Method for improving high temperature stress resistance of crops

Abstract

The invention discloses a method for improving high temperature stress resistance of crops, and belongs to the technical field of gene engineering. According to the method, the expression of the OsUVR8bS177A gene in a crop plant is up-regulated by utilizing a biological technical means, so that the tolerance of the plant to high-temperature stress is improved. The invention provides the gene OsUVR8bS177A capable of regulating and controlling the high-temperature stress tolerance of the crops, the OsUVR8b has natural variation among rice varieties, the 177 site of the OsUVR8b in a high-temperature sensitive variety is serine, the 177 site of the OsUVR8b in a high-temperature resistant variety is alanine, the survival rate and the maturing rate of the plants in a high-temperature environment can be remarkably increased by utilizing the overexpression of the OsUVR8bS177A gene, and the survival rate of the plants in a high-temperature environment can be remarkably increased. The invention provides a molecular basis for cultivating and screening high-temperature-resistant varieties. As the variation site of the type exists in the nature, the variation site has important practical application value in the aspect of cultivating high-temperature-resistant rice varieties in the future.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to rice OsUVR8b S177A Application of genes in improving crop tolerance to high temperature stress. Background Art

[0002] Rice (Oryza sativa L.) is an annual aquatic herb in the Poaceae family and one of the most important food crops for humans. The growth of rice is closely related to the environmental temperature. High temperature is one of the most important environmental factors affecting crop yields. Excessive temperature will affect the flowering period and pollen fertility of crops, thereby reducing rice yields. In addition, excessive accumulation of intracellular ROS caused by high temperature will also damage the plant's photosynthetic system and thylakoid membrane, causing energy deficiency. Therefore, it is of great significance to develop new varieties of high temperature-resistant crops.

[0003] When plants face extreme conditions such as high temperatures, they will activate a series of regulatory mechanisms to maintain normal growth. Generally speaking, the higher the temperature, the stronger the ultraviolet rays. The UVR8 in Arabidopsis has been identified as a receptor that mediates UV-B radiation response. In Arabidopsis, the UV-B light receptor AtUVR8 can receive UV-B light signals, transform from dimers to monomers, and initiate the transduction of UV-B light signals, thereby achieving Arabidopsis' adaptation and tolerance to 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 an adversity signal and an important light signal for plant morphology. UVR8, as a UV photoreceptor in plants, can sense and absorb UV light to transmit light signals and thus regulate plant growth and development. At present, most of the research on the UV light receptor UVR8 is focused on the morphology of Arabidopsis. For Arabidopsis, UV-B signals are mostly related to growth, development and morphology. 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-andOsCOP1-independent for UV-B responses. Nature Communication, 2024, 15: 6396.). Studies have shown that UVR8 in Arabidopsis and rice responds differently to ultraviolet light (Yu-Long Chen.etal.OsUVR8b, rather than OsUVR8a, plays a predominant role in rice UVR8-mediated UV-B response.Physiol Plant,2024,176(4):14471.).

[0005] Ultraviolet rays are closely related to temperature. Generally speaking, rice grown at low latitudes will receive stronger ultraviolet rays and have a higher growth environment temperature, but there have been no studies reporting whether UVR8 is also involved in high temperature regulation. Therefore, it is of great significance to gain a deeper understanding of the role of light receptors in responding to temperature regulation. Summary of the invention

[0006] The purpose of the present invention is to clone genes with high temperature stress resistance from rice, so as to provide a theoretical basis and candidate gene resources for the creation of new germplasm of high temperature resistant crops.

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

[0008] The present invention uses sequence comparison analysis in the NCBI genome database to search for homologous genes of the Arabidopsis thaliana UVR8 gene sequence in rice, and finds two UVR8 genes in rice, named OsUVR8a and OsUVR8b, which have 74% and 75% similarity to AtUVR8, respectively. Through sequence comparison, it was found that OsUVR8b has natural variation among rice varieties. OsUVR8b is composed of 453 amino acids. In the early-maturing japonica rice variety kitaake and the high-temperature sensitive variety IR64, the 177th position of OsUVR8b was found to be serine (S), while in the high-temperature resistant rice variety N22, the 177th position of OsUVR8b was alanine (A). Then, the transgenic overexpression technology was used to regulate the expression of rice OsUVR8b. S177 and OsUVR8b S177A Through gene overexpression, the study found that OsUVR8b S177 Overexpressed rice showed poor survival under high temperature conditions, while OsUVR8b S177A Overexpressing rice showed higher resistance to high temperature and the fruit set rate under high temperature also increased, 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 Application of genes in improving crop resistance to high temperature stress, specifically, the OsUVR8b 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 phosphorylation site is at position 177 in the amino acid sequence of OsUVR8b. When the 177th amino acid is serine (S), it can be phosphorylated by OsSnRK1.1, and when this site mutates to alanine (A), the phosphorylation effect is significantly reduced.

[0011] Western blot analysis of OsUVR8b S177 OsUVR8b S177A The protein stability of OsUVR8b was determined under heat stress. S177A The protein stability is stronger than OsUVR8b S177 The results of ROS content determination showed that OsUVR8b S177A The ROS accumulation was less than that of OsUVR8b S177 These results suggest 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: using biological technology to make OsUVR8b S177A Gene expression is upregulated in crop plants to improve the plants' tolerance to high temperature stress.

[0013] The high temperature stress refers to the environmental temperature being higher than the suitable temperature for plant growth and development, and the temperature range of heat stress is 35-45°C. For example, the suitable temperature for rice growth is 20-35°C, and the temperature that causes heat stress is generally above 35°C. If it exceeds 35°C, it will have a negative impact on the growth and yield of rice.

[0014] In the present invention, the biological technology means may include but is not limited to transgenic technology.

[0015] Furthermore, the application includes: S177A The gene fragment is cloned into a plant overexpression vector, and the target fragment is introduced into the recipient plant using Agrobacterium-mediated genetic transformation technology to cultivate high-temperature resistant transgenic plants.

[0016] The present invention can synthesize a gene fragment for encoding a protein having an amino acid sequence such as that shown in SEQ ID NO. 1 according to the codon preference of a specific crop.

[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 crop resistance to high temperature stress indicators include: plant survival rate, pollen fertility at maturity, and fruit setting rate. S177A The survival rate of plants with gene overexpression under high temperature stress conditions at the seedling stage and the fruit setting rate at the mature stage were significantly improved.

[0020] The present invention also provides a breeding method for improving the resistance of rice to high temperature stress, comprising: modifying the nucleotide sequence OsUVR8b shown in SEQ ID NO.2 S177A The gene fragment is cloned into a plant overexpression vector, and the target fragment is introduced into the recipient rice using Agrobacterium-mediated genetic transformation technology to cultivate transgenic rice plants that are resistant to high temperature stress.

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

[0022] The plant overexpression vector in the present invention can be a vector known in the art and suitable for rice for Agrobacterium-mediated genetic transformation technology, and can be but not limited to pCAMBIA1300 vector.

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

[0024] The present invention has the following beneficial effects:

[0025] The present invention provides a gene OsUVR8b capable of regulating crop tolerance to high temperature stress S177A OsUVR8b has natural variation among rice varieties. The 177th position of OsUVR8b in heat-sensitive varieties is serine, while the 177th position of OsUVR8b in heat-resistant varieties is alanine. S177A Gene overexpression can significantly improve the survival rate and fruiting rate of plants in high temperature environments. The present invention provides a molecular basis for breeding and screening high temperature resistant varieties. Since this type of mutation site exists in nature, it has important practical application value in breeding high temperature resistant rice varieties in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the sequence alignment of OsUVR8b gene in kitaake, N22, IR64 and Arabidopsis.

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

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

[0029] Figure 4 OsUVR8b S177 The results of transgenic rice construction, where A is the vector schematic diagram and B is the expression level detection result.

[0030] Figure 5 OsUVR8b S177A The results of transgenic rice construction, where A is the vector schematic diagram and B is the expression level detection result.

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

[0032] Figure 7 OsUVR8b S177 and OsUVR8b S177A Fertility analysis results, where A is the pollen iodine staining result and B is the fruit set rate result.

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

[0034] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

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

[0037] By using the amino acid sequence corresponding to the Arabidopsis 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, according to sequence similarity analysis, the homology of the UVR8 protein sequence in Arabidopsis to OsUVR8a and OsUVR8b was 74% and 75%, respectively. OsUVR8b in rice had site variation at position 177 in kitaake, N22 and IR64. N22 variety is a heat-resistant rice variety, while IR64 is a heat-sensitive variety.

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

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

[0041] To investigate the function of rice OsUVR8b 177 locus, we constructed S177 and OsUVR8b S177A Overexpression plasmid recombinant vector is used to obtain overexpression plants using Agrobacterium-mediated genetic transformation method.

[0042] 1. Build 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 of the CDS of the gene is 1362bp (Os04t0435700-01), and the molecular weight of its protein is approximately 49.83kDa (Q7XRB6).

[0044] Primers were designed based on the sequence, F1: 5'-ATGGACGCGGTCATGTCCGC-3'; R1: 5'-AACGAATGCACACGCATCCTCT-3', RNA was extracted from rice kitaake and N22 varieties and reverse transcribed into cDNA as templates, and OsUVR8b was amplified using high-fidelity enzymes. S177 and OsUVR8b A177 The fragments were recovered and purified after agarose gel electrophoresis confirmed that the band size was correct.

[0045] 1.2 Primers were designed according to 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 connected to the pCAMBIA1300 overexpression vector through homologous recombination. After transformation and colony PCR analysis, sequencing was verified to be correct. The vector schematic diagram is shown in the figure. Figure 2 and Figure 3 shown.

[0046] Specifically, OsUVR8bS177A The CDS nucleotide 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 nucleotide 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 used Agrobacterium-mediated genetic transformation of rice callus. After obtaining T0 transgenic plants, mRNA was extracted and reverse transcription and qPCR analysis were performed.

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

[0050] The internal reference gene was Expro, and the sequences were F: 5′-ATCTGCACCACCTCTGAGAC-3′, R: 5′-TTCTTGAGGTGAGGGAAGCAC-3′.

[0051] The results are as follows Figure 4 , Figure 5 As shown, OsUVR8b S177 Independently transformed strains were numbered 1, 4, 18, OsUVR8b S177A The independent transformed plants were numbered 8, 14, and 30. The above plants had higher expression levels than the wild type and were used for subsequent phenotypic identification and fruit setting rate detection.

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

[0053] 1. To explore OsUVR8b S177 and OsUVR8b S177A The heat tolerance phenotype of overexpressing rice at the seedling stage, taking OsUVR8b S177 and OsUVR8b S177A Three families of overexpressing rice were subjected to heat tolerance experiments. This paper mainly explored the survival rate of rice under high temperature stress, so the extreme high temperature of 45°C was selected as the heat treatment condition.

[0054] First, soak the above two materials at 37℃, and plant them in black hydroponic boxes after the seeds turn white. After growing for about 3 weeks, select rice seedlings with consistent growth for seedling phenotypic experiments. Place the rice seedlings in a 45℃ light culture box for stress treatment. The growth conditions are 14h of light and 10h of darkness, normal white light conditions, no additional ultraviolet rays, and grow in normal conditions after the leaves curl up, and calculate the survival rate.

[0055] After statistical analysis, it was found that OsUVR8b S177A -OE material has a higher survival rate than wild type ( Figure 6 A), while OsUVR8b S177 -OE survival rate is lower than that of wild type ( Figure 6 B), OsUVR8b under heat stress S177A -OE had a significantly higher survival rate than OsUVR8b S177 -OE.

[0056] 2. In order to explore the fertility of OsUVR8b 177 locus at maturity, the booting material OsUVR8b 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 ear stage were taken under normal conditions and after stress treatment, stained with I2-KI, and the pollen fertility was observed under a stereo microscope, and the fruit setting rate was counted.

[0057] Pollen fertility and fruit setting rate experiments showed that under high temperature, OsUVR8b S177A -OE had higher pollen fertility and fruiting rate than OsUVR8b S177 -OE( Figure 7 A, B), the results show that the mutation of site 177 to A is beneficial to improving the fruit setting rate of rice under high temperature.

[0058] 3. In order to explore how locus 177 affects the high temperature resistance of rice, OsUVR8b S177 and OsUVR8b S177A The protein stability and ROS content were detected.

[0059] First, OsUVR8b was detected by Western blot. S177 -GFP and OsUVR8b S177A -GFP protein was detected. Rice seedlings with a growth period of 3 weeks were placed at 45°C for 0, 12h, 24h and 48h, and OsUVR8b was detected using GFP antibody. S177 and OsUVR8b S177AIn addition, rice seedlings with a growth period of 3 weeks were subjected to heat stress for 3 days for DAB staining and determination of hydrogen peroxide content.

[0060] The results showed that OsUVR8b S177 The stability of proteins decreased under heat stress ( Figure 8 A), while OsUVR8b S177A The protein can always exist relatively stably in rice cells ( Figure 8 B) This situation should be caused by the phosphorylation of OsUVR8b by OsSnRK1.1.

[0061] DAB staining and hydrogen peroxide content assay showed that after 3 days of heat stress, OsUVR8b S177A The amount of ROS accumulation was less than that of OsUVR8b S177 ( Figure 8 C, D), which indicates that the 177 position has a stronger ability to clear ROS content after changing to A, which may be the reason why the 177 position has a higher high temperature tolerance after changing to A.

[0062] The above results indicate that when site 177 in OsUVR8b is A, the survival rate and fruit set rate of rice under high temperature can be significantly improved. The discovery and functional characterization of this gene site will provide molecular and application value for future rice breeding and cultivation of near-isogenic lines of high-temperature-resistant varieties.

Claims

1. OsUVR8b S177A The application of the gene in improving the resistance of crops to high temperature stress is characterized in that: OsUVR8b S177A The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The application includes: using biological technology to make OsUVR8b S177A Gene expression is upregulated in crop plants to improve the plants' tolerance to high temperature stress.

3. The use according to claim 2, characterized in that The application includes: S177A The gene fragment is cloned into a plant overexpression vector, and the target fragment is introduced into the recipient plant using Agrobacterium-mediated genetic transformation technology to cultivate high-temperature resistant transgenic plants.

4. The use according to claim 1, characterized in that The crop is rice.

5. The use according to claim 1 or 4, characterized in that: OsUVR8b S177A The coding sequence of the gene is shown as SEQ ID NO.

2.

6. The use according to claim 1 or 4, characterized in that: The indicators of crop resistance to high temperature stress include: plant survival rate, pollen fertility at maturity, and fruit setting rate.

7. A breeding method for improving rice resistance to high temperature stress, characterized in that: include: The nucleotide sequence of OsUVR8b is shown in SEQ ID NO.2 S177A The gene fragment is cloned into a plant overexpression vector, and the target fragment is introduced into the recipient rice using Agrobacterium-mediated genetic transformation technology to cultivate transgenic rice plants that are resistant to high temperature stress.

8. The breeding method according to claim 7, characterized in that The plant overexpression vector is pCAMIBA1300 vector.

9. The breeding method according to claim 7, characterized in that The recipient rice is the japonica rice variety kitaake.

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