Application of TaPMT-L gene in improving drought resistance of plants

Abstract

The invention provides an application of a TaPMT-L gene in improving drought resistance of plants. A nucleotide sequence of the gene is shown as SEQ ID No.1; the invention also provides a specific plant breeding method. Compared with the prior art, the application has the following advantages: the wheat gene TaPMT-L is identified, and overexpression and RNAi interference expression vectors of the wheat gene TaPMT-L are constructed, so that a stably inherited TaPMT-L transgenic wheat material is obtained. Phenotypic and physiological analysis under drought stress treatment shows that the drought tolerance in a TaPMT-L overexpression strain is remarkably enhanced, and the drought tolerance in an RNAi silent strain is reduced, so that the TaPMT-L has a function of positively regulating and controlling the drought resistance in wheat. The drought-resistant regulatory gene which is clear in function and can be stably expressed provides a new molecular tool for genetic improvement of the drought-resistant character of wheat, and has important theoretical value and application prospect for cultivation of new varieties of drought-resistant wheat.

Description

Technical Field

[0001] This application relates to the field of agricultural biotechnology, specifically to the genetic basis and molecular improvement of crop stress resistance traits, particularly the functional study and application of p-coumaroyl-CoA:monolignol transferase family genes in the regulation of drought resistance traits in wheat, involving the cloning, expression regulation, and utilization methods of the TaPMT-L gene in improving wheat drought resistance; specifically, the application of the TaPMT-L gene in improving plant drought resistance. Background Technology

[0002] Wheat (Triticum aestivum L.), as one of the world's most important food crops, plays a crucial role in ensuring national food security and addressing climate change. In recent years, frequent droughts and water scarcity have severely impacted wheat's normal growth and yield formation, particularly in arid and semi-arid regions, where water stress has become a key limiting factor for high and stable wheat yields. To enhance wheat's drought resistance, it is urgent to deeply explore and utilize functional genes closely related to drought tolerance at the genetic level.

[0003] p-coumaroyl-CoA:monolignol transferase (PMT) is a key enzyme in the phenylpropanoid metabolic pathway in plants, widely involved in the synthesis of bioactive metabolites such as lignin, flavonoids, and antioxidants. Numerous studies have shown that PMT genes play a crucial role in plant responses to various abiotic and biotic stresses, including drought, high temperatures, and pathogen infection, potentially enhancing plant resilience through strengthening cell wall structure, regulating antioxidant systems, and participating in signal transduction. However, the number of PMT gene members varies across different species, their functions are significantly differentiated, and the regulatory mechanisms of specific genes under specific stress conditions differ considerably.

[0004] In existing wheat drought resistance research and breeding practices, although some functional genes related to drought response have been identified, there are still significant shortcomings in the following aspects: 1. Limited sources of functional genes: Many reported drought-resistant genes are derived from model plants or heterologous species, and their adaptability and stability in wheat have not been fully verified. There is a lack of target genes derived from hexaploid wheat itself that have good genetic background compatibility.

[0005] 2. The functions of key regulatory genes are unclear: The PMT gene family has many members with significant functional differentiation. Currently, there are no publicly available studies that have conducted in-depth functional analysis and verification of the role of specific PMT genes in the drought resistance mechanism of wheat, which makes it difficult to effectively utilize these genes in actual breeding.

[0006] 3. Weak application and translational research: Most existing research is limited to the analysis of gene expression changes, lacking systematic functional verification (such as transgenic, overexpression, knockout, etc.) and genetic engineering breeding pathways, which makes it difficult to efficiently translate scientific research results into variety improvement.

[0007] 4. Lack of targeted breeding tools: At present, there is no mature technical solution or application method for constructing drought-resistant wheat germplasm based on PMT genes, which limits the in-depth application of molecular design breeding in improving drought resistance traits.

[0008] Therefore, how to screen out PMT genes with specific regulatory capabilities and verify their drought resistance function, and then apply them to genetic improvement, remains an important technical challenge in the field of wheat molecular breeding. Summary of the Invention

[0009] To address the aforementioned technical limitations, this application proposes the application of the TaPMT-L gene in improving plant drought resistance; it overcomes the deficiencies and defects mentioned in the background art.

[0010] To achieve the above objectives, this application adopts the following technical solution: The invention of this application provides the application of the TaPMT-L gene in improving plant drought resistance, and the nucleotide sequence of the TaPMT-L gene is shown in SEQ ID No. 1.

[0011] SEQ ID No. 1:

[0012] Optionally, the above application involves constructing a TaPMT-L overexpression vector to obtain drought-resistant transgenic plants.

[0013] Optionally, in the above applications, the drought resistance is manifested in the fact that, under water deficit conditions, the yield per plant of the TaPMT-L gene-overexpressing line is higher than that of the wild type.

[0014] Alternatively, in the above application, the plant is wheat.

[0015] The second inventive point of this application is to provide a plant breeding method, which obtains plants with stronger drought resistance than the target plant by increasing the activity of TaPMT-L protein in the target plant; the amino acid sequence of the TaPMT-L protein is shown in SEQ ID No. 2.

[0016] SEQ ID No. 2: MGFAVVRTNREFVRPSAATPPSSGELLELSIIDRVVGLRHLVRSLHIFSAAAPSGGDAKPSPARVIKEALGKALVDYYPFAGRFVDGGGGPGSARVECTGEGAWFVEAAA GCSLDDVNGLDHPLMIPEDDLLPDAAPGVHPLDLPLMMQVTEFSCGGFVVGLISVHTMADGLGAGQFINAVGDYARGLDRPRVSPVWAREAIPSPPKLPPGPPPELKMFQ LRHVTADLSLDSINKAKSAYFAATGHRCSTFDVAIAKTWQARTRALRLPEPTSRVNLCFFANTRHLMAGAAAWPAPAAGGNGGNGFYGNCFYPVSVVAESGAVEAADVAG VVGMIREAKARLPADFARWAVADFREDPYELSFTYDSLFVSDWTRLGFLEADYGWGPPSHVIPFAYYPFMAVAIIGAPPVPKTGARIMTQCVEDDHLPAFKEEIKAFDK.

[0017] The third inventive point of this application is to provide another plant breeding method, which obtains plants with stronger drought resistance than the target plant by promoting the expression of the TaPMT-L gene in the target plant; the nucleotide sequence of the TaPMT-L gene is shown in SEQ ID No.1.

[0018] The fourth inventive point of this application is to provide another plant breeding method, which obtains plants with lower drought resistance than the target plant by inhibiting the expression of the TaPMT-L gene in the target plant; the nucleotide sequence of the TaPMT-L gene is shown in SEQ ID No. 1.

[0019] Optionally, the above-mentioned plant breeding methods promote the expression of the TaPMT-L gene in the target plant by overexpressing the TaPMT-L gene; and inhibit the expression of the TaPMT-L gene in the target plant by silencing / interfering with the expression of the TaPMT-L gene.

[0020] Optionally, in the above-described plant breeding method, the target plant is wheat.

[0021] The fifth inventive point of this application is to provide the application of the aforementioned TaPMT-L gene in increasing wheat yield.

[0022] Compared with the prior art, this application has the following advantages: This application identified the wheat gene TaPMT-L and constructed its overexpression and RNAi interference expression vectors, obtaining stably inherited TaPMT-L transgenic wheat materials. Phenotypic and physiological analyses under drought stress treatment showed that drought tolerance was significantly enhanced in TaPMT-L overexpression lines, while drought tolerance decreased in RNAi-silenced lines, indicating that TaPMT-L has a positive regulatory function on drought resistance in wheat. This application provides a well-defined and stably expressed drought resistance regulatory gene, offering a new molecular tool for the genetic improvement of drought resistance traits in wheat, and has significant theoretical value and application prospects for breeding new drought-resistant wheat varieties.

[0023] The TaPMT-L gene involved in this application, as a member of the PMT family, was found for the first time to be significantly upregulated under drought stress and closely associated with drought-resistant traits. Functional validation showed that TaPMT-L plays an active role in regulating wheat drought resistance, possessing a clear biological function and application potential. The identification and application of this gene not only fills a gap in existing technologies but also provides a new molecular tool for constructing drought-resistant wheat germplasm resources, possessing significant theoretical and practical value. Attached Figure Description

[0024] Figure 1 The image shown is a detection pattern of agarose gel electrophoresis in one embodiment of this application.

[0025] Figure 2 The figure shows the relative expression levels of PMT-L in different tissues under drought treatment in one embodiment of this application.

[0026] Figure 3The diagram shown is an example of Agrobacterium-mediated transformation of wheat seedlings in one embodiment of this application.

[0027] Figure 4 The figure shown is a comparison of the performance of different strains under normal and drought treatment conditions in one embodiment of this application.

[0028] Figure 5 The image shown is a comparison of the performance of different strains of material under PEG treatment conditions in one embodiment of this application.

[0029] Figure 6 The figure shown is a statistical comparison of physiological indicators (relative water content, soluble sugar content, malondialdehyde content, and chlorophyll content) of different strains under drought treatment conditions in one embodiment of this application. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0032] All statistical analyses in the following examples were performed using GraphPad Prism 9 software (GraphPad Software, La Jolla, CA, USA) and SPSS 26.0 software (IBM, Chicago, IL, USA). Quantitative data are expressed as mean ± standard deviation (SD) of three independent experiments. Statistical differences were determined by unpaired independent samples t-tests for comparisons between two groups or by one-way ANOVA combined with Tukey's post-hoc test for comparisons among multiple groups. Pearson correlation analysis was used to investigate associations, and a p-value <0.05 was considered statistically significant.

[0033] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0034] Example 1 Core mechanism based on experimental verification: 1. To clarify the positive regulatory function of the TaPMT-L gene in drought response. A drought-induced PMT family candidate gene, named TaPMT-L, was identified through screening using prior RNA-seq data. Cloning, sequencing, and sequence annotation revealed that this gene is located on wheat chromosome 1B, encoding 449 amino acids, and exhibits typical BAHD acyltransferase structural characteristics. Subcellular localization prediction showed that it is located in the cell membrane and cytoplasm, possessing intracellular activity potential.

[0035] Expression pattern analysis showed that TaPMT-L was significantly upregulated (~9-fold) in wheat leaves under 20% PEG6000 simulated drought stress, and also exhibited stress response characteristics in roots and stems. Expression profiles in different tissues indicated a certain degree of spatiotemporal expression selectivity. qRT-PCR combined with normalization analysis confirmed that this gene showed a continuous upregulation trend under drought stimulation, suggesting that it may play a positive regulatory role in wheat drought tolerance.

[0036] 2. Functional validation experiments support TaPMT-L's regulation of wheat drought tolerance. Using the wheat variety “Xinchun 9” as a background, pCAMBIA1302-TaPMT-L overexpression lines and pSTARGATE-TaPMT-L-RNAi interference lines were constructed and obtained, and used for functional enhancement and loss analysis, respectively. The T2 generation transgenic lines showed significant differences in both PEG treatment and soil water control experiments.

[0037] Under soil drought treatment, TaPMT-L overexpression lines maintained higher leaf water content and stronger uprightness, with significantly lower MDA content and conductivity than WT; RNAi lines, on the other hand, exhibited severe curling, drying, and other damage symptoms. Under PEG treatment, overexpression lines had more developed root systems and slower leaf water loss, while interference lines showed severe water loss and overall deterioration of physiological indicators.

[0038] qRT-PCR validation showed that the expression level of TaPMT-L in different strains was highly positively correlated with the drought stress response phenotype, further confirming its core role in regulating drought tolerance.

[0039] 3. Speculation on regulatory mechanisms and metabolic pathways Based on research on PMT in other gramineous plants, it is speculated that TaPMT-L may enhance the plant's resistance to water transpiration under drought stress by participating in the binding reaction between monomeric lignin and p-coumaryl groups, thereby regulating the lignin molecular structure and strengthening the cell wall's hydrophobicity and mechanical strength. Furthermore, its cell membrane localization characteristics suggest that it may be involved in transmembrane regulation of signaling molecules or antioxidant metabolic linkages, possessing the potential for multi-pathway cross-functionality.

[0040] 4. Design of drought-resistant application strategies based on functional verification A TaPMT-L expression regulatory system was constructed to enhance lignin accumulation and water conduction capacity through overexpression, thereby improving wheat's water retention and tissue homeostasis adaptation in drought environments. This strategy can be used for: 1) Develop new transgenic wheat materials resistant to drought; 2) Use TaPMT-L as a molecular marker to screen for drought-resistant genotypes; 3) Construct a network map of wheat lignin regulation based on the PMT pathway to provide molecular breeding support for improving adaptability to drought environments.

[0041] This application systematically elucidates the core function of TaPMT-L in regulating wheat drought adaptation, from gene discovery and expression verification to transgenic material creation and functional evaluation, and establishes an applicable molecular breeding tool based on its regulatory pathway. This technical solution fills the gap in the application of wheat lignin transferase genes in drought-resistant breeding, and has clear scientific value and application potential.

[0042] The purpose of this application is: (1) Screen and clone a TaPMT-L that responds to drought stress from hexaploid wheat and clarify its expression characteristics; (2) Through functional verification, the positive regulatory role of TaPMT-L in improving wheat drought resistance was confirmed, and its molecular mechanism in the drought stress response process was revealed. (3) Construct expression vectors that can be used for genetic transformation and develop their application methods in wheat molecular breeding.

[0043] In summary, this application provides a drought-resistant functional gene with a clear origin, well-defined function, and great application potential, along with its usage plan, which is of great significance for breeding new drought-resistant wheat germplasm.

[0044] Example 2 I. Materials 1. The wheat variety is "Xinchun No. 9", a local variety developed in Xinjiang.

[0045] 2. All reagents, expression / cloning vectors, and competent cells used were conventional materials.

[0046] II. Methods and Results 1. TaPMT-L gene cloning and sequence analysis 1) Cloning of the TaPMT-L gene RNA was extracted from 200 mg of frozen wheat leaves using the TransZol Up Plus RNA kit (TransGen), and RNA integrity was assessed by 1% agarose gel electrophoresis. RNA purity was evaluated using a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific), and its concentration was quantified using an Agilent Bioanalyzer 2100 (Agilent Technologies). Reverse transcription was performed using EasyScript One-Step gDNA Removal and cDNA Synthesis Super Mix (TransGen) as a template for subsequent cloning. Based on bioinformatics analysis of the previous RNA-seq data, a drought-inducible PMT gene was identified and named TaPMT-L. PCR amplification was performed using the extracted DNA as a template based on its sequence information.

[0047] The PCR primer sequences are: TaPMT-L F (SEQ ID No. 3): 5'-CCCCACCAAACCCAGAC-3'; TaPMT-L R (SEQ ID No. 4): 5'-CTTCAACCCACAATGCTAAACT-3'.

[0048] The PCR reaction system consisted of: 1.0 μL template, 1.0 μL forward primer, 1.0 μL reverse primer, 10.0 μL 5×TransStart FastPfu Buffer, 4.0 μL dNTPs, 1.0 μL TransStart FastPfu DNA Polymerase, and 32.0 μL water. The PCR reaction conditions were: 95 ℃ for 2 min; 95 ℃ for 20 sec; 57 ℃ for 20 sec; 72 ℃ for 1 min, 33 cycles; and 72 ℃ for 5 min. After confirming the PCR product was correct via agarose gel electrophoresis, it was purified and recovered. The recovered fragment was ligated into the pEASY®-T1 Cloning Kit vector, transformed into competent E. coli cells, and positive clones were selected for sequencing after antibiotic selection.

[0049] 2) TaPMT-L gene sequence analysis After sequencing verification, the TaPMT-L gene sequence was homology-aligned using the NCBI BLAST tool to confirm its conservation in wheat and other gramineous plants. Its open reading frame (ORFfinder) was predicted to determine its coding region. GSDS analysis was used to analyze its gene structure, clarifying the distribution characteristics of exons and introns. Plant-mSubP was used to predict the subcellular localization of its protein product, inferring its functional location. CPC was used to assess its coding potential, and ProtParam was used to analyze the physicochemical properties of its encoded protein. RegRNA 2.0 was used to predict functional RNA elements in its sequence, including ribosome binding sites and miRNA target sites, providing a reference for subsequent functional verification and expression regulation studies.

[0050] The results are as follows: PCR amplification was performed using wheat cDNA as a template to obtain the target fragment of 1350 bp, which was then recovered and sequenced. Figure 1 GSDS platform analysis revealed that the gene consists of two exons and one intron, with a relatively compact structure and a typical eukaryotic expression framework. Based on genome sequence alignment with a reference genome, TaPMT-L was precisely located on wheat chromosome 1B. ORFfinder analysis showed that the gene possesses a complete open reading frame, encoding a polypeptide chain of 449 amino acids. ProtParam predicted its theoretical molecular mass to be 48771 Da and its isoelectric point to be 5.48. CPC assessment results indicated strong coding potential, supporting its status as a functional structural gene. Plant-mSubP predicted that the protein is mainly located in the cytoplasm and cell membrane, suggesting a possible role in intracellular signal transduction or metabolic regulation. Further analysis of its mRNA sequence using RegRNA 2.0 revealed a typical ribosome binding site in the 5' untranslated region and multiple potential miRNA recognition sites at the 3' end, suggesting that its expression regulation may be influenced by multiple factors. These results provide fundamental information for subsequent functional validation and in-depth research into its molecular mechanisms of drought resistance.

[0051] 2. TaPMT-L gene expression analysis 1) Cultivation and treatment of experimental materials Plump and uniform Xinchun No. 9 wheat seeds were selected, disinfected with sodium hypochlorite solution, washed with sterile water, and sown in germination boxes lined with three layers of filter paper. The seeds were cultured at 22℃ (16 h light / 8 h dark). When the seedlings reached two weeks of age, drought stress was applied. The experimental treatment used 20% PEG6000 solution to simulate drought conditions for 48 hours, while the control group used an equal volume of distilled water. Each group had three biological replicates. Leaf and root materials were collected after treatment. Furthermore, to further clarify the spatiotemporal expression characteristics of TaPMT-L in different tissues, roots, stems, leaves, young spikes, glumes, and grains of potted wheat were collected under normal and drought conditions (soil moisture content 40%, lasting 3 days). RNA was extracted from each tissue using the TRIzol method, and after testing for purity and integrity, it was reverse transcribed for subsequent expression analysis.

[0052] 2) qRT-PCR detection TaPMT-L gene expression was quantitatively analyzed using the TransScript® Green One-Step qRT-PCR SuperMix kit on a Roche LightCycler 480 real-time quantitative PCR instrument to evaluate its expression response in different tissues and under drought stress conditions. Primers, analyzed by melting curve analysis and 1.5% agarose gel electrophoresis, demonstrated good amplification specificity and produced single amplified products, meeting the requirements for qPCR analysis. Each reaction system consisted of three biological replicates and three technical replicates, with TaActin used as an internal control gene.

[0053] qRT-PCR primers: TaPMT-L-qPCR-F (SEQ ID No.5): 5'-GTTCCTGGTCGCTCTGTG-3'; TaPMT-L-qPCR-R (SEQ ID No. 6): 5'-GCCGCCCATGTTGTT-3'.

[0054] qRT-PCR data processing was performed through 2 −ΔΔCt Relative expression levels were calculated. In the drought treatment expression analysis, the expression level of the control sample was normalized to 1, and the expression changes of TaPMT-L in the PEG stress group were compared. In the tissue expression pattern analysis, the expression level of grain tissue under normal moisture conditions was used as a reference, and the relative expression levels of other tissues were normalized. The final result was the average of three replicates. Error bars represent standard errors, and the statistical significance level was set as follows: * indicates P < 0.05, ** indicates P < 0.01.

[0055] The results showed that TaPMT-L significantly induced expression under drought conditions, especially in leaves, where the expression level was upregulated by 9 times compared to the control group. Meanwhile, significant spatiotemporal differences in expression were observed in different tissues, with significantly increased expression levels in roots and stems, while expression levels in young spikes, glumes, and grains remained relatively stable. This suggests that TaPMT-L may play a role in the wheat's response to water stress. Figure 2 ).

[0056] 3. Creation of wheat TaPMT-L overexpression and RNAi lines 1) Construction of overexpression vectors The expression vector pCAMBIA1302 was digested with Spe I and BstE II, and the large fragment was recovered. Specific amplification primers were designed based on the TaPMT-L gene sequence. The primer sequences are as follows: TaPMT-L-All-F (SEQ ID No.7): 5'-accatggtagatctgactagtATGGGGTTCGCGGTGACG-3'; TaPMT-L-All-R (SEQ ID No.8): 5'-ggggaaattcgagctggtcaccTTACTTGCCAGCGGCGAA-3'.

[0057] In this diagram, lowercase letters represent the 15bp overlapping sequence of the linear vector, and lowercase italic letters represent the Spe I and BstE II restriction sites, respectively. PCR amplification was performed using the recombinant vector pEASY®-T1-PMT-L, a positive clone selected for sequencing after resistance screening in section 1 of Part II (1) of the aforementioned TaPMT-L gene clone. The PCR reaction system consisted of: 1.0 μL Template, 1.0 μL Forward Primer, 1.0 μL Reverse Primer, 25.0 μL 2×TransStart® FastPfu FlyReaction Mix, 1.0 μL TransStart® FastPfu Fly DNA Polymerase, and 21.0 μL Water. The PCR reaction conditions were: 98 ℃ for 1 min; 98 ℃ for 10 sec; 70 ℃ for 5 sec; 72 ℃ for 1 min, 33 cycles; 72 ℃ for 1 min. The reaction product was seamlessly cloned into the recovered linearized vector fragment, transformed into competent E. coli cells, and positive clones were screened. Single colonies were picked, expanded, and verified by PCR followed by sequencing. Positive clones were labeled as pCAMBIA1302-PMT-L transformants.

[0058] 2) Construction of RNAi vectors Specific primers were designed using the conserved domain (200-400 bp) of the TaPMT-L gene. GatewayBP recombinant attB1 and attB2 sequence tags were added to both ends of the primers, and the interfering fragment was obtained by PCR amplification. After purification of the PCR product by agarose gel electrophoresis, a BP reaction was performed using BP Clonase™ to recombine the fragment into the pDONR™ vector. This vector was then transformed into *E. coli* DH5α, and positive clones were screened using antibiotic plates. Entryclones verified by restriction enzyme digestion and sequencing were then reacted with the RNAi expression vector pSTARGATE using LR Clonase™ to recombine the target fragment into the inverted repeat frame of the pSTARGATE vector, forming an ihpRNA structure. The recombinant product was transformed into *E. coli* DH5α, and positive clones were screened on kanamycin-containing plates. Single clones were picked, plasmid DNA was extracted, and the recombination was verified by restriction enzyme digestion, electrophoresis, and sequencing. The final pSTARGATE-TaPMT-L-RNAi recombinant plasmid was used to transform *Agrobacterium* EHA105 competent cells.

[0059] 3) Agrobacterium-mediated transformation The recombinant expression vector and RNAi interference vector, verified by enzyme digestion, ligation, and sequencing, were transformed into Agrobacterium EHA105 competent cells using a freeze-thaw method. Procedure: The frozen competent cells were slowly thawed on ice. 100 μL of bacterial culture was aspirated, and 2 μL of recombinant plasmid was added. The mixture was gently tapped to mix, and the cells were incubated on ice for 5 min. Then, the cells were rapidly frozen in liquid nitrogen for 5 min, followed immediately by heat shock in a 37°C water bath for 5 min. Next, 700 μL of antibiotic-free YEP liquid medium was added, and the cells were cultured at 28°C and 200 rpm with shaking for 2 h to promote cell growth. The cells were then centrifuged at 3000 rpm for 4 min, the supernatant was discarded, and the remaining bacterial culture was used to re-mix the precipitated cells. The mixture was then evenly spread onto YEP plates containing antibiotics (50 μg / mL Kan and 50 μg / mL Rif) and incubated at 28°C for approximately 36 h. After the resistant colonies grew, single colonies were randomly selected for PCR verification. Amplification was performed using TaPMT-L specific primers, and positive strains with clear bands and good amplification specificity were screened, numbered, and stored for subsequent genetic transformation experiments in wheat. Simultaneously, the verified positive Agrobacterium transformants were cryopreserved at -80℃ with glycerol.

[0060] 4) Agrobacterium-mediated transformation of wheat embryos Transformants were inoculated into YEP liquid medium containing kanamycin (50 μg / mL) and rifampin (50 μg / mL) and cultured with shaking at 28℃ and 200 rpm until the OD600 value of the bacterial culture reached approximately 0.5-2. The bacterial cells were collected by centrifugation (4000 rpm, 10 min), and the supernatant was discarded. Infection medium (MS + 100 μM acetylsylcholine + 0.02% Silwet L-77) was prepared. Grains of Xinchun No. 9 wheat, approximately 14 days after flowering, were selected. After removing the husks, immature seeds were aseptically removed, and the endosperm was removed to separate the embryos. Fresh embryos were placed in the infection medium, gently shaken, and incubated at room temperature for 10-15 min. After incubation, the surface bacterial culture was blotted dry with sterile filter paper, and the embryos were transferred to a co-culture medium, laid flat with the embryos facing upwards, and cultured in the dark at 25℃ for 2-3 days. After co-culture, the embryos were transferred to an induction medium to induce callus formation. After the callus tissue gradually expands, it is transferred to differentiation medium to induce shoot differentiation, and finally transferred to rooting medium to obtain a complete plant. Figure 3 After rooting, the plants were transplanted into nutrient pots and acclimatized in a greenhouse. When the seedlings reached the three-leaf stage, leaves were collected, DNA was extracted, and PCR testing was performed to screen for T0 transgenic plants that were positive for the target gene. Plants transgenic with the pCAMBIA1302 empty vector were used as negative controls.

[0061] When T2 generation wheat seedlings reached the three-leaf stage, they were irrigated with a 20% PEG6000 solution to simulate drought stress for 7 consecutive days. After treatment, plant phenotypes were observed, the survival rates of each transgenic line and the control were recorded, and the relative water content, chlorophyll content, and soluble sugar levels of leaves were measured to assess the physiological adaptability of each line under drought conditions. Simultaneously, leaf tissues were collected, total RNA was extracted, and cDNA was reverse transcribed. The expression level of TaPMT-L was detected by qRT-PCR, as described in section 2, point 2) of Part II, qRT-PCR detection.

[0062] The results are as follows: A plant expression vector and RNAi interference vector containing the TaPMT-L gene were successfully constructed. After verification by sequencing, they were introduced into Agrobacterium EHA105 competent cells using a freeze-thaw method. The recombinant strain was then used to infect the immature embryos of Xinchun 9 wheat using Agrobacterium-mediated transformation. After tissue culture stages including co-culture, callus induction, shoot differentiation, and rooting, a batch of normally growing wheat regenerated plants were obtained. Gene-specific PCR and qPCR detection of the T0 generation plants confirmed the acquisition of multiple positive plants. Through continuous self-pollination and selection, genetically stable T2 generation lines were obtained.

[0063] 4. Drought treatment and phenotypic identification Healthy TaPMT-L overexpressing lines, RNAi interference lines, and wild-type (WT) wheat plants with uniform growth were selected and transplanted into identical substrate and uniformly sized nutrient pots at the three-leaf stage. Each plant was kept under the same light, temperature, and humidity conditions for two weeks to acclimatize. Drought treatment was conducted in an artificial climate chamber (photoperiod 16h light / 8h dark, 22℃ / 18℃). Drought stress was implemented using two methods: 1) Soil drought treatment When the plants reached the jointing stage, normal irrigation was stopped, allowing the soil moisture content to naturally decrease to 40% of field capacity, and this drought lasted for 5 days. The control group maintained soil moisture content at 80% of field capacity. During this period, each plant was irrigated using the same water supply standard to ensure consistent treatment. The appearance of the plant leaves, including curling, wilting, and yellowing, was recorded regularly and photographed for record-keeping.

[0064] 2) PEG simulated drought treatment Plump and uniform seeds were sown in germination boxes containing three layers of filter paper. At the three-leaf stage, the seeds were irrigated with a 20% (w / v) PEG6000 solution for 2 days. After treatment, the leaf condition and root growth were compared with the control group (without PEG).

[0065] After treatment, leaf samples were collected from different strains, and drought-related physiological indicators such as relative water content (RWC), chlorophyll content, soluble sugar, and malondialdehyde (MDA) were measured. The measurement methods followed standard physiological indicator testing, with at least three biological replicates for each indicator. Simultaneously, leaf RNA was collected, and qRT-PCR was used to detect the expression level of TaPMT-L in each strain. The drought-resistant physiological indicators, morphological characteristics, and gene expression levels were comprehensively compared to evaluate the regulatory effect of TaPMT-L overexpression or interference on wheat drought resistance.

[0066] The results are as follows: In soil drought treatment, WT plants showed significant leaf curling and wilting after 5 days of drought; RNAi-interferenced lines exhibited more severe drought damage, with greater leaf curling, severe wilting, and even partial leaf tip drying; while overexpression lines showed stronger drought resistance, with leaves maintaining higher uprightness, darker color, and significantly reduced wilting. Figure 4 ).

[0067] In PEG-simulated drought treatment, the root growth of seedlings from WT and RNAi interference lines was significantly inhibited, and leaves showed rapid water loss, curling, and yellowing; while the overexpression lines maintained relatively green and plump leaves, and their root growth was relatively longer and more vigorous. Figure 5 ).

[0068] Significant differences were also observed in the drought tolerance physiological indicators of different strains under drought stress. Figure 6 Compared with WT, the overexpression lines showed significantly increased relative water content (RWC) in leaves, significantly increased soluble sugar content, significantly decreased malondialdehyde (MDA) content and electrolyte leakage rate, and maintained a high level of chlorophyll content; while the RNAi interference lines showed the opposite trend, with significantly decreased RWC and chlorophyll, increased MDA content, and lower soluble sugar content than WT.

[0069] qRT-PCR results showed that under drought conditions, the expression level of TaPMT-L in the overexpression lines was significantly higher than that in the WT lines, while the expression level in the RNAi interference lines was significantly reduced, consistent with the trends of phenotypic and physiological changes.

[0070] Comprehensive analysis showed that high expression of TaPMT-L significantly enhanced the drought resistance of wheat, while reduced expression of TaPMT-L weakened the drought resistance of the plants, indicating that TaPMT-L plays a positive regulatory role in the formation of drought resistance in wheat.

[0071] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of the TaPMT-L gene in improving plant drought resistance, characterized by, The nucleotide sequence of the TaPMT-L gene is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, Drought-resistant transgenic plants were obtained by constructing the TaPMT-L overexpression vector.

3. The application according to claim 1, characterized in that, The drought resistance is reflected in the fact that, under water deficit conditions, the yield per plant of the TaPMT-L gene-overexpressing line is higher than that of the wild type.

4. The application according to any one of claims 1-3, characterized in that, The plant in question is wheat.

5. A plant breeding method, characterized in that, The goal is to obtain plants with stronger drought resistance than the target plant by increasing the activity of the TaPMT-L protein in the target plant; the amino acid sequence of the TaPMT-L protein is shown in SEQ ID No.

2.

6. A plant breeding method, characterized in that, The goal is to obtain plants with stronger drought resistance than the target plant by promoting the expression of the TaPMT-L gene in the target plant; the nucleotide sequence of the TaPMT-L gene is shown in SEQ ID No.

1.

7. A plant breeding method, characterized in that, The method involves inhibiting the expression of the TaPMT-L gene in the target plant to obtain plants with lower drought resistance than the target plant; the nucleotide sequence of the TaPMT-L gene is shown in SEQ ID No.

1.

8. The plant breeding method according to claim 6 or 7, characterized in that, The way to promote TaPMT-L gene expression in the target plant is to overexpress the TaPMT-L gene; the way to inhibit TaPMT-L gene expression in the target plant is to silence / interfere with TaPMT-L gene expression.

9. The plant breeding method according to any one of claims 5-8, characterized in that, The target plant is wheat.

10. The application of the TaPMT-L gene as described in claim 1 in increasing wheat yield.

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