Application of tasA protein in improving stress resistance of rice

Abstract

The application belongs to the field of agricultural biotechnology, and more particularly relates to application of a TasA protein in improving stress resistance of rice. The amino acid sequence of the TasA protein is shown as SEQ ID NO. 1, and the rice tolerance to high temperature and drought stress can be significantly improved. Experiments show that the use of a rice stress resistance regulator containing the TasA protein can effectively alleviate the inhibition of stress on plant growth. Compared with the untreated group, the rice seedlings to which the rice stress resistance regulator containing the TasA protein is applied have a significantly increased plant height, fresh weight and dry weight under high temperature stress, and also show a better growth state under drought stress. The rice stress resistance regulator provided by the application has a simple use method and low cost. The application provides a new technical path for improving crop stress resistance by using an exogenous TasA protein, and is suitable for stress resistance improvement of rice and other crops.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, and more specifically, relates to the application of TasA protein in improving the stress resistance of rice. Background Technology

[0002] Climate change is exacerbating the problem by increasing the frequency and intensity of extreme heat events, severely threatening the yields of major food crops such as rice. Intermittent or persistent high temperatures not only inhibit the vegetative growth of rice but also lead to pollen abortion and reduced seed setting rate during the reproductive stage, ultimately resulting in yield reduction. It is estimated that for every 1°C increase in average temperature, rice yield decreases by 3.2%. Over the past few decades, the average temperature in major rice-growing regions worldwide has risen by approximately 1°C, and this trend may intensify further. Therefore, exploring new heat-resistant pathways has become an urgent need to ensure the stability of rice production.

[0003] Plants possess a variety of mechanisms to resist high-temperature stress, such as producing heat shock proteins to maintain protein homeostasis, activating antioxidant enzyme systems to scavenge reactive oxygen species, and accumulating osmotic regulators like proline and soluble sugars to maintain cellular homeostasis. These responses collectively form a coordinated regulatory network that can reduce cell damage under high-temperature conditions. Although genetic improvement measures have made some progress in enhancing crop heat tolerance, these methods often require multiple generations of breeding due to the complex regulatory networks and the intensification of global warming, making it difficult to meet production demands in the short term. Combining existing technologies with biotechnologies such as beneficial microorganisms may open up new, more flexible, and sustainable pathways to enhance plant heat tolerance.

[0004] Microorganisms, due to their unique metabolites and regulatory functions, are considered one of the important exogenous pathways to improve plant stress resistance. Currently, the industrial production, transportation, and field application of microbial agents often face the problem of high-temperature environments, and thermophilic microorganisms have limited heat resistance and unstable performance. Thermophilic microorganisms, on the other hand, are naturally adapted to high-temperature environments, and their unique genetic resources hold the promise of overcoming the "heat intolerance" problem in the application of microbial preparations.

[0005] Thermophilic microorganisms are often found in extreme environments such as hot springs and volcanic soils. Prolonged exposure to high temperatures endows them with unique physiological mechanisms, such as the production of stable extracellular enzymes, heat-resistant proteins, and various secondary metabolites. These characteristics not only ensure their own survival but may also translate into advantages that enhance the host's heat tolerance during interactions with plants. Among the extracellular proteins secreted by Bacillus subtilis, TasA is a core functional amyloid protein produced by bacteria such as Bacillus subtilis, playing a crucial structural scaffold role in biofilm formation. Existing research has found that TasA protein can directly inhibit various plant pathogenic fungi and synergistically enhance the destructive effects of antimicrobial substances on fungal cell structures; however, the role of TasA protein in plant heat tolerance remains unknown. Summary of the Invention

[0006] The purpose of this invention is to provide the application of TasA protein in improving the stress resistance of rice, so as to solve the above-mentioned technical problems.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the application of TasA protein in improving the stress resistance of rice, and the amino acid sequence of TasA protein is shown in SEQ ID NO.1.

[0008] This invention utilizes the TasA protein as an exogenous active factor to activate rice's defense response to high temperature and drought stress through brief root immersion treatment. Unlike traditional genetic improvement, this strategy does not rely on transgenic manipulation; instead, it rapidly and flexibly enhances rice's stress resistance through exogenous protein addition. Experiments have verified that the TasA protein significantly promotes the growth of rice seedling height, fresh weight, and dry weight under both high temperature and drought stress, demonstrating an enhanced stress resistance effect. This concept expands the application boundaries of exogenous proteins in plant protection and provides a novel biotechnological solution for addressing extreme environments caused by climate change.

[0009] Furthermore, the stress resistance includes resistance to high temperatures and drought.

[0010] Furthermore, the application is to increase the biomass of rice under high temperature and / or drought stress.

[0011] Furthermore, the biomass includes plant height, fresh weight, and dry weight.

[0012] Furthermore, the application involves soaking rice seeds in an aqueous solution of the TasA protein for 1-3 hours, followed by germination to obtain seedlings, and then soaking the roots of the seedlings in the aqueous solution of the TasA protein for 0.5-1.5 minutes.

[0013] Furthermore, the concentration of the TasA protein in the aqueous solution is 0.5~1.5 μg / mL.

[0014] The present invention provides a rice stress resistance regulator prepared by mixing the TasA protein described in claim 1 with water.

[0015] Furthermore, the concentration of TasA protein in the rice stress resistance regulator is 0.5~1.5 μg / mL.

[0016] The present invention has at least the following beneficial effects: (1) This invention provides a new use of TasA protein in improving the stress resistance of rice and expands the application scope of microbial functional proteins in crop stress resistance improvement.

[0017] (2) The present invention uses the method of exogenous application of TasA protein, which does not require genetic transformation of plants. The treatment method is simple, flexible in application, and easy to promote in production practice.

[0018] (3) The experimental results showed that TasA protein could significantly increase the plant height, fresh weight and dry weight of rice seedlings under high temperature stress and improve the growth status under drought stress, indicating that it has a good stress resistance and promotion effect.

[0019] (4) The protein composition provided by the present invention is simple in composition, easy to use and low in cost, providing a new technical path for the development of novel crop stress-resistant biological agents. Attached Figure Description

[0020] Figure 1 Transcriptome analysis of Bacillus subtilis LSG20 under different temperature conditions is shown in Figure A. Figure A is a heatmap of sample correlations, displaying the clustering relationships between biological replicates at 28 ℃, 45 ℃, and 60 ℃. The value on each colored block in the heatmap represents the correlation between the two samples corresponding to that block on the horizontal and vertical axes; the larger the value, the higher the correlation. Figure B is a bar chart of differentially expressed genes. The horizontal axis represents different sets of differentially expressed genes: blue represents all differentially expressed genes, orange represents upregulated genes, and green represents downregulated genes. The vertical axis represents the number of differentially expressed genes.

[0021] Figure 2 The graphs show the effects of Bacillus subtilis LSG20 metabolite on the heat tolerance of Nipponbare rice. A represents the phenotypic pattern after metabolite treatment. Scale bar = 13cm. B shows plant height, C shows fresh weight, and D shows dry weight.

[0022] Figure 3 This is a statistical graph of Tasa expression in transcriptome analysis.

[0023] Figure 4 Figure 1 shows the expression and purification of TasA protein. In Figure 2, A represents the SDS-PAGE analysis of TasA protein expression and purification results in *E. coli* BL21(DE3), M is the protein marker, UI is the uninduced bacterial culture, WC is the whole-cell lysate after induction, P is the precipitate, S is the supernatant, FT is the flow-through fraction, and 20 mM and 500 mM are the elution buffers for 20 mM and 500 mM imidazole, respectively. Figure 3 shows the finally purified recombinant TasA protein.

[0024] Figure 5The graphs show the effects of exogenous TasA protein on Nipponbare rice under high temperature and drought stress. A represents the phenotypic distribution of Nipponbare seedlings under normal temperature (NS), normal temperature with TasA (NS-TasA), high temperature stress (HS), high temperature stress with TasA (HS-TasA), drought stress (DS), and drought stress with TasA (DS-TasA), with a scale bar of 13 cm. B represents plant height under control and high temperature stress, C represents fresh weight under control and high temperature stress, D represents dry weight under control and high temperature stress, E represents plant height under control and drought stress, F represents fresh weight under control and drought stress, and G represents dry weight under control and drought stress. Detailed Implementation

[0025] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0026] Example 1 I. Experimental Methods.

[0027] 1. Strains and culture conditions The tested strain LSG20 was isolated from a hot spring in Lingshui, Hainan Province, and is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34029. For long-term preservation, the strain was stored in a 40% (v / v) glycerol solution at -80°C. For routine experiments, the strain was recovered by streaking with glycerol tubes and cultured on LB agar plates at 45°C to obtain single colonies, which were then used as experimental materials.

[0028] 2. Transcriptome sequencing at different temperatures using LSG20 The strains were cultured in LB liquid medium at different temperatures (28℃, 45℃, and 60℃, 180 rpm) for 12 h. After culturing, the supernatant was discarded, and the bacterial cells were collected and washed with sterile distilled water. Total RNA was extracted from the bacterial cells using an RNA extraction kit and used for library construction after quality testing. rRNA was removed using the RiboCop rRNA Depletion Kit for Mixed Bacterial Samples (Lexogen, USA), and then a library was constructed using Illumina® Stranded mRNA Prep, Ligation Kit (Illumina, USA). Paired-end sequencing was performed on the Illumina NovaSeq 6000 platform. After quality control to remove adapters and low-quality reads, high-quality clean data was obtained. The effective data were aligned to the reference genome using Bowtie2, and expression levels were quantified using RSEM (TPM / FPKM). Differential expression analysis was performed using DESeq2. GO enrichment analysis of differentially expressed genes was performed using clusterProfiler, while KEGG enrichment analysis was performed using a combination of KOBAS and clusterProfiler. Transcriptional structure and sRNA prediction were performed using Rockhopper, sRNA secondary structure prediction was performed using RNAfold, and target gene prediction was performed using IntaRNA.

[0029] 3. High-temperature stress treatment with Bacillus subtilis LSG20 One week prior to the high-temperature stress treatment (day 14), pretreatment was performed: the roots of Nipponbare (japonica rice) seedlings were immersed in metabolic solutions of Bacillus subtilis LSG20 at different temperatures (prepared under culture conditions of 28℃, 45℃, and 60℃, and filtered through a 0.22μm filter membrane) for 1 min (the control treatment consisted of an equal volume of sterile water). Immediately after the pretreatment, the seedlings were returned to their original hydroponic conditions and cultured for another 7 days. High-temperature stress treatment was then initiated on day 21. The high-temperature stress conditions were: 14 h light exposure, 42°C, and 90% relative humidity. Phenotypic data were recorded after the high-temperature stress treatment.

[0030] II. Experimental Results.

[0031] like Figure 1 As shown, transcriptomic differences were analyzed under normal temperature (28℃), medium-high temperature (45℃), and extreme high temperature (60℃). Figure 1 The results showed that LSG20 underwent large-scale transcriptional regulation at different temperatures, indicating that LSG20 employs a temperature-dependent regulatory strategy during high-temperature adaptation. Figure 1 B).

[0032] like Figure 2 As shown, under high-temperature stress, plant height, fresh weight, and dry weight of both rice varieties were significantly inhibited. However, the 28℃ metabolite solution of LSG20 significantly alleviated the damage caused by high-temperature stress to both rice varieties. In Nipponbare rice, treatment with the 28℃ metabolite solution significantly increased plant height, fresh weight, and dry weight, by 20.97%, 20.53%, and 34.54%, respectively. In JLYHZ rice, the 28℃ metabolite solution also had a mitigating effect on high-temperature stress. In contrast, the protective effects of treatments with metabolites at 45℃ and 60℃ were limited, with an overall effect lower than that of 28℃.

[0033] The above experimental results indicate that metabolites at 28℃ may be key factors in enhancing the heat resistance of rice. Combined with transcriptome analysis, such as... Figure 3 As shown, TasA expression was highest at 28℃, but decreased significantly at 45℃ and 60℃, consistent with the results of the metabolic fluid effect. Based on this, TasA was selected as a representative functional protein for validation, and its effects on rice seedlings under high temperature and drought stress were evaluated.

[0034] Example 2 I. Heterologous expression and purification of TasA protein.

[0035] The nucleotide sequence of the TasA gene was obtained based on transcriptome analysis, as shown in SEQ ID No. 1. Primers were designed based on this sequence for amplification, and the gene was then cloned into the pET-28a(+) vector (EMD Biosciences (Novagen)). BamH I and Xho Between the I sites, the plasmid carries a His tag, indicating resistance to Kanamycin. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells and cultured in LB medium containing 50 μg / mL kanamycin. When the bacterial culture OD... 600 When the concentration reached 0.6, 0.5 mM IPTG was added for induction, and the cells were cultured overnight in a shaker at 16°C. After collecting the cells, they were lysed by sonication, and the precipitate was removed by centrifugation. The supernatant was purified into a His-tagged fusion protein using a Ni-NTA affinity chromatography column (Qiagen, Germany). After dialysis to remove imidazole, the protein molecular weight and purity were verified by SDS-PAGE, and the concentration was determined using a BCA protein quantification kit (ThermoFisher Scientific, USA). Finally, the purified protein was dissolved in sterile PBS to prepare a concentration of 1 μg / mL for use in rice treatment experiments.

[0036] SEQ ID No.1: GGATCCGCATTTAACGACATTAAATCAAAGGATGCTACTTTTGCATCAGGTACGCTTGATTTATCTGCTAAAGAGAATTCAGCGAGTGTGAACTTATCAAATCTAAAGCCGGGAGATAAGTTGACAAAGGATTTCCAATTTGAAAATAACGGATCACTTGCGATCAAAGAAGTTTTAATGGCGCTTAATTATGGAGATTTTAAAGCAAACGGCGGCAGCAATACATCTCCAGAAGATTTCCTCAGCCAGTTTGAAGTGACATTGTTGACAGTTGGAAAAGAGGGCGGCAATGGTTACCCGAAAAACATTATTTTAGATGATGCGAACCTTAAAGACTTGTATTTGATGTCTGCTAAAAATGATGCAGCGGCTGCTGAAAAAATCAAAAAACAAATAGACCCTAAATTCTTACATGCAAGCGGTAAAGTCAATGTAGCAACAATTGACGGTAAAACCGCTCCTGAATATGATGGTGTTCCAAAAACACCAACTGACTTCGATCAGGTTCAAATGGAAATCCAATTCAAAGATGATAAAACAAAAGATGAAAACGGGCTTATGGTTCAAAATAAATATCAAGGCAACTCCATTAAGCTTCAATTCTCGTTCGAAGCTACACAGTGGAACGGCTTGACAATCAAAAAGGACCATACTGATAAAGACGGTTATGTGAAAGAAAATGAAAAAGCGCACAGCGAGGATAAAAATTAACTCGAG。

[0037] The amino acid sequence of the TasA protein is shown in SEQ ID No.2.

[0038] SEQ ID No.2: MGMKKKLSLGVASAALGLALVGGGTWAAFNDIKSKDATFASGTLDLSAKENSASVNLSNLKPGDKLTKDFQFENNGSLAIKEVLMALNYGDFKANGGSNTSPEDFLSQFEVTLLTVGKEGGNGYPKNI ILDDANLKDLYLMSAKNDAAAAEKIKKQIDPKFLHASGKVNVATIDGKTAPEYDGVPKTPTDFDQVQMEIQFKDDKTKDENGLMVQNKYQGNSIKLQFSFEATQWNGLTIKKDHTDKDGYVKENEKAHSEDKN.

[0039] II. Plant materials and growing conditions.

[0040] The rice variety used in the experiment was Nipponbare (japonica rice). Nipponbare was purchased from Xiantao Jiarui Agricultural Supplies Store. Rice seeds with uniform maturity, plumpness, and freedom from pests, diseases, and mechanical damage were selected for the experiment. The seeds were placed in a glass dish and soaked in sterile water (121℃, 20 min), and floating seeds were removed. Subsequently, they were disinfected with 70% ethanol for 3 min, then with 2% sodium hypochlorite solution for 10 min, rinsed thoroughly with sterile water, and dried on sterile filter paper for later use.

[0041] III. High temperature and drought stress treatment with TasA protein solution.

[0042] After sterilization, the seeds were soaked in 200 mL of 1 μg / mL TasA protein solution and sterile water (control, NS) for 2 hours. After soaking, they were placed in a dark climate chamber at a constant temperature of 28℃ and a relative humidity of 70% for 3 days to germinate. Once the seeds showed signs of sprouting, they were transferred to 96-well hydroponic boxes, one seed per well, for a total of 48 seedlings. The rice seedlings were grown at 28℃ / 24℃ (light / dark), with a photoperiod of 12h / 12h (light / dark) and a relative humidity of 70%. Yoshida rice nutrient solution was used during seedling growth, and the solution was changed every 7 days, maintaining a pH of 5.7.

[0043] One week prior to drought and heat stress treatment (i.e., day 14), pretreatment was performed: the roots of seedlings were immersed in a 1 μg / mL TasA protein solution for 1 min (control group seedlings were immersed in an equal volume of sterile water). Immediately after treatment, the seedlings were returned to their original hydroponic conditions and cultured for another 7 days. Drought and heat stress treatments were then initiated on day 21. Drought stress (DS) conditions were: 20% (w / v) PEG6000 was added to Yoshida nutrient solution (CHINOOK CN260806), stirred thoroughly until completely dissolved, and normal growth conditions (28 / 24℃, 12 / 12h, 70% relative humidity) were maintained for 72 consecutive hours. Heat stress (HS) conditions were: 14h light, 42℃, and 90% relative humidity. Phenotypic images were taken after treatment with high temperature and drought stress. NS-TasA indicates that the patient was not subjected to high temperature and drought stress after soaking in TasA protein solution. HS-TasA indicates that the patient was subjected to high temperature stress after soaking in TasA protein solution. DS-TasA indicates that the patient was subjected to drought stress after soaking in TasA protein solution.

[0044] IV. Measurement of rice seedling indicators.

[0045] After the high temperature and drought stress treatments were completed, the growth indicators of rice seedlings were measured. Each treatment was set up with 3 biological replicates, and each box contained 48 seedlings. For plant height measurement, 48 seedlings were randomly selected from 3 pots and measured individually. Fresh weight and dry weight were measured on a box-by-box basis. The fresh weight of 48 seedlings in each box was collected, and then the seedlings were dried at a constant temperature of 60℃ until constant weight and then measured again. A total of 3 replicates were performed.

[0046] All tests were performed in triplicate. Data were statistically analyzed using SPSS software (version 26.0, SPSS Inc., Chicago, IL, USA). Two-tailed Student's t-tests were used to analyze statistical significance, with a significance level set at P < 0.05. Figures and tables were generated using GraphPad Prism version 8.0.2.

[0047] V. Experimental Results.

[0048] 1. The TasA gene was cloned into the pET-28a(+) vector and heterologously expressed in Escherichia coli BL21(DE3). Figure 4 (A). SDS-PAGE results showed that a distinct protein band appeared at approximately 31 kDa after IPTG induction. After purification with Ni²⁺-NTA, a single band (31 kDa) with a purity exceeding 90% was obtained. Figure 4 The B indicates that the recombinant TasA has been successfully expressed and can be used for subsequent functional analysis.

[0049] 2. High temperature stress treatment caused a significant decrease in the plant height, fresh weight, and dry weight of rice seedlings. Figure 5 Seedlings treated with exogenous TasA protein showed stronger heat resistance, with a significant increase in plant height of 10.66%, and increases in fresh weight and dry weight of 15.88% and 19.44%, respectively. Figure 5 (B, C, and D). High temperature and drought stress often occur simultaneously in the natural environment, and the two are strongly associated. In addition to high temperature stress, the role of TasA protein under drought stress was also investigated. Using PEG6000 to simulate a drought environment, it was found that the overall growth of the control group was severely inhibited ( Figure 5 TasA protein was added exogenously. Rice seedlings with this added protein maintained good growth even under drought conditions. Figure 5 (E and F).

[0050] The above results demonstrate that exogenous application of TasA protein can alleviate high temperature and drought stress in rice seedlings. This finding not only validates that TasA is a key factor in enhancing rice heat tolerance but also indicates that TasA possesses broad-spectrum stress regulation potential. This discovery expands our understanding of extracellular protein function and provides new materials for the application of microbial factors in crop stress tolerance improvement.

[0051] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0052] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0053] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of a TasA protein to improve stress resistance in rice, characterized in that, The amino acid sequence of the TasA protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The aforementioned resistance includes resistance to high temperatures and drought.

3. The application according to claim 1, characterized in that, The application aims to increase the biomass of rice under high temperature and / or drought stress.

4. The application according to claim 3, characterized in that, The biomass includes plant height, fresh weight, and dry weight.

5. The application according to claim 1, characterized in that, The application involves soaking rice seeds in an aqueous solution of the TasA protein for 1-3 hours, followed by germination to obtain seedlings, and then soaking the roots of the seedlings in the aqueous solution of the TasA protein for 0.5-1.5 minutes.

6. The application according to claim 4, characterized in that, The concentration of the TasA protein in aqueous solution is 0.5~1.5 μg / mL.

7. A rice stress resistance regulator, characterized in that, It is prepared by mixing the TasA protein described in claim 1 with water.

8. The rice stress resistance regulator according to claim 7, characterized in that, The concentration of TasA protein in the rice stress resistance regulator is 0.5~1.5 μg / mL.

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