A method to improve crop tolerance to high temperature stress

Abstract

This invention discloses a method for improving crop tolerance to high-temperature stress, belonging to the field of genetic engineering technology. The method includes using biological techniques to enhance the tolerance of OsUVR8b... S177A Genes are upregulated in crop plants to enhance their tolerance to heat stress. This invention provides a gene, OsUVR8b, that can regulate crop tolerance to heat stress. S177A OsUVR8b exhibits natural variation among rice varieties. In heat-sensitive varieties, the 177th position of OsUVR8b is serine, while in heat-resistant varieties, it is alanine. This variation is utilized in rice varieties where OsUVR8b... S177A Gene overexpression can significantly improve the survival rate and seed setting rate of plants under high temperature conditions. This invention provides a molecular basis for breeding and screening heat-resistant varieties. Since this type of variation site exists in nature, it has important practical application value in the future breeding of heat-resistant rice varieties.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to rice OsUVR8b. S177A Application of genes in improving crop tolerance to high temperature stress. Background Technology

[0002] Rice (Oryza sativa L.) is an annual aquatic herbaceous plant belonging to the Poaceae family and is one of the most important food crops for humankind. Rice growth is inextricably linked to environmental temperature; high temperatures are one of the most significant environmental factors affecting crop yield. Excessively high temperatures can affect the flowering period and pollen fertility, thus reducing rice yield. Furthermore, excessive accumulation of intracellular ROS caused by high temperatures can damage the plant's photosynthetic system and thylakoid membranes, leading to energy deficiency. Therefore, developing new heat-resistant crop varieties is of great importance.

[0003] When plants face extreme conditions such as high temperatures, they activate a series of regulatory mechanisms to maintain normal growth. Generally, the higher the temperature, the stronger the ultraviolet radiation. The UVR8 site in Arabidopsis thaliana has been identified as a receptor that mediates the UV-B radiation response. In Arabidopsis thaliana, the UV-B photoreceptor AtUVR8 can receive UV-B light signals, transform from a dimer to a monomer, and initiate the transduction of UV-B light signals, thus enabling Arabidopsis thaliana to adapt to and tolerate UV-B (Rizzini, L. et al. Perception of UV-B by the Arabidopsis UVR8 Protein. Science, 2011, 332(6025):103-106).

[0004] Ultraviolet light is both a stress signal and an important light signal for plant morphogenesis. UVR8, as an ultraviolet photoreceptor in plants, can sense and absorb ultraviolet light to transmit light signals and thus regulate plant growth and development. At present, most of the research on the ultraviolet photoreceptor UVR8 focuses on the morphogenesis of Arabidopsis thaliana. For Arabidopsis thaliana, UV-B signals are mostly related to growth, development and morphogenesis. However, in rice, UV-B is more of a stress signal that affects rice growth (Hu, S. et al. Nuclear accumulation of rice UV-B photoreceptors is UV-B-and OsCOP1-independent for UV-B responses. Nature Communication, 2024, 15:6396.). Studies have shown that UVR8 in Arabidopsis thaliana and rice responds differently to ultraviolet light (Yu-Long Chen. et al. OsUVR8b, rather than OsUVR8a, plays a predominant role in rice UVR8-mediated UV-B response. Physiol Plant, 2024, 176(4): 14471.).

[0005] Ultraviolet radiation is closely related to temperature. Generally speaking, rice grown at low latitudes receives stronger ultraviolet radiation and grows in warmer environments. However, whether UVR8 also participates in high-temperature regulation has not yet been reported in studies. Therefore, a deeper understanding of the role of photoreceptors in temperature-responsive regulation is of great significance. Summary of the Invention

[0006] The purpose of this invention is to clone genes with heat stress resistance from rice, providing a theoretical basis and candidate gene resources for the creation of new heat-resistant crop germplasm.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention uses sequence alignment analysis in the NCBI genome database to search for homologous genes of the Arabidopsis thaliana UVR8 gene in rice. Two UVR8 genes were found in rice, named OsUVR8a and OsUVR8b, with similarities of 74% and 75% to AtUVR8, respectively. Sequence alignment revealed natural variation in OsUVR8b among rice varieties. OsUVR8b consists of 453 amino acids. In the early-maturing japonica rice variety Kitaake and the heat-sensitive variety IR64, the 177th position of OsUVR8b was serine (S), while in the heat-resistant rice variety N22, the 177th position was alanine (A). Furthermore, transgenic overexpression technology was used to express OsUVR8b in rice. S177 and OsUVR8b S177A Through gene overexpression, studies have found that OsUVR8b S177 Overexpression of OsUVR8b resulted in a lower survival rate under high temperature conditions, while OsUVR8b... S177A Overexpression of rice showed higher high-temperature resistance and increased seed setting rate under high temperature, indicating that this site in OsUVR8b plays an important role in regulating rice's high-temperature tolerance.

[0009] Therefore, the present invention provides OsUVR8b S177A The application of genes in improving crop resistance to high temperature stress, specifically, the OsUVR8b gene. S177A The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1.

[0010] Mechanistic studies have shown that OsUVR8b can be phosphorylated by OsSnRK1.1 protein kinase, and the phosphate site is position 177 of the OsUVR8b amino acid sequence. When the amino acid at position 177 is serine (S), it can be phosphorylated by OsSnRK1.1, while when this site is mutated to alanine (A), the phosphorylation effect is significantly reduced.

[0011] Using Western blot technology to analyze OsUVR8b S177 and OsUVR8b S177A Protein stability was determined, and it was found that OsUVR8b under heat stress showed [stability / stability]. S177A The protein stability is stronger than that of OsUVR8b S177 ROS content determination results revealed OsUVR8b S177A ROS accumulation is less than OsUVR8b S177 These results indicate that OsUVR8b S177A It is beneficial to improve protein stability and ROS scavenging ability, and has important application value in improving the high temperature resistance of rice.

[0012] Furthermore, the application includes: utilizing biological techniques to make OsUVR8b S177A Genes are upregulated in crop plants to enhance their tolerance to high-temperature stress.

[0013] High-temperature stress refers to an ambient temperature higher than the optimal temperature for plant growth and development, with the temperature range of heat stress being 35–45℃. For example, the optimal temperature for rice growth is 20–35℃, and the temperature that causes heat stress to rice is generally above 35℃. Temperatures exceeding 35℃ will have a negative impact on the growth and yield of rice.

[0014] In this invention, the biological techniques described may include, but are not limited to, transgenic technology.

[0015] Furthermore, the application includes: using the OsUVR8b S177A Gene fragments are cloned into plant overexpression vectors, and the target fragments are introduced into recipient plants using Agrobacterium-mediated genetic transformation technology to cultivate heat-resistant transgenic plants.

[0016] This invention can synthesize gene fragments that encode proteins with amino acid sequences as shown in SEQ ID NO. 1, based on the codon preferences of specific crops.

[0017] Furthermore, the crop may be, but is not limited to, rice.

[0018] Furthermore, the OsUVR8b S177A The coding sequence of the gene is shown in SEQ ID NO.2.

[0019] Furthermore, the indicators of the crop's resistance to high-temperature stress include: plant survival rate, pollen fertility at maturity, and seed setting rate. Compared to wild-type plants, OsUVR8b S177A Plants with overexpressed genes showed significantly improved seedling survival rate and fruit setting rate at maturity under high temperature stress.

[0020] This invention also provides a breeding method for improving the heat stress tolerance of rice, comprising: using the nucleotide sequence OsUVR8b as shown in SEQ ID NO.2. S177A Gene fragments were cloned into plant overexpression vectors, and the target fragments were introduced into recipient rice using Agrobacterium-mediated genetic transformation technology to cultivate transgenic rice plants resistant to high-temperature stress.

[0021] OsUVR8b in transgenic rice plants S177A Overexpression of the gene-encoded protein enhances protein stability and ROS scavenging ability under heat stress, thereby improving heat resistance and increasing the survival rate and seed setting rate of rice under high temperature conditions.

[0022] In this invention, the plant overexpression vector can be any vector known in the art that is suitable for rice to be used in Agrobacterium-mediated genetic transformation technology, and can be, but is not limited to, the pCAMBIA1300 vector.

[0023] Furthermore, the recipient rice is the japonica rice variety Kitaake.

[0024] The beneficial effects of this invention are as follows:

[0025] This invention provides a gene, OsUVR8b, that can regulate crop tolerance to high temperature stress. S177A OsUVR8b exhibits natural variation among rice varieties. In heat-sensitive varieties, the 177th position of OsUVR8b is serine, while in heat-resistant varieties, it is alanine. This variation is utilized in rice varieties where OsUVR8b... S177A Gene overexpression can significantly improve the survival rate and seed setting rate of plants under high temperature conditions. This invention provides a molecular basis for breeding and screening heat-resistant varieties. Since this type of variation site exists in nature, it has important practical application value in the future breeding of heat-resistant rice varieties. Attached Figure Description

[0026] Figure 1 Sequence alignment of the OsUVR8b gene in kitaake, N22, IR64 and Arabidopsis thaliana.

[0027] Figure 2 OsUVR8b S177 Schematic diagram of a gene overexpression vector.

[0028] Figure 3 OsUVR8b S177A Schematic diagram of a gene overexpression vector.

[0029] Figure 4 OsUVR8b S177 The results of constructing transgenic rice are shown in Figure A, which is a schematic diagram of the vector, and Figure B is the result of expression level detection.

[0030] Figure 5 OsUVR8b S177A The results of constructing transgenic rice are shown in Figure A, which is a schematic diagram of the vector, and Figure B is the result of expression level detection.

[0031] Figure 6 OsUVR8b S177 and OsUVR8b S177A Phenotypic results at the seedling stage under heat stress, where A represents OsUVR8b S177 -OE seedling phenotype, B is OsUVR8b S177A -OE seedling phenotype.

[0032] Figure 7 OsUVR8b S177 and OsUVR8b S177A The fertility analysis results are shown, where A represents the pollen iodine staining results and B represents the seed set rate results.

[0033] Figure 8 OsUVR8b S177 and OsUVR8b S177A Results of protein stability and ROS content detection under heat stress, where A represents OsUVR8b. S177 Protein stability under heat stress at different times, B is OsUVR8b S177A Protein stability under heat stress at different times, C is OsUVR8b S177 and OsUVR8b S177A The DAB staining results, D is OsUVR8b S177 and OsUVR8b S177A The results of the hydrogen peroxide content determination. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0036] Example 1: Sequence alignment of UVR8 protein in rice and Arabidopsis thaliana

[0037] By using the amino acid sequence corresponding to the Arabidopsis thaliana AtUVR8 protein sequence number Q9FN03, a homology search was performed in NCBI (https: / / www.ncbi.nlm.nih.gov / protein / ) to find the homologous OsUVR8 gene in rice, and the sequences of OsUVR8b in rice varieties kitaake, N22 and IR64 were compared respectively.

[0038] The results are as follows Figure 1 As shown, sequence similarity analysis revealed that the UVR8 protein sequence in Arabidopsis thaliana shared 74% and 75% homology with OsUVR8a and OsUVR8b, respectively. In rice, OsUVR8b showed a site variation at position 177 in kitaake, N22, and IR64. N22 is a heat-resistant rice variety, while IR64 is a heat-sensitive variety.

[0039] The above results suggest that the natural variation sites of OsUVR8b may be related to temperature adaptability.

[0040] Example 2: Rice OsUVR8b S177 and OsUVR8b S177A Obtaining overexpression plants

[0041] To investigate the function of the OsUVR8b 177 site in rice, we constructed an OsUVR8b... S177 and OsUVR8b S177A Overexpression plasmid recombinant vectors were used to obtain overexpression plants via Agrobacterium-mediated genetic transformation.

[0042] 1. Construct OsUVR8b S177 and OsUVR8b S177A Overexpression recombinant vector

[0043] 1.1 Obtain OsUVR8b from the NCBI Genome Database S177 CDS sequence, OsUVR8b S177 The full-length CDS of the gene is 1362 bp (Os04t0435700-01), and its protein molecular weight is approximately 49.83 kDa (Q7XRB6).

[0044] Primers were designed based on the sequence: F1: 5'-ATGGACGCGGTCATGTCCGC-3'; R1: 5'-AACGAATGCACACGCATCCTCT-3'. RNA was extracted from rice varieties kitaake and N22 and reverse transcribed into cDNA. OsUVR8b was then amplified using a high-fidelity enzyme. S177 and OsUVR8b A177 After confirming the correct band size by agarose gel electrophoresis, the fragments were recovered and purified.

[0045] 1.2 Primers were designed based on the vector. The primer sequences were F2: 5'-agaacacgggggactctagaATGGACGCGGTCATGTCCGC-3'; R2: 5'-ctcaccattgttggatccAACGAATGCACACGCATCCTCT-3'. The fragment amplified in step 1.1 was used as a template for amplification. Homologous arms were added to both ends of the OsUVR8b gene, and then ligated into the pCAMBIA1300 overexpression vector via homologous recombination. After transformation and colony PCR analysis, sequencing confirmed the correctness of the vector. A schematic diagram of the vector is shown below. Figure 2 and Figure 3 As shown.

[0046] Specifically, OsUVR8bS177A The CDS sequence of the gene is shown in SEQ ID NO.2, and the encoded amino acid sequence is shown in SEQ ID NO.1; OsUVR8b S177 The CDS sequence of the gene is shown in SEQ ID NO.4, and the encoded amino acid sequence is shown in SEQ ID NO.3.

[0047] 2. OsUVR8b S177 and OsUVR8b S177A Creation of overexpression rice

[0048] The japonica rice variety kitaake was used as the recipient material for transformation. The transformation method was Agrobacterium-mediated genetic transformation of rice callus. After obtaining transgenic plants with T0 bands, mRNA was extracted and analyzed by reverse transcription and qPCR.

[0049] The qPCR primer sequences are F: 5'-CGGGTGGAGGCATACTCTTG-3', R: 5'-AGTTGTCCGCTAGTACCCCT-3';

[0050] The internal reference gene is Extro, with the sequence F: 5'-ATCTGCACCACCTCTGAGAC-3', R: 5'-TTCTTGAGGTGAGGGAAGCAC-3'.

[0051] The results are as follows Figure 4 , Figure 5 As shown, OsUVR8b S177 The independent transformation plants were numbered 1, 4, 18, and OsUVR8b. S177A The independent transformed plants were numbered 8, 14, and 30. These plants showed higher expression levels compared to the wild type and were used for subsequent phenotypic identification and seed setting rate detection.

[0052] Example 3: Functional identification of rice OsUVR8b 177 site under heat stress

[0053] 1. In order to investigate OsUVR8b S177 and OsUVR8b S177A Overexpression of the heat tolerance phenotype in rice seedlings was achieved using OsUVR8b. S177 and OsUVR8b S177A Heat tolerance experiments were conducted on three families of rice that overexpressed the gene. This study mainly investigated the survival rate of rice under high temperature stress, therefore, an extreme high temperature of 45℃ was selected as the heat treatment condition.

[0054] First, the two materials mentioned above were soaked at 37℃. After the seeds showed signs of sprouting, they were planted in black hydroponic boxes. After about 3 weeks of growth, rice seedlings with uniform growth were selected for seedling phenotypic experiments. The rice seedlings were then placed in a 45℃ light incubator for stress treatment, with growth conditions of 14 hours of light and 10 hours of darkness under normal white light conditions without additional ultraviolet light. After the leaves curled, the seedlings were placed in a normal environment for growth, and the survival rate was recorded.

[0055] Statistical results revealed that OsUVR8b S177A -OE materials have a higher survival rate than wild-type materials. Figure 6 A), while OsUVR8b S177 -OE has a lower survival rate than wild type ( Figure 6 B) OsUVR8b under heat stress treatment S177A -OE has a significantly higher survival rate than OsUVR8b. S177 -OE.

[0056] 2. To investigate the fertility of OsUVR8b at site 177 at maturity, OsUVR8b materials with consistent growth status during the booting stage were compared. S177 -OE and OsUVR8b S177A -OE was placed in a 45℃ climate chamber for 2 days and then moved to a normal environment for growth. During this period, florets at the spike stage under normal conditions and stress treatment were taken respectively, stained with I2-KI, and their pollen fertility was observed under a stereomicroscope. The seed set rate was also counted.

[0057] Pollen fertility and seed setting rate experiments showed that, under high temperatures, OsUVR8b S177A -OE has higher pollen fertility and seed set rate than OsUVR8b. S177 -OE( Figure 7 The results (A, B) indicate that the mutation at site 177 to A is beneficial for increasing the seed setting rate of rice under high temperature.

[0058] 3. To investigate how locus 177 affects high-temperature resistance in rice, OsUVR8b was studied. S177 and OsUVR8b S177A The protein stability and ROS content were detected.

[0059] First, use Western blot on OsUVR8b S177 -GFP and OsUVR8b S177A -GFP protein was detected. Three-week-old rice seedlings were treated at 45℃ for 0, 12, 24, and 48 hours, and OsUVR8b was detected using a GFP antibody. S177 and OsUVR8b S177AProtein content; in addition, rice seedlings with a growth period of 3 weeks were subjected to DAB staining and hydrogen peroxide content determination 3 days after heat stress treatment.

[0060] The results showed that OsUVR8b S177 Protein stability decreases under heat stress. Figure 8 A), while OsUVR8b S177A The protein can remain relatively stable in rice cells. Figure 8 B) This situation is likely caused by the phosphorylation of OsUVR8b by OsSnRK1.1.

[0061] DAB staining and hydrogen peroxide content determination experiments showed that after 3 days of heat stress treatment, OsUVR8b S177A The ROS accumulation is less than that of OsUVR8b S177 ( Figure 8 (C, D) This indicates that the 177 site, when changed to A, has a stronger ability to scavenge ROS, which may be the reason for the higher high-temperature tolerance after the 177 site is changed to A.

[0062] The above results indicate that when the 177th locus in OsUVR8b is A, it can significantly improve the survival rate and seed setting rate of rice under high temperature. The discovery and functional characterization of this gene locus provides molecular and application value for future rice breeding and the development of heat-resistant varieties with near-isogenic lines.

Claims

1. OsUVR8b S177A The application of genes in improving crop resistance to high temperature stress is characterized by, The applications include: utilizing biological technologies to enable... OsUVR8b S177A The gene is upregulated in crop plants to enhance their tolerance to high-temperature stress; OsUVR8b S177A The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.1; the crop is rice.

2. The application as described in claim 1, characterized in that, The application includes: [the following] OsUVR8b S177A Gene fragments are cloned into plant overexpression vectors, and the target fragments are introduced into recipient plants using Agrobacterium-mediated genetic transformation technology to cultivate heat-resistant transgenic plants.

3. The application as described in claim 1, characterized in that, The OsUVR8b S177A The coding sequence of the gene is shown in SEQ ID NO.

2.

4. A breeding method for improving the heat stress tolerance of rice, characterized in that, include: The nucleotide sequence is as shown in SEQ ID NO.

2. OsUVR8b S177A Gene fragments were cloned into plant overexpression vectors, and the target fragments were introduced into recipient rice using Agrobacterium-mediated genetic transformation technology to cultivate transgenic rice plants resistant to high-temperature stress.

5. The breeding method as described in claim 4, characterized in that, The plant overexpression vector was pCAMIBA1300.

6. The breeding method as described in claim 4, characterized in that, The recipient rice is the japonica rice variety Kitaake.

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