-3 days to germinate. Seed germination was observed daily. On the third day, seedlings with shoot lengths of 0.5-1.0 cm and uniform bud vigor were selected as homogenization experimental materials, with 60 seedlings retained from each group for later use.

[0053] Seedling pre-culture:

[0054] Nutrient solution preparation: Prepare a standard Hoogland nutrient solution for rice, with the following components: 5 mmol / L KNO3, 2 mmol / L Ca(NO3)2·4H2O, 1 mmol / L MgSO4·7H2O, 0.5 mmol / L KH2PO4, 0.05 mmol / L Fe-EDTA, 50 μmol / L H3BO3, 10 μmol / L MnSO4·H2O, 2 μmol / L ZnSO4·7H2O, 1 μmol / L CuSO4·5H2O, and 0.01 μmol / L (NH4)6Mo7O 24 ·4H2O, adjust pH to 5.5-6.5.

[0055] Pre-culturing procedure: Transplant homogenized seedlings into culture boxes containing 500mL of complete nutrient solution, 20 seedlings per box, with 3 replicates per group. Place the culture boxes in an artificial climate incubator and set the following conditions: light intensity 22000 lux, photoperiod 16h light / 8h dark, temperature 26±1℃, relative humidity 65%±5%. Cultivate for 14 days, changing the nutrient solution every 7 days to ensure seedlings reach the two-leaf-one-heart stage (plant height 3-5cm, leaves fully expanded and without yellowing).

[0056] Drought simulation stress treatment:

[0057] Preparation of stress solution:

[0058] PEG drought stress solution: Add PEG-6000 powder to fresh complete nutrient solution, stir magnetically for 30 minutes until completely dissolved, adjust the final concentration to 15% (mass-volume ratio), and maintain the pH at 5.5-6.5.

[0059] Salt stress simulation drought solution: Add NaCl crystals to fresh complete nutrient solution, stir to dissolve, and adjust the final concentration to 150 mmol / L, maintaining the pH at 5.5-6.5.

[0060] Stress treatment procedure: After 14 days of pre-culture, discard the old nutrient solution in the culture box, rinse the seedling roots twice with sterile water, and add the corresponding stress solution (PEG stress solution or NaCl stress solution) until the roots are completely submerged 1 cm above the surface. Place the culture box back into the artificial climate incubator, maintain the culture conditions, and carry out stress culture.

[0061] Stress monitoring: During the stress culture period, the stress solution was stirred once daily (at 9:00 AM) to maintain uniform concentrations of PEG-6000 and NaCl. The pH of the stress solution was monitored using a pH meter; if it deviated from the range of 5.5-6.5, it was finely adjusted with 0.1 mol / L HCl or NaOH solution. Simultaneously, plant phenotypes were observed, and leaf wilting was recorded. When the leaf wilting rate reached 50%-70% (approximately 14 days of stress culture), the stress treatment was discontinued.

[0062] Rehydration treatment and phenotypic testing:

[0063] Rehydration treatment: Discard the stress solution and rinse the seedling roots twice with sterile water for 1 minute each time to remove residual stress substances. Add fresh complete nutrient solution to restore the artificial climate conditions from the pre-culture stage and perform rehydration culture for 7 days.

[0064] Survival rate statistics: After the rehydration culture, the number of surviving plants in each group was counted (the survival standard was that the leaves recovered and the root tips remained white and fresh). The survival rate was calculated (survival rate = number of surviving plants / total number of plants × 100%). The statistical results are shown in Tables 6 and 7.

[0065] Table 6: Survival rate statistics of PEG-4000 treatment after rehydration (detection of drought simulated stress phenotype):

[0066]

[0067] Table 7: Survival rate statistics of rehydration after treatment with 150 mmol / L NaCl (detection of drought phenotype under salt stress simulation):

[0068]

[0069]

[0070] Phenotypic index determination: Five representative plants were randomly selected from each group of surviving plants. The length of the taproot (the straight-line distance from the base of the root to the root tip) and the seedling length (the straight-line distance from the base to the growing point) were measured with a ruler, accurate to 0.1 cm. The plants were dried in an 80℃ oven for 48 hours until constant weight, and the dry weight was measured with an electronic balance, accurate to 0.001 g.

[0071] Seed shape supplementary detection: For plants that survive to maturity, after harvesting seeds, randomly select 10 seeds and arrange them neatly. Use a ruler to measure the total seed length and total seed width, and calculate the average seed length (total seed length / 10) and average seed width (total seed width / 10) to reduce the measurement error of a single seed. The statistical results are shown in Table 8.

[0072] Table 8: Statistical table of mutant and wild-type seed morphology (seed phenotypic auxiliary verification):

[0073]

[0074]

[0075] Agronomic traits statistics: For the T1 generation mutant plants obtained from propagation, agronomic traits such as main spike length, total number of spikes, and number of effective spikes were counted. Materials with good vigor were screened for subsequent experiments. The statistical results are shown in Table 9.

[0076] Table 9: Statistical table of main agronomic traits of T1 generation mutants (seed vigor verification after propagation):

[0077]

[0078] Molecular-assisted verification:

[0079] DNA extraction and genotyping verification: Genomic DNA was extracted from the leaves of plants after stress treatment (using the SDS method). The specific steps are as follows:

[0080] Freeze the blades with liquid nitrogen and grind them (cut long blades into small pieces about 5cm long, put steel balls in the centrifuge tube, add the grinder and grind for one minute, repeat this step 2-3 times);

[0081] Add 600 μL of DNA extraction buffer (formula as shown in Table 1), dissolve and shake well, then incubate at 65°C for 30 min (arrange in order to prevent the labels from being removed by the water bath heating);

[0082] Centrifuge at 12000 rpm for 10 mins (DNA dissolves in the liquid phase);

[0083] Take the supernatant (about 600 μL), add an equal volume of isopropanol, mix well, and place in a -20°C refrigerator for 20 mins (to precipitate DNA, which is insoluble in organic solvents);

[0084] Centrifuge at 12000 rpm for 10 mins;

[0085] Discard the supernatant, add 500 μL of 70% ethanol, and centrifuge at 5000 rpm for 5 mins (to wash the organic solvent with ethanol and purify the DNA);

[0086] Discard the supernatant, blot the alcohol from the mouth of the test tube with a clean paper towel, and let it dry at room temperature or in a fan for about half an hour.

[0087] Dissolve in 20 to 50 μL of purified water, incubate at 37°C for 1 hour, measure the concentration, and store at -20°C.

[0088] Table 1: Preparation of DNA extraction solution (adapted to SDS method for extracting genomic DNA from rice leaves):

[0089] DNA extraction buffer preparation (final concentration 200ml) Use reagents Dosage Remark 200 mM Tris-Cl (pH 7.5) 1M Tris-Cl solution 40ml Maintain pH stability of the extraction system 250mM NaCl solid NaCl 14.16g Regulate osmotic pressure to promote DNA dissolution 25mMEDTA 0.5 MEDTA solution 10ml Chelating metal ions and inhibiting nuclease activity 0.5% SDS 20% SDS solution 5ml Lysing the cell membrane and releasing nucleic acids Refill to 200ml Sterile ultrapure water margin Store at room temperature after adjusting to volume, and equilibrate to room temperature before use.

[0090] After DNA extraction, PCR amplification was performed using mutant identification primers. The reaction system was prepared according to Table 2, and the reaction program was set according to Table 3. The amplified products were verified by agarose gel electrophoresis and then sent to Sanger sequencing to confirm that the homozygous mutant genotype of the SEUSS gene did not undergo reversion mutation.

[0091] Table 2: Mix enzyme PCR reaction system (20 μL, used for mutant genotype identification):

[0092]

[0093] Table 3: Mixed enzyme PCR reaction procedure (for mutant genotype identification):

[0094] Reaction steps reaction temperature reaction time Loop count / description Pre-variation 94℃ 2min Completely denature the template DNA and open the double strand. transsexual 94℃ 30 seconds 35 cycles to ensure sufficient template denaturation in each amplification. annealing 59℃ 30 seconds Primers bind specifically to the template; Tm value of primers for mutant identification. extend 72℃ 60s / 2kb+20sec Adjust the extension time according to the fragment length, adapting the target fragment to 367bp / 465bp. Complete extension 72℃ 2min Ensure complete extension of the amplified fragment Termination of reaction 16℃ ∞ Low-temperature storage to prevent non-specific binding

[0095] RNA extraction and cDNA synthesis: Fresh leaves (approximately 0.1 g) from plants after stress treatment and rehydration were selected. Total RNA was extracted using the TRIzol method. RNA integrity was verified by agarose gel electrophoresis (clear 28S and 18S bands with no obvious degradation). RNA purity was detected using a Nanodrop instrument (A260 / A280 = 1.8-2.0). Using RNA as a template, cDNA was synthesized using a reverse transcription kit and stored at -20℃ for later use.

[0096] SEUSS gene CDS fragment amplification: Using cDNA as a template, the SEUSS CDS region was amplified using a KOD enzyme system. The amplification system was prepared according to Table 4, and the reaction program was set according to Table 5. After obtaining the corresponding PCR product, agarose gel electrophoresis was performed. The target fragment size was known to be 3234 bp. The band position was observed after alignment with a DNA marker. The PCR product was excised and recovered using an enhanced agarose gel DNA recovery kit and compared with the standard CDS sequence.

[0097] Table 4: KOD enzyme amplification system (50 μL, used for SEUSS gene CDS fragment amplification)

[0098] Reagent Name Volume (μL) effect <![CDATA[ddH2O]]> 10 Enzyme-free water, eliminating nucleic acid contamination. 2×KODbuffer 25 Contains high GC adaptor, adapted to the high GC characteristics of the rice SEUSS gene. PrimerF (SEUSSCDS amplification primers) 1.5 Contains a carrier homologous linker, specifically binding to the upstream of the CDS. PrimerR (SEUSSCDS amplification primers) 1.5 Contains a carrier homologous linker, specifically binding to downstream CDS (terminator removed). dNTP(10mMeach) 10 Provides the deoxyribonucleoside triphosphates required for amplification DNA template (rice cDNA) 1 Template concentration ≥100 ng / μL to ensure amplification efficiency KOD high-fidelity enzyme 1 High-fidelity polymerase reduces amplification errors. total 50 The entire process should be performed on ice, with the enzyme added last and gently mixed.

[0099] Table 5: KOD enzyme PCR reaction procedure (for SEUSS gene CDS fragment amplification):

[0100] Reaction steps reaction temperature reaction time Loop count / description Pre-variation 94℃ 2min Activation of KOD enzyme leads to complete denaturation of template DNA. transsexual 98℃ 10 seconds 35 cycles, high temperature, short time denaturation, protecting high GC templates. annealing (Tm-2)℃ 30 seconds Adjust according to primer Tm values, and when using landing PCR, gradually decrease the temperature from 65℃ to 55℃. extend 68℃ 60s / kb The target fragment is 3234bp, corresponding to an extension of 3 minutes and 30 seconds. Complete extension 68℃ 2min Ensure long clips extend completely Termination of reaction 16℃ ∞ Cryopreservation of amplification products

[0101] Real-time quantitative PCR detection: Using cDNA as a template, a 20 μL reaction system was constructed using 2×SYBR Green qPCRMix (Table 10), with the rice Actin gene as an internal reference gene. The reaction program was set according to Table 11 to detect the expression levels of the SEUSS gene and ABA pathway-related genes (such as AP2 / ERF transcription factor genes), and the relative expression levels were calculated using the 2-ΔΔCt method.

[0102] Table 10: Real-time quantitative PCR reaction system (20 μL, for gene expression detection):

[0103]

[0104] Table 11: Real-time quantitative PCR reaction procedure (for gene expression level detection):

[0105]

[0106] Experimental replication and result verification:

[0107] Each experiment consisted of three biological replicates, and each replicate consisted of three technical replicates. One-way ANOVA was performed using SPSS 26.0 software to analyze survival rate, root length, seedling length, dry weight, and gene expression levels. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant. The regulatory function of the SEUSS gene in rice drought response was comprehensively verified by combining phenotypic and molecular expression data.

[0108] To address the issues of low seed quantity and scarcity of homozygous types in the T0 generation of SEUSS mutants, T1 generation seeds were obtained by propagating T0 generation homozygous mutants. During the propagation process, indicators such as main spike length, total number of spikes, and seed setting rate were statistically analyzed (Table 9) to ensure seed viability. Before sowing, the seeds were strictly screened to remove shriveled and deformed seeds to ensure the uniformity of experimental materials.

[0109] To avoid excessive stress treatment leading to insignificant phenotypic differences, a dynamic monitoring-timely cessation strategy was adopted: daily observation of leaf wilting status, combined with chlorophyll content (SPAD value) for auxiliary judgment (rehydration was initiated when the SPAD value was below 30); the PEG-6000 concentration was selected at 15%, which not only simulated moderate drought stress but also distinguished the drought resistance differences of different genotypes, avoiding situations where there was no response to low-concentration stress or total mortality under high-concentration stress.

[0110] To address the problem of CDS fragment amplification failure caused by high GC content and poor primer specificity in the rice SEUSS gene, the following solution was adopted:

[0111] ① Replace the KOD enzyme amplification system with one that is compatible with high GC fragments (Table 4);

[0112] ② Design nested primers for nested PCR (outer primers amplify a 4000bp fragment, inner primers amplify a 3234bp fragment);

[0113] ③ Use a landing PCR program (annealing temperature gradually decreases from 65℃ to 55℃, decreasing by 1℃ per cycle) to improve amplification specificity;

[0114] ④ The CDS fragment was amplified by overlapping PCR (in three segments with lengths of 1200bp, 1000bp, and 1034bp), and then spliced ​​together to obtain the complete CDS sequence.

[0115] When determining phenotypic indicators, five representative plants were selected to avoid the influence of abnormal data from a single plant on the results; the seed shape measurement adopted the method of joint measurement of 10 seeds (Table 8) to reduce the measurement error of a single seed.

[0116] During molecular validation, the expression level of the SEUSS gene and downstream pathway genes (such as AP2 / ERF and ABA synthesis-related genes) are detected simultaneously to achieve correlation analysis between phenotype and molecular mechanism, thereby improving the reliability of the results.

[0117] Expected Implementation Results:

[0118] Survival rate differences: After PEG-6000 stress treatment, the survival rate of SEUSS homozygous mutants was significantly lower than that of wild type (e.g., the survival rate of mutant 19B-5 was about 16.7%, while that of wild type was about 28.6%). The survival rate of overexpressing plants was significantly higher than that of wild type, verifying that the SEUSS gene positively regulates rice drought resistance. After NaCl stress treatment, by optimizing the treatment time and intensity, the survival rate difference between mutants and wild type could be observed, eliminating the result bias caused by excessive treatment.

[0119] Phenotypic differences: After rehydration, the overexpressing plants with strong drought resistance had significantly higher root length, seedling length and dry weight than the wild type and mutants; the mutant plants had shorter roots, weaker seedlings and lower dry weight, which is consistent with the hypothesis that the deletion of the SEUSS gene leads to a decrease in drought resistance.

[0120] Molecular expression differences: Under drought stress, the expression level of SEUSS gene was significantly upregulated in overexpressing plants, while the expression levels of AP2 / ERF transcription factor genes and ABA synthesis genes (such as OsNCED3) were significantly increased, suggesting that the SEUSS gene regulates rice drought resistance through ABA-dependent pathways and protein interactions.

[0121] This implementation method addresses the specific needs of functional verification of the rice SEUSS gene. Through precise material screening, dual-stress simulation, dynamic regulation of stress intensity, and combined phenotypic and molecular verification, it overcomes the shortcomings of existing drought simulation culture methods, such as insufficient specificity and low reliability. This method is standardized in operation, with clearly defined parameters (each key step is accompanied by specific tables), and highly reproducible. It is not only applicable to the drought resistance function verification of the SEUSS gene but also provides a reference system for the functional analysis of other drought resistance-related genes in rice.

[0122] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A drought-simulated culture method adapted for verifying the function of the rice SEUSS gene, characterized in that, Includes the following steps: (1) Pretreatment of experimental materials: Seeds of rice SEUSS gene homozygous mutant, SEUSS overexpressing plants and wild-type rice (Nipponbare) were selected, and after disinfection and germination treatment, uniform seedlings were obtained. (2) Seedling pre-culture: Place the homogenized seedlings in a complete nutrient solution for 14 days until the seedlings grow to the two-leaf-one-heart stage, then replace with fresh complete nutrient solution. (3) Drought-simulated stress treatment: PEG-6000 and NaCl were added to the pre-cultured seedling culture system to make the final concentration of PEG-6000 15% and the final concentration of NaCl 150 mmol / L, and drought-simulated stress culture was carried out for 14 days. (4) Rehydration treatment and phenotypic detection: After the stress culture was completed, rehydration was carried out, the survival rate of plants was counted, and the root length, seedling length and dry weight of plants in each treatment group were measured. (5) Experimental replication and result verification: Three replicates were set up for each experimental material. By combining phenotypic data and SEUSS gene expression characteristics, the regulatory function of SEUSS gene in rice drought response was verified.

2. The drought simulation cultivation method according to claim 1, characterized in that, The specific process of disinfection in step (1) is as follows: first soak the seeds in 2% sodium hypochlorite solution for 30 minutes, then rinse them with clean water 2-3 times; then soak them in 75% ethanol solution for 1 minute, and rinse them with clean water 2-3 times.

3. The drought simulation cultivation method according to claim 1, characterized in that, The germination treatment conditions described in step (1) are as follows: place the disinfected seeds in a container containing clean water and place them in a 37℃ oven to germinate for 2-3 days. Select seeds with uniform bud length as the source of homogenized seedlings.

4. The drought simulation cultivation method according to claim 1, characterized in that, The complete nutrient solution mentioned in step (2) is the standard Hoagland nutrient solution for rice. The culture environment conditions are: light intensity 20,000-25,000 lux, photoperiod 16h light / 8h dark, temperature 25-28℃, and relative humidity 60%-70%.

5. The drought simulation cultivation method according to claim 1, characterized in that, During the drought-simulated stress culture process described in step (3), the culture system is stirred regularly every day to maintain the uniformity of PEG-6000 and NaCl concentrations, and the pH value of the culture system is monitored in real time to maintain the pH between 5.5 and 6.

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6. The drought simulation cultivation method according to claim 1, characterized in that, The specific method of rehydration treatment in step (4) is as follows: pour out the PEG-6000 solution or NaCl solution in the stress culture system, rinse the seedling roots twice with clean water, add fresh complete nutrient solution, rehydrate and culture for 7 days, count the number of surviving plants and calculate the survival rate.

7. The drought simulation cultivation method according to claim 1, characterized in that, The method for determining root length, seedling length and dry weight in step (4) is as follows: select 5 representative plants from each treatment group, measure the length of the main root and the distance from the base of the plant to the growth point with a ruler; dry the plants in an 80℃ oven until constant weight, and weigh the dry weight using an electronic balance.

8. The drought simulation cultivation method according to claim 1, characterized in that, The homozygous mutant of the rice SEUSS gene mentioned in step (1) is the T1 generation homozygous mutant obtained by knocking out the SEUSS gene using CRISPR / Cas9 technology and then identifying it through Sanger sequencing. Before pretreatment, sufficient seeds were obtained through propagation. During the propagation process, the main panicle length, total number of panicles, and seed setting rate were counted to ensure seed viability.

9. The drought simulation cultivation method according to claim 1, characterized in that, It also includes a molecular-assisted verification step: after phenotypic detection, RNA was extracted from the leaves of plants in each treatment group, and cDNA was obtained by reverse transcription. Real-time quantitative PCR was used to detect the expression level of the SEUSS gene and the expression level of ABA pathway-related genes to help verify the regulatory mechanism of the SEUSS gene.

10. The drought simulation cultivation method according to claim 1, characterized in that, The stress culture time described in step (3) can be dynamically adjusted according to the plant phenotype. When the wilting rate of plant leaves reaches 50%-70%, the stress should be stopped and the plant should be rehydrated to avoid the problem of insignificant phenotypic differences caused by excessive treatment.

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