Application of rice OsHT gene in improving drought resistance of rice

Abstract

The invention belongs to the technical field of plant genetic engineering, and discloses application of a rice OsHT gene in improvement of drought resistance of rice. Clustered regularly interspaced short palindromic repeats (CRISPR) mutant phenotype identification is carried out on candidate genes, and the OsHT gene for controlling rice drought response is successfully cloned. The drought stress phenotype identification results in the seedling stage and the adult-plant stage show that when the gene segment is deleted, the drought stress resistance of the rice is obviously enhanced, so that the function of the gene and the potential of the gene in practical application are proved.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of the rice OsHT gene in improving rice drought resistance. This invention employs a candidate gene screening method to clone an OsHT gene that controls rice drought resistance. Biological function verification shows that it can improve rice drought tolerance. Co-segregation detection experiments show that the OsHT mutant is closely associated with the drought-resistant phenotype. This invention confirms the biological function of this gene and its application pathways and methods. Background Technology

[0002] Rice (Oryza sativa L.) is one of the world's major food crops and the staple food for more than half of the global population. Drought stress is one of the environmental factors affecting crop yield, and elucidating its mechanisms has always been a hot topic of research for scientists. Drought at any developmental stage of a plant can have a significant impact on yield. For example, drought during the reproductive growth stage of rice can directly lead to a yield loss of about 50% (Bernardo et al., Molecular Markers and Selection for Complex Traits in Plants: Learning from the Last 20 Years. Crop Science, 2008, 48: 1649-1664). Drought resistance (DR) in rice is a complex quantitative trait, and drought stress is often accompanied by high temperature or other abiotic stresses (Hu and Xiong, Genetic engineering and breeding of drought-resistant crops. Annual review of plant biology, 2014, 65: 715-741). Over a long period of evolution, plants have developed complex response mechanisms to adapt to the changing natural environment. For example, plants can adapt to water-scarce environments by shortening their life cycle through earlier flowering, reducing water loss (stomatal closure), or enhancing water absorption. Additionally, plants can regulate drought resistance by influencing the accumulation of osmotic substances, such as antioxidants and reactive oxygen species (ROS). The plant response to drought generally includes drought stress perception, signal transduction, and corresponding adaptations at the morphological, physiological, and molecular levels. With the rapid development of sequencing technology and genomics, a large number of genes involved in drought stress responses have been discovered. Based on the differences in the functions of drought-related genes, they are mainly divided into three categories: (1) protein kinases, currently rice protein kinases related to drought stress mainly include MAPK, SnRK, and CDPK subfamily members (Hadiarto et al., Progress studies of drought-responsive genes in rice. Plant Cell Reports, 2011.30:297-310); (2) transcription factor genes, such as AP2 / EREBP, bZIP, zinc finger proteins, MYB / MYC and NAC transcription factors (Calkhoven CF, Ab G. Multiple steps in the regulation of transcription-factor level and activity).Biochemical Journal, 1996, 317: 329-342; Shinozaki et al. Gene networks involved in drought stress response and tolerance. Journal of Experimental Botany, 2007, 58: 221-227; Hu et al. Genetic engineering and breeding of drought-resistant crops. Annual Review of Plant Biology, 2014, 65: 715-741); (3) Functional protein-coding genes, such as genes encoding osmotic regulatory substances and membrane transport proteins (You et al. An ornithine δ-aminotransferase gene OsOAT confersdrought and oxidative stress tolerance in rice. Plant Science, 2012, 197: 59-69; Jang et al. Expression of abifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without (Stunting growth. Plant Physiool, 2003, 131:516-524). These genes complete drought adaptive responses through processes such as participation in drought signal response, signal transduction, and gene expression regulation.

[0003] In Arabidopsis, the HT1 (HIGH LEAF TEMPERATURE 1) gene encodes a serine / threonine protein kinase that negatively regulates CO2-induced stomatal closure. HT1 phosphorylates and inhibits the downstream kinase CBC1 / 2 (OPEN STOMATA 1), thereby affecting the activity of the anion channel SLAC1 and ultimately regulating stomatal opening and closing (Takahashi et al. Stomatal CO2 / bicarbonate sensor consists of two interacting protein kinases, Raf-like HT1 and non-kinase-activity requiring MPK12 / MPK4. Sci Adv, 2022, 8: eabq6161). Stomatal closure is a core drought resistance mechanism in plants to reduce water loss. HT1, as an upstream regulator of CO2 signaling pathway and water use efficiency (Xiao et al. A module involving HIGH LEAF TEMPERATURE1 controls instantaneous water use efficiency. Plant Physiol. 2024, 196(2): 1579-1594), directly regulates stomatal movement in drought response.

[0004] Rice is an important food crop and model plant. In today's world, where extreme weather conditions are frequent, breeding rice varieties with enhanced stress resistance is of great significance. Isolating the OsHT gene from rice and identifying its function in improving rice stress resistance will be of great importance for breeding new stress-resistant rice varieties. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the rice OsHT gene in improving the drought resistance of rice, wherein the protein encoded by the OsHT gene is shown in SEQ ID NO.3.

[0006] To achieve the above objectives, the present invention adopts the following technical measures:

[0007] This invention employs a candidate gene screening method to clone a gene OsHT that controls drought resistance in rice. The loss of function of this fragment enhances the drought resistance of rice under drought conditions. The OsHT gene is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2, and the encoded amino acid sequence is shown in SEQ ID NO.3.

[0008] The scope of protection of this invention also includes:

[0009] The application of reducing the expression level of the OsHT gene in improving the drought resistance of rice, wherein the protein encoded by the OsHT gene is shown in SEQ ID NO.3.

[0010] The reduction described above is achieved through suppression or knockout.

[0011] The above-described applications involve introducing substances that reduce or prevent the expression of the OsHT gene into rice.

[0012] In the above-described applications, preferably, the substance is an expression cassette, recombinant vector, or recombinant microorganism that reduces the expression level of the OsHT gene.

[0013] The above-described applications involve knockout using homologous recombination or CRISPR gene editing methods. The knockout gene translates into a protein that has no original function or cannot be translated into a protein.

[0014] In the above-described applications, preferably, the CRISPR gene editing method uses the CRISPR / Cas9 method, with the target site being: GCAACAAGGGCACCTACCGATGG.

[0015] The above-described applications involve inhibition using antisense RNA technology or interfering RNA technology.

[0016] In the applications described above, the OsHT gene is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0017] Application of reagents for detecting the gene encoding the protein shown in SEQ ID NO.3 in the screening or breeding of drought resistance in rice.

[0018] The method for determining the application of the above-mentioned technology is as follows: rice that does not detect the gene or whose expression level is significantly reduced compared to normal is drought-resistant rice.

[0019] The expression cassette that reduces the expression level of the OsHT gene, the application of recombinant vectors or recombinant microorganisms in the creation of drought-resistant rice, wherein the protein encoded by the OsHT gene is shown in SEQ ID NO.3.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] This invention employs a candidate gene screening method to clone a gene, OsHT, that controls drought resistance in rice. Biological function verification shows that it can improve the drought tolerance of rice. Co-segregation detection experiments show that the Osht mutant is closely related to the drought-resistant phenotype, providing a theoretical basis for the creation of drought-resistant rice. Attached Figure Description

[0022] Figure 1 This document describes the gene editing process of the Osht CRISPR mutant in rice.

[0023] Figure 2 This is a schematic diagram of leaf temperature measured by an infrared thermometer during the seedling stage of rice Osht CRISPR mutant.

[0024] Figure 3 This is a schematic diagram of the drought stress phenotype of rice Osht CRISPR mutant seedlings. Detailed Implementation

[0025] The following embodiments define the present invention and describe the method of constructing Osht CRISPR mutants and verifying OsHT gene function. Based on the following description and these embodiments, those skilled in the art can determine the essential features of the present invention, and various changes and modifications can be made to the present invention to suit different uses and conditions without departing from the spirit and scope thereof.

[0026] Example 1:

[0027] Constructing Osht CRISPR mutants

[0028] This invention employs a candidate gene screening method to clone a gene OsHT that controls drought resistance in rice. The loss of function of this fragment enhances the drought resistance of rice under drought conditions. The OsHT gene is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2, and the encoded amino acid sequence is shown in SEQ ID NO.3.

[0029] The OsHT gene sequence was obtained from the Rice Data (http: / / www.ricedata.cn / gene / ) rice gene database. A target site was selected using CRISPR-P v2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ). Vector construction for CRISPR mutant lines can be referred to relevant literature (Xie Kabin et al. Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA-processing system. PNAS. 2015, 112:3570-3575.), and will not be described in detail here due to space limitations. The target site selected from the CRISPR-Pv2.0 website is as follows:

[0030] Target site: GCAACAAGGGCACCTACCGATGG.

[0031] The constructed CRISPR vector OsHT-CRISPR was transferred into the rice variety "Zhonghua 11" (a publicly available rice variety provided by the China National Rice Research Institute) using the Agrobacterium-mediated rice genetic transformation method (the specific steps of which are described below). After pre-culture, infection, co-culture, screening for hygromycin-resistant callus, differentiation, rooting, hardening-off, and transplanting, transgenic plants were obtained.

[0032] Based on the aforementioned gene editing target sites, primers were designed to detect the editing status of the OsHT gene in the mutant. The detection primers were: HT-F: TGGTGAGCAATGCGCG; HT-R: gGATCGGAGCAAATCTCATTTCTC. Specific PCR amplification of the OsHT gene was performed, and the amplified PCR products were sequenced, simultaneously detecting the presence of Cas9. Sequencing results showed that in the Osht CRISPR homozygous Cas9-free mutant family, the OsHT gene had a one-base insertion at target site 1. Figure 1 The OsHT gene in the mutant family is mutated, and the inserted Osht undergoes a frameshift mutation, resulting in loss of protein function. The obtained homozygous mutant Osht contains the polynucleotide shown in SEQ ID NO.4 and is used for the following drought resistance detection.

[0033] Example 2:

[0034] Identifying the temperature phenotype of Osht CRISPR mutant seedlings

[0035] The homozygous mutant (Osht) and Zhonghua 11 (ZH11), whose genotypes had been identified in Example 1, were directly sown into small cylindrical containers after germination. The soil used in the experiment was a mixture of paddy soil from southern China and coarse sand at a volume ratio of 2:3. An equal volume of water was added to each container with an equal amount of uniformly mixed sand, allowing the water to seep out naturally to ensure consistent soil compaction. Open-field photographs were taken of the rice at the 4-leaf stage, and leaf temperature was measured using an infrared thermometer.

[0036] The results showed that, compared with the ZH11 control, the ht mutant plants exhibited higher leaf temperatures. Figure 2 This indicates that its water loss has decreased.

[0037] Example 3:

[0038] Identification of drought stress phenotypes in Osht CRISPR mutant seedlings

[0039] The homozygous mutant (Osht) and Zhonghua 11 (ZH11), whose genotypes had been identified in Example 1, were directly sown into small cylindrical containers after germination. The soil used in the experiment was a mixture of paddy soil from southern China and coarse sand at a volume ratio of 2:3. An equal volume of water was added to each container with an equal amount of uniformly mixed sand, and the water was allowed to seep out to ensure consistent soil compaction. The experiment was repeated in three replicates.

[0040] Healthy plants at the 4-leaf stage were subjected to drought stress for 6-10 days (depending on the planting conditions), followed by 7 days of rehydration. Photos were taken and the survival rate of the plants was assessed. Compared to the ZH11 control, the CRISPR homozygous mutant plants exhibited a drought-resistant phenotype, with a survival rate approximately 30% higher after drought and rehydration.

Claims

1. Reduce OsHT The application of gene expression levels in improving drought resistance in rice, as described above. OsHT The protein encoded by the gene is shown in SEQ ID NO.

3.

2. In the application according to claim 1, the reduction is achieved by suppression or knockout.

3. The application according to claim 1, wherein the process of the application is to reduce OsHT The expression level of genes or substances that prevent gene expression are introduced into rice.

4. The application according to claim 3, wherein the substance is for reducing... OsHT Gene expression cassettes, recombinant vectors, or recombinant microorganisms.

5. In the application according to claim 2, the knockout is performed using homologous recombination or CRISPR gene editing methods, and the protein translated from the knocked-out gene has no original function or cannot be translated into a protein.

6. In the application according to claim 5, the CRISPR gene editing method uses the CRISPR / Cas9 method, and the target site is: GCAACAAGGGCACCTACCGATGG.

7. The application according to claim 2, wherein the inhibition is performed using antisense RNA technology or interfering RNA technology.

8. Application of reagents for detecting the gene encoding the protein shown in SEQ ID NO.3 in the screening or breeding of drought resistance in rice.

9. The application according to claim 8, wherein the determination method in the application is: drought-resistant rice is rice in which the gene is not detected or the expression level of the gene is significantly reduced compared with normal.

10. Reduce OsHT The application of gene expression cassettes, recombinant vectors, or recombinant microorganisms in the creation of drought-resistant rice, as described above. OsHT The protein encoded by the gene is shown in SEQ ID NO.3.

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