Use of osspt38 and / or osspt38d protein or encoding gene thereof in stress resistance and yield increase of oryza sativa l

Abstract

Provided is the use of an OsSPT38D protein and an encoding gene OsSPT38D thereof in improving the stress resistance and / or yield of dicotyledonous or monocotyledonous plants.

Description

Application of OsSPT38 and / or OsSPT38D proteins or their encoding genes in rice stress resistance and yield increase Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of OsSPT38 and / or OsSPT38D proteins or their encoding genes in improving the drought and salt resistance and / or yield of plants such as rice. Background Art

[0002] Rice (Oryza sativa L.) is an important food crop, and about 60% of the population in my country relies on rice as their staple food. Rice is also a high-water-consuming crop, and its water consumption accounts for more than 65% of my country's total agricultural water consumption. It is known as the largest consumer of agricultural water. With the change of global climate, the increase in the frequency and intensity of extreme climate events such as droughts poses a serious threat to the growth, development and yield of rice. Developing water-saving and drought-resistant rice and enhancing the drought resistance of rice are effective ways to alleviate the shortage of water resources in my country and promote the sustainable development of my country's agriculture. In addition, the secondary salinization of soil caused by unreasonable irrigation has greatly restricted the production of rice, and there are 2.34 million hm2 of land in my country. 2 Coastal mudflats and 100 million hm 2 Inland saline-alkali land represents a vast potential resource for utilization. Rice, a moderately salt-sensitive crop, is considered the preferred food crop for developing coastal tidal flats and saline-alkali land. Therefore, identifying drought- and salt-tolerant genes and improving rice's resistance to drought and salt stress are crucial for promoting sustainable agricultural development and safeguarding national food security.

[0003] Ferredoxins (Fd) are a class of small proteins containing iron-sulfur clusters. They are ubiquitous in a variety of organisms, including bacteria, algae, plants and animals. They function as electron carriers, participating in electron transport in a variety of metabolic processes. In plants, photosynthetic Fd, located in chloroplasts, receive photosynthetic electrons from the PSI and transfer them to downstream Fd-dependent metabolic processes, such as carbon, nitrogen, and sulfur assimilation, chlorophyll synthesis and degradation, and fatty acid synthesis. They play a vital role in plant photosynthesis and the regulation of plant growth and development (Hanke, GUY, and Mulo, P. (2013). Plant type ferredoxins and ferredoxin-dependent metabolism. Plant Cell Environ. 36, 1071-1084). The rice genome contains seven ferredoxin genes, among which OsFd1 has the highest expression level and its function is also the most important. The loss-of-function mutant of OsFd1, fd1, rapidly dechlorosis after the two-leaf stage, with a significant decrease in photosynthetic capacity, significantly reduced chlorophyll a, b and carotenoid contents, and dies at the three-leaf stage (He, L., Li, M., Qiu, Z., Chen, D., Zhang, G., Wang, X., Chen, G., Hu, J., Gao, Z., Dong, G., Ren, D., Shen, L., Zhang, Q., Guo, L., Qian, Q., Zeng, D., and Zhu, L. (2020). Primary leaf-type ferredoxin 1 participates in photosynthetic electron transport and carbon assimilation in rice. Plant J 104, 44-58).

[0004] In addition to participating in photosynthesis and regulating plant growth and development, Fd also plays an important role in resisting adverse stress. For example, overexpression of the sweet pepper Fd1 (Plant Ferredoxin Like Protein, PFLP) gene in tobacco, rice and orchids improves the plants' resistance to carrot Erwinia subsp. Carotovora, Pseudomonas syringae pv. tabaci, Xanthomonas oryzae pv. oryzae and bacterial blight (Tang K, Sun X, Hu Q, Wu A, Lin C, Lin H, Twyman RM, Christou P, Feng T. (2001). Transgenic rice plants expressing the ferredoxin-like protein (AP1) from sweet pepper show enhanced resistance to Xanthomonas oryzae pv. oryzae. Plant Sci. 160, 1035-1042), indicating that Fd is involved in plant resistance to disease stress. In addition, Fd is also involved in the removal of excessive reactive oxygen species (ROS) in plants. The water-water cycle in which Fd participates is a pathway for removing chloroplast ROS. Specifically, Fd participates in the water-water cycle through the regeneration of ascorbic acid (AsA) to protect the photosynthetic system from photooxidative damage (Liu Cong, Dong Lamie, Lin Jianzhong, Liu Xuanming, 2019. Research progress on reactive oxygen metabolism and regulatory mechanisms in plants under adverse stress. Life Science Research, 23: 253-258). T-DNA insertion mutants of Arabidopsis thaliana AtFd2 accumulate ROS in their chloroplasts, and transient expression of OsFd4 alleviates hydrogen peroxide (H2O2)-induced rice cell death, indicating that OsFd4 is involved in regulating rice tolerance to exogenous oxidative stress (Lu, M., Chen, J., Meng, H., Mo, G., Liu, Y., Chen, F., Wang, Z., and Wang, M. (2023). Rice ferredoxin OsFd4 contributes to oxidative stress tolerance but compromises defense against blight bacteria. Crop J. 11, 1937-1942). Both drought and salt stress can dramatically increase ROS levels in plant cells, causing oxidative stress.Fd is involved in the removal of excess ROS, suggesting that Fd may be involved in regulating plant drought resistance and salt tolerance. For example, transgenic rice overexpressing the sweet pepper Fd1 (Plant Ferredoxin Like Protein, PFLP) gene in rice showed higher antioxidant enzyme activity, more ABA accumulation, and significantly upregulated expression of stress-related genes under salt stress treatment, thereby improving the salt tolerance of transgenic rice (Huang, HE, Ho, MH, Chang, H., Chao, HY, and Ger, MJ (2020). Overexpression of plant ferredoxin-like protein promotes salinity tolerance in rice (Oryza sativa). Plant Physiol Biochem. 155, 136-146).

[0005] SUMOylation, a post-translational modification of proteins, plays a crucial role in regulating protein stability, protein-protein interactions, and subcellular localization. SUMO E3 ligases, which recognize and select substrates, directly promote the binding of SUMO proteins to target proteins and have been shown to have both positive and negative regulatory functions in plant responses to abiotic stresses.

[0006] Both drought and high salt concentrations can cause osmotic and oxidative stress in plants. Furthermore, high salt concentrations can also lead to secondary stresses such as ion toxicity. Plants perceive drought and salt stress signals and initiate responses, including but not limited to increasing root water uptake, closing stomata to reduce water loss, regulating tissue osmotic potential, promoting the production of stress-protective metabolites such as proline and trehalose, and triggering antioxidant systems to maintain redox homeostasis. Furthermore, plant responses to salt stress include regulating ion balance. Both drought resistance and salt tolerance in rice are quantitative traits controlled by multiple genes and have a complex genetic basis. Hundreds of QTLs (Quantitative Trait Loci) or genes associated with drought resistance have been cloned in rice, but the genetic resources available for drought and salt tolerance in rice breeding remain limited. Therefore, the discovery and identification of new, high-quality drought and salt tolerance genes or genetic loci provides an important theoretical foundation and genetic resources for breeding new rice varieties with superior stress tolerance traits or improving the traits of major cultivated varieties in different regions, and has significant research and application value.

[0007] Summary of the Invention

[0008] One objective of the present invention is to obtain a rice drought-resistant and salt-tolerant mutant spt38D, as well as the gene OsSPT38 involved in rice drought and salt tolerance identified from the mutant, its gain-of-function mutant gene OsSPT38D, and its encoded protein, as well as its role in improving the resistance of plants such as rice to drought and salt stress.

[0009] In this field, "salt tolerance" generally refers to the ability of a plant to tolerate salt damage during growth; "drought resistance" generally refers to the ability of a plant to adapt to and resist water loss caused by drought during growth.

[0010] The present invention is applied to the genetic improvement of drought and salt tolerance traits in crops such as rice, which is of great significance to my country's food security. The 212D mutation involved in the present invention is extremely rare in natural variations in rice varieties, and its application potential is great and the prospects are broad.

[0011] Specifically, the present invention provides the following technical solutions.

[0012] In one aspect, the present invention provides a gain-of-function mutant spt38D that improves drought resistance and salt tolerance in rice, as well as a drought-resistance / salt-tolerance gene OsSPT38 cloned from the mutant, a gain-of-function mutant gene OsSPT38D thereof, and its encoded protein OsSPT38D, for use in cultivating or breeding plants with enhanced drought resistance / salt tolerance compared to wild-type plants. The amino acid sequence of the OsSPT38 protein encoded by the OsSPT38 gene is shown in SEQ ID NO:2, and the amino acid sequence of the OsSPT38D protein encoded by the OsSPT38D gene is shown in SEQ ID NO:4. Compared with the amino acid sequence of the OsSPT38 protein shown in SEQ ID NO:2, the amino acid sequence of the OsSPT38D protein encoded by the OsSPT38D gene is shown in SEQ ID NO:4. ...4, the amino acid sequence of the OsSPT38 protein encoded by the OsSPT38D gene is shown in SEQ ID NO:4. Compared with the amino acid sequence of the OsSPT38 protein shown in SEQ ID NO:4, the amino acid sequence of the OsSPT38 protein encoded by the OsSPT38D gene is shown in SEQ ID NO:4.

[0013] In some embodiments, the rice drought and salt tolerance gain-of-function mutant spt38D is caused by a gain-of-function mutation in OsSPT38, specifically a mutation of amino acid 212 in the amino acid sequence of OsSPT38 to aspartic acid (212D). Furthermore, the gain-of-function mutant gene of gene OsSPT38 is OsSPT38D.

[0014] In some embodiments, the gain-of-function mutation site in the rice drought and salt tolerance gain-of-function mutant spt38D is a rare natural variation, found in only 7 of 4,774 rice germplasms examined, all of which belong to the temperate japonica rice group.

[0015] In some embodiments, OsSPT38 encodes a plant SUMO E3 ligase that interacts with OsSCE3 (a SUMO E2 conjugating enzyme) and various stress-related substrate proteins, promoting SUMOylation of these substrate proteins and enhancing their stability. Compared to OsSPT38, OsSPT38D exhibits increased SUMOylation activity.

[0016] In some embodiments, the present invention relates to an amino acid sequence of a derivative protein that has undergone substitution, deletion and / or addition of one or more amino acid residues compared to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and has the same activity as the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: 4.

[0017] In some embodiments, the amino acid sequence of the derived protein has at least 50%, such as 52%-91%, homology with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Preferably, the amino acid sequence of the derived protein is an amino acid sequence as shown in any one of SEQ ID NOs: 5 to 27.

[0018] In some embodiments, the nucleotide sequence of the gene OsSPT38D encoding the protein OsSPT38D is selected from any one of the following:

[0019] 1) the sequence shown in SEQ ID NO: 3;

[0020] 2) sequences that have conservative substitutions, deletions, or additions of one or more bases compared to the sequence shown in SEQ ID NO: 3 and have the same function as the sequence shown in SEQ ID NO: 3;

[0021] 3) A sequence that can hybridize to the sequence shown in SEQ ID NO: 3 under highly stringent conditions.

[0022] In some embodiments, the sequence that has the same function as the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 has at least 50% identity with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 52%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0023] On the other hand, the present invention provides the use of OsSPT38 and / or OsSPT38D proteins or their encoding genes in improving the stress resistance and / or yield of plants, preferably the stress resistance is salt tolerance and / or drought resistance, wherein the amino acid sequence of the OsSPT38 protein is shown in SEQ ID NO:2, and the amino acid sequence of the OsSPT38D protein is shown in SEQ ID NO:4, which has an amino acid mutation 212D at position 212 compared with the amino acid sequence of the OsSPT38 protein shown in SEQ ID NO:2.

[0024] The present invention also provides a method for improving plant drought resistance and / or salt tolerance and / or yield, the method comprising: mutating the amino acid at position 212 of the amino acid sequence of the OsSPT38 protein to Asp (OsSPT38D, 212D) in a wild-type plant or plant cell to enhance the function of OsSPT38; or increasing the protein content of OsSPT38 and its gain-of-function mutant OsSPT38D, thereby enhancing the plant's drought resistance and / or salt tolerance, wherein the amino acid sequence of the protein encoded by the OsSPT38D gene is as follows:

[0025] 1) the amino acid sequence shown in SEQ ID NO: 4; or

[0026] 2) An amino acid sequence of a derivative protein that has undergone substitution, deletion and / or addition of one or more amino acid residues compared to the amino acid sequence shown in SEQ ID NO: 4 and has the same activity as the amino acid sequence shown in SEQ ID NO: 4.

[0027] In some embodiments, the amino acid sequence of the derived protein has at least 50%, such as 52%-91%, homology with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Preferably, the amino acid sequence of the derived protein is an amino acid sequence as shown in any one of SEQ ID NOs: 5 to 27.

[0028] The present invention also provides a recombinant expression vector or host cell comprising the OsSPT38D gene, preferably the host cell is an Escherichia coli cell or an Agrobacterium cell.

[0029] The present invention also provides use of a recombinant expression vector or host cell comprising the OsSPT38D gene in improving stress resistance and / or yield of a plant, preferably the stress resistance is salt tolerance and / or drought resistance.

[0030] In some embodiments, mutating amino acid 212 of the amino acid sequence of OsSPT38 to Asp (OsSPT38D, 212D); or increasing the protein content of OsSPT38 and its gain-of-function mutant OsSPT38D is achieved by:

[0031] 1) transferring the OsSPT38D gene carrying the point mutation (212Asp) into other rice varieties or plants by breeding (e.g., hybridization with the spt38D mutant); or

[0032] 2) mutating the amino acid at position 212 of the OsSPT38 amino acid sequence and its homologous sequence to Asp by gene editing (e.g., site-directed gene editing, promoter editing); or editing the promoter region of the OsSPT38 or OsSPT38D gene; or knocking in relevant elements (e.g., enhancers) in the promoter region of the OsSPT38 or OsSPT38D gene, to increase the content and / or activity of the OsSPT38 or OsSPT38D protein; or

[0033] 3) Enhancing the content and / or activity of the protein encoded by the gene and its homologous gene, or its active fragment in a transgenic plant or hybrid offspring plant by transgenic means (e.g., genetic transformation of a recombinant vector), wherein the enhancement of the protein content encoded by the gene preferably involves overexpression of the gene.

[0034] In another aspect, the present invention provides a method for improving plant stress resistance and / or yield, preferably the stress resistance is salt tolerance and / or drought resistance, the method comprising any one of the following or a combination thereof:

[0035] 1) Using gene editing methods, the amino acid 212 of the OsSPT38 protein in plants was mutated to Asp (212D), and plant mutants carrying the 212D mutation were obtained.

[0036] 2) introducing the 212D mutation into the recipient plant by crossing a plant carrying the OsSPT38D protein with a recipient plant,

[0037] 3) overexpressing the gene encoding the OsSPT38 protein or the OsSPT8D protein in plants, and

[0038] 4) Transfecting the gene encoding OsSPT38 protein or OsSPT38D protein into recipient plants.

[0039] In some embodiments, the plant or its progeny obtained by the method has a growth advantage under drought or high-salt growth conditions in the field, and / or the plant or its progeny has increased yield.

[0040] Furthermore, in some embodiments of the present invention, an OsSPT38 nucleotide sequence or an active fragment thereof is introduced into the model plant Arabidopsis thaliana cells to generate transgenic Arabidopsis thaliana cells. Transgenic Arabidopsis thaliana plants expressing the isolated nucleotide sequence or active fragment thereof are generated from the transgenic Arabidopsis thaliana cells. Compared to control Arabidopsis thaliana plants not introduced with the isolated nucleotide sequence or active fragment thereof, the transgenic Arabidopsis thaliana plants exhibit improved resistance to osmotic stress and salt tolerance. This result demonstrates that the OsSPT38 gene is functionally conserved in model plants, providing strong evidence for its application in the breeding of various crops.

[0041] In some embodiments, the plant is a dicotyledonous or monocotyledonous plant, preferably rice, corn, wheat, soybean, sorghum, millet, rye, barley, oat, rapeseed, cotton, potato, grape, pepper, tobacco, alfalfa and Arabidopsis, most preferably rice and Arabidopsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1. Identification and mapping of the associated genes for the drought- and salt-tolerant rice mutant spt38D. Figures 1A-B show the phenotypes of the wild-type (LG31) and the M3 mutant (spt38D) after treatment with 25% PEG4000 (Figure 1A) or 150 mM NaCl (Figure 1B). Figure 1C shows the schematic structure of the OsSPT38 gene and the sequencing results of the mutation site in the spt38D mutant.

[0043] Figure 2. Drought and salt tolerance phenotypes of the spt38D allelic mutant and natural variation at the mutation site. Figure 2A shows the phenotypes of 18 spt38D allelic mutants before and after treatment with 25% PEG4000 or 150 mM NaCl. Figure 2B shows the schematic structure of the OsSPT38 gene and the sequencing results of the mutation sites of 18 spt38D allelic mutants. Figure 2C shows the variation of nucleotide position 635 of the OsSPT38 gene in natural rice populations.

[0044] Figure 3. Drought and salt tolerance phenotypes of the spt38D mutant. Figures 3A-B show the phenotypes of LG31 and spt38D before and after treatment with 25% PEG4000 (Figure 3A) and survival statistics (Figure 3B). Figures 3C-D show the phenotypes of LG31 and spt38D before and after treatment with 150 mM NaCl (Figure 3C) and survival statistics (Figure 3D). Figures 3E-F show the phenotypes of LG31 and spt38D before and after soil drought treatment at the seedling stage (Figure 3E) and survival statistics (Figure 3F). Figures 3G-J show the phenotypes of LG31 and spt38D during field drought treatment (Figure 3G) and yield (Figure 3H), effective tiller number (Figure 3I), and seed set rate (Figure 3J).

[0045] Figure 4. Screening and validation of the interaction between OsSPT38 and OsSPT38D and the E2 conjugating enzyme OsSCE3. Figures 4A-B show yeast two-hybrid screening for interactions with OsSPT38 (Figure 4A) and OsSPT38D (Figure 4B). Figures 4C-E further validate the interaction between OsSPT38 and OsSPT38D and the E2 conjugating enzyme OsSCE3 using yeast two-hybrid (Figure 4C), LCI (Figure 4D), and BiFC (Figure 4E).

[0046] Figure 5. Domain structure comparison of OsSPT38 with known SUMO ligases from Arabidopsis and rice.

[0047] Figure 6. Interaction between OsSPT38 and OsFd1. Figures 6A-D show the interaction between OsSPT38 and OsFd1 verified by yeast two-hybrid (Figure 6A), pull-down (Figure 6B), BiFC (Figure 6C), and LCI (Figure 6D).

[0048] Figure 7. OsSPT38 promotes SUMOylation of the substrate protein OsFd1, while OsSPT38D further enhances SUMOylation. Figure 7A shows a schematic diagram of the two SUMO sites and their mutations in OsFd1. Figure 7B shows an in vitro SUMOylation experiment of OsFd1 in recombinant Escherichia coli. Figure 7C shows an in vitro SUMOylation experiment of OsFd1 in recombinant Escherichia coli using OsSPT38 and OsSPT38D. Figure 7D shows an in vitro SUMOylation experiment of OsFd1 in tobacco leaves.

[0049] Figure 8. Effect of OsSPT38 on OsFd1 protein stability. Figures 8A-B show cell-free protein degradation assays for OsFd1. Figure 8A shows a Western blotting image of GST-OsFd1, and Figure 8B shows changes in relative protein abundance of GST-OsFd1. Figure 8C shows changes in OsFd1 protein abundance in LG31 and spt38D cells following PEG4000 or salt treatment, as assessed by Western blotting.

[0050] Figure 9. Drought and salt tolerance phenotypes of OsSPT38 knockout mutants and overexpression materials. Figure 9A-B shows the drought and salt tolerance phenotypes of wild type (LG31), OsSPT38 knockout mutants (ko-10, ko-12), OsSPT38 overexpression (OE W -2, OE W -28) and OsSPT38D overexpression (OE M -5, OE M-27) rice lines before and after treatment with 25% PEG4000 (Figure 9A) and before and after treatment with 150mM NaCl (Figure 9B). Figures 9C-D are the survival rate statistics after treatment with 25% PEG4000 (Figure 9C) and 150mM NaCl (Figure 9D). Figure 9E shows two homozygous knockout mutants (ko-10, ko-12) of OsSPT38 obtained by CRISPR technology in the LG31 background and their editing types. Figure 9F shows qRT-PCR detection of OsSPT38 overexpression (OE W -2, OE W -28) and OsSPT38D(OE M -5, OE M -27) relative gene expression levels of rice lines.

[0051] Figure 10. Drought and salt tolerance phenotypes of Arabidopsis overexpressing the OsSPT38 gene. Figure 10A shows the growth of wild-type (LG31) and OsSPT38-overexpressing (OE-1, OE-2) strains grown on MS, MS medium containing 200 mM mannitol, or MS medium containing 150 mM NaCl. Figure 10B shows the biomass statistics of Arabidopsis grown on MS and MS medium containing 200 mM mannitol. Figure 10C shows the survival statistics of Arabidopsis grown on MS and MS medium containing 150 mM NaCl.

[0052] Figure 11. A diagram showing the similarity and evolutionary relationship between the amino acid sequences of OsSPT38 from different species and rice. Figure 11A shows an alignment of the amino acid sequences of OsSPT38 proteins from different species, highlighting the site of the OsSPT38 mutation in the spt38D mutant. Figure 11B shows the evolutionary relationship.

[0053] Figure 12. OsSPT38 promotes SUMOylation modification of substrate proteins in the maize homologous protein Zm00001eb032750 in an in vitro recombinant Escherichia coli system.

[0054] Figure 13. OsSPT38 positively regulates drought resistance and yield traits in rice. Figures 13A-F show the relationship between LG31, knockout mutant (ko-12) and overexpression material (OE W -2, OE W Figure 13A shows the yield per plant, Figure 13B shows the yield per unit area, Figure 13C shows the number of effective tillers, Figure 13D shows the seed setting rate, Figure 13E shows the number of grains per ear, and Figure 13F shows the plant height.

[0055] Preservation Instructions

[0056] Taxonomy: Rice (Japonica)

[0057] Name: spt38D

[0058] Latin name: Oryza sativa L.ssp.japonica

[0059] Depository: General Microbiology Center of China Culture Collection Administration

[0060] Abbreviation of depository institution: CGMCC

[0061] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0062] Deposit date: March 15, 2024

[0063] CGMCC registration number: CGMCC NO.45892 DETAILED DESCRIPTION

[0064] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific examples and with reference to the accompanying drawings. However, it should be understood that the embodiments are for illustrative purposes only and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. The methods used in the following examples are all conventional methods unless otherwise specified, and the reagents used are all commercially available reagents unless otherwise specified.

[0065] Example 1 Obtaining the Drought-Resistant and Salt-Tolerant Rice Mutant spt38D and Mapping the Related Genes

[0066] Wild-type seeds of the rice variety Longjing 31 (LG31, the leading cultivar in Northeast China) (Rice Research Institute, Heilongjiang Academy of Agricultural Sciences) were mutagenized with ethyl methyl sulfonate (EMS) (CAS: 62-50-0). The mutagenesis procedure involved soaking Longjing 31 seeds in a 0.5% EMS solution for 16 hours, rinsing repeatedly, and sowing them in experimental fields (M1 generation). The M1 generation plants were self-pollinated and harvested to create a pool of M2 generation rice mutants (approximately 320,000 M1 seeds, with seeds from 1,000-1,200 M1 plants pooled together, for a total of 103 pools). The mutant pool was screened using a 25% PEG4000 (Lotte, Korea) solution as the screening condition. The specific operation is as follows: about 10,000 seeds (M2 generation) are selected from each pool, soaked and germinated for 2-3 days, and then evenly spread in seedling pots. After hydroponics for 12 days using Yoshida nutrient solution (nutrient solution formula see below), they are treated with 25% PEG4000 or 150mM NaCl (prepared with Yoshida nutrient solution), and the surviving seedlings are selected as candidate mutants for subsequent research. Among them, a mutant with an excellent PEG-resistant phenotype was screened from pool numbered 38 and transplanted to the field for normal growth. The harvested M3 generation mutant seeds were germinated and transferred to a 96-well PCR plate with a removed bottom. After 12 days of hydroponic growth in Yoshida nutrient solution, they were treated with 25% PEG4000 and 150mM NaCl. The results showed that compared with the wild-type LG31, the mutant exhibited excellent PEG-resistance and salt-tolerance phenotypes (Figure 1A-B). This mutant was named spt38D (salt- and PEG4000-tolerant 38D) (Deposit Number CGMCC NO. 45892).

[0067] The BC1F1 progeny were obtained by hybridizing spt38D with the wild type Longjing 31, and the BC1F1 progeny were further self-pollinated to construct the BC1F2 population. The BC1F2 generation was also treated with 150mM NaCl. The results showed that the ratio of surviving seedlings (tolerant) to dead seedlings (sensitive) was 413:160 [χ 2 =2.1421<χ 2(P < 0.05) = 3.84], a segregation ratio close to 3:1, indicating that the gene associated with drought and salt tolerance in spt38D is a single dominant gene. Using the MutMap method combined with whole-genome resequencing (methods described below), we located the locus associated with drought and salt tolerance in spt38D at Chr12:27027394 on chromosome 12, where a single base mutation from G to A occurs. The gene containing this mutation is LOC_Os12g43560, which we named OsSPT38. The nucleotide sequence of the OsSPT38 gene coding region (LOC_Os12g43560 / OsSPT38 gene coding region is shown in SEQ ID NO:1, and the encoded amino acid sequence is shown in SEQ ID NO:2) contains a guanine to adenine mutation (G635A) at position 635, resulting in a glycine to aspartic acid mutation (Gly212Asp) at position 212 of the gene-encoded protein (Figure 1C). The gene with this base mutation was named OsSPT38D. Furthermore, first-generation sequencing of the OsSPT38 genomic sequences of other drought- and salt-tolerant mutants revealed that 18 mutants with excellent drought and salt tolerance phenotypes also had a guanine to adenine mutation (G635A) at position 635 in the OsSPT38 gene (Figures 2A-B). These 18 additional independent allelic mutations further confirmed that OsSPT38 is an associated gene for drought and salt tolerance in spt38D.

[0068] To investigate the variation in nucleotide position 635 of the OsSPT38 gene across natural rice populations, we analyzed the polymorphism of nucleotide position 635 in 4726 Asian cultivated rice accessions using RiceVarMap v2.0 (https: / / ricevarmap.ncpgr.cn / ). The results showed that 4719 of the 4726 Asian cultivated rice accessions carried a G / C base at position 635 of the OsSPT38 gene, representing 99.85%. However, only 7 accessions, all belonging to the temperate japonica group, carried an A / T base at position 635 of the OsSPT38 gene, representing only 0.15% (Figure 2C). Furthermore, analysis of 27 common wild rice (Oryza rufipogon), 11 African cultivated rice (Oryza glaberrima), and 10 wild rice (Oryza barthii) germplasm accessions using RiceSuperPIRdb (http: / / www.ricesuperpir.com / ) revealed no A / T mutation at position 635 of the OsSPT38 gene. These results suggest that the A / T mutation at position 635 of the OsSPT38 gene is extremely rare, possessing significant domestication potential and promising applications for the utilization of this rare variant in improving cultivated varieties.

[0069] The MutMap method combined with whole genome resequencing technology to quickly locate mutation sites is as follows: the control LG31 and BC1F2 populations (constructed by LG31 and OsSPT38D) were cultured in Yoshida nutrient solution for 12 days and then treated with 150mM NaCl. After all the control LG31s died, there were still a few surviving resistant plants in the BC1F2 isolation population, and normal nutrient solution was restored for 4 days. Thirty surviving and best-growing seedlings were selected from the BC1F2 segregating population, and genomic DNA was extracted from each individual seedling. Equal amounts of genomic DNA from these 30 samples were mixed and analyzed by genome resequencing using the MutMap method (Abe, A., Kosugi, S., Yoshida, K., Natsume, S., Takagi, H., Kanzaki, H., Matsumura, H., Yoshida, K., Mitsuoka, C., Tamiru, M., Innan, H., Cano, L., Kamoun, S., and Terauchi, R. (2012). Genome sequencing reveals agronomically important loci in rice using MutMap. Nat Biotechnol. 30, 174-178) (Paisonno Biotechnol., Ltd.).

[0070] The rice growth and processing of the seedling hydroponic experiment were completed in an artificial climate chamber. The growth conditions were set to 14 hours of light / 10 hours of darkness, and the light intensity was 300 μmol / m 2 / s, the temperature was 30°C in light and 28°C in dark, and the humidity was set to 70%.

[0071] Yoshida nutrient solution formula: NH4NO3 (1mM), NaH2PO4·2H2O (0.6mM), K2SO4 (0.3mM), CaCl2 (0.3mM), MgCl2·6H2O (0.6mM), Fe(II)-EDTA (10μM), H3BO3 (48.7μM), MnSO4 (9μM), CuSO4·5H2O (0.3μM), ZnSO4·7H2O (0.7μM), NaMoO4·2H2O (0.1μM), and the final nutrient solution pH was adjusted to 5.8.

[0072] Example 2 Drought and salt tolerance and yield-increasing phenotypes of mutant spt38D

[0073] Using the same method as in Example 1, wild-type (LG31) and M5-generation mutant (spt38D) seeds were germinated and transferred to bottom-less PCR plates. After 12 days of hydroponic growth, they were treated with 25% PEG4000 or 150 mM NaCl. The results showed that compared to LG31, the mutant spt38D had less leaf curling, slower leaf dehydration and withering after PEG or NaCl treatment, and a significantly higher survival rate after returning to normal nutrient solution than the control LG31 (Figures 3A-D). The results of the seedling soil drought experiment (methods described below) also showed that compared to LG31, the mutant spt38D had stronger drought resistance at the seedling stage, and its survival rate after drought treatment and rehydration was significantly higher than that of the control LG31 (Figures 3E-F). Furthermore, field trials throughout the entire growth period (methods described below) showed that under normal paddy field management, the main agronomic traits of the spt38D mutant were not significantly different from those of the control LG31, with the exception of increased effective tillers and earlier heading. Yield data indicated that the spt38D mutant exhibited a 15.5% yield increase compared to the LG31 control. Under drought conditions, the spt38D mutant exhibited later leaf curling, less leaf dehydration, curling, and wilting than the LG31 control, and significantly increased effective tillering and seed set rates, resulting in a significantly higher yield than the LG31 control (44.5% increase) (Figures 3G-J).

[0074] Seedling stage soil drought experiment: After soaking and germination of LG31 and spt38D seeds, uniformly germinated seeds were evenly distributed in the same pot filled with nutrient soil, placed in a sink with appropriate amount of water, and grown in the greenhouse for 12 days. The pots were removed and placed on a growth rack to drain. After several days, the soil gradually dried out, and the rice seedlings gradually showed signs of leaf curling and wilt. After the wilt symptoms of the control LG31 and the mutant spt38D showed significant differences (approximately 12 days), the pots were returned to the sink for rehydration. Survival rates were calculated one day after rehydration. Greenhouse growth conditions were set at 30°C during the day and 28°C at night, with a humidity of 50-70%.

[0075] Field drought test: The field drought experiment was completed in Bengbu City, Anhui Province from May to October 2022. LG31 and spt38D seeds were soaked and germinated and then sown in the nursery of the rice field. The seedlings were transplanted at the 3-leaf stage (about 25 days after sowing). Three independent plots were set up for each material, and 6 rows were planted in each plot, with 10 plants per row, 20 cm between rows, and 17 cm between plants. The paddy field control group was managed according to the normal rice cultivation mode. The drought treatment group was cut off from water after the seedlings turned green, and grew under natural conditions until maturity (watering was done once on the 55th day after transplanting). After the rice matured, the side rows were removed, and 30 plants were selected from each plot for harvesting and yield measurement.

[0076] Example 3. OsSPT38 and OsSPT38D promote SUMOylation of substrate proteins

[0077] A yeast two-hybrid library screening approach identified a series of OsSPT38-interacting proteins, including OsFd1, OsDIP1, and ASC1. Using OsFd1 as an example, it was confirmed that OsSPT38 promotes SUMOylation of the substrate protein OsFd1, while OsSPT38D further enhances SUMOylation activity.

[0078] First, yeast two-hybrid (Y2H), BiFC, LCI, and pull-down assays (see below) confirmed that both OsSPT38 and OsSPT38D interact with OsFd1 (Figure 6). Furthermore, using the SUMOylation site prediction website (https: / / www.abcepta.com / sumoplot), we identified two SUMOylation sites in OsFd1: lysine residues (K) at positions 125 and 134 (Figure 7A). SUMOylation experiments in Escherichia coli (see below) revealed that mutating either lysine residue (K) at positions 125 and 134 of OsFd1 to arginine residues (R) reduced SUMOylation of OsFd1 (Figure 7B). Furthermore, SUMOylation experiments conducted in Escherichia coli revealed that lysine residues (K) at positions 125 and 134 of OsFd1 are essential for SUMO modification. Addition of OsSPT38 promoted SUMOylation of the substrate protein OsFd1, while addition of OsSPT38D further enhanced SUMOylation (Figure 7C). Similar results were obtained in tobacco (N. benthamiana) leaves expressing OsFd1, OsSPT38, and OsSPT38D (see methods below) (Figure 7D). These experiments demonstrate that OsSPT38 promotes SUMOylation of the substrate protein OsFd1, and that OsSPT38D is more potent than OsSPT38 in promoting SUMOylation of the substrate protein.

[0079] Yeast two-hybrid (Y2H) assay: The CDS sequence of OsSPT38 or OsSPT38D was inserted into the pGBKT7 vector (Shanghai Ouyi Biomedical Technology Co., Ltd.) using the ClonExpress recombination method (ClonExpress II One Step Cloning Kit, Cat. No. C112, manufactured by Nanjing Novozymes Biotechnology Co., Ltd.) to construct the pGBKT7-OsSPT38 and pGBKT7-OsSPT38D fusion vectors. Similarly, the CDS sequence of OsSCE3 (from the RGAP database, gene number LOC_Os04g49130) was inserted into the pGADT7 vector (Shanghai Ouyi Biomedical Technology Co., Ltd.) to construct the pGADT7-OsSCE3 fusion vector. pGBKT7-OsSPT38 or pGBKT7-OsSPT38D was co-transformed with pGADT7-OsSCE3 into the Y2H Gold yeast strain. Transformants were screened on yeast double-deficient medium (Beijing Fanjinuo Technology Co., Ltd., Catalog No. YGM003A-7). Positive clones were serially diluted with deionized water (0-, 10-, 100-, and 1000-fold) and inoculated onto yeast quadruple-deficient medium (Beijing Fanjinuo Technology Co., Ltd., Catalog No. YGM003A-9) to detect interactions between the two. Specific experimental procedures were performed according to the "Yeast Protocols Handbook (PT3024-1; Clontech, TaKaRa, China)".

[0080] Bimolecular fluorescence complementation (BiFC) experiments: The CDS sequence of OsSPT38 was inserted into the pAS054-nYFP vector (SEQ ID NO: 28) using the ClonExpress recombination method (ClonExpress II One-Step Cloning Kit, Catalog No. C112, produced by Nanjing Novozymes Biotechnology Co., Ltd.) to construct the pAS054-OsSPT38-nYFP fusion vector. Similarly, the CDS sequence of OsSCE3 was inserted into the pAS058-cYFP vector (SEQ ID NO: 29) to construct the pAS058-OsSCE3-cYFP fusion vector. Both pAS054-OsSPT38-nYFP and pAS054-OsSPT38-nYFP were co-transfected into tobacco (N. benthamiana) leaves using Agrobacterium tumefaciens GV3101. After 48 hours of induction in the dark, images were obtained using a laser confocal microscope (Carl Zeiss, Model LSM980). The specific experimental steps were completed with reference to “Yuan Meng, Xu Chunjue. Tobacco System BiFC. (2018). Bio-101:e1010133. Doi:10.21769 / BioProtoc.1010133”.

[0081] Split-luciferase complementation imaging (LCI) assay: The CDS sequence of OsSPT38 was inserted into the JW771 vector (see “Gou, JY, Felippes, FF, Liu, CJ, Weigel, D., and Wang, JW (2011). Negative regulation of anthocyanin biosynthesis in Arabidopsis by amiR156-targeted SPL transcription factor. Plant Cell 23, 1512-1522.”) using the ClonExpress recombination method (ClonExpress II One Step Cloning Kit, Cat. No. C112, produced by Nanjing Novozymes Biotechnology Co., Ltd.) to construct the JW771-OsSPT38-nLUC fusion vector. The OsSCE3 CDS sequence was inserted into the JW772 vector using the same method (see "Gou, JY, Felippes, FF, Liu, CJ, Weigel, D., and Wang, JW (2011). Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell 23, 1512-1522") to construct the JW772-OsSCE3-cLUC fusion vector. JW771-OsSPT38-nLUC and JW772-OsSCE3-cLUC were co-transfected into tobacco (N. benthamiana) leaves using Agrobacterium tumefaciens GV3101. After 48 hours of induction under dark conditions, 1 mM D-luciferin (Shanghai MacLean Biochemical Technology Co., Ltd., Cat. No. D797559) was injected into tobacco leaves, and then images were observed using a low-light cooled charge-coupled device (CCD) imaging system (Shanghai Tianneng Life Sciences Co., Ltd.). The specific experimental steps were completed with reference to the experimental methods section in "Huang J, Huang J, Feng Q, et al. SUMOylation facilitates the assembly of a Nuclear Factor-Y complex to enhance thermotolerance in Arabidopsis. Journal of Integrative Plant Biology, 2023, 65(3): 692-702".

[0082] Pull-down experiment: The same method as above was used to construct pGEX-6P-1-OsFd1 (containing a GST tag) and pV29-H-OsSPT38 (containing an MBP tag) fusion vectors using the pGEX-6P-1 vector (HonorGene, Cat. No. HG-VYA0225) and pV29-H vector (SEQ ID NO: 30). GST-OsFd1 and MBP-OsSPT38 recombinant proteins were expressed and purified in Escherichia coli BL21 (DE3) strains. GST or GST-Fd1 was incubated with GSH agarose beads (Changzhou Tiandi Renhe Biotechnology Co., Ltd., Cat. No. SM002005) at 4°C for 3 hours, followed by the addition of purified MBP-OsSPT38 protein and incubation for 3 hours. Wash three times with wash buffer (BBI Life Sciences Co., Ltd., Cat. No. C600326). After eluting the proteins from the beads using elution buffer (BBI Life Sciences Co., Ltd., Catalog No. C600325), GST or GST-Fd1 was detected using anti-GST antibody (Abimat Pharmaceutical Technology (Shanghai) Co., Ltd., Catalog No. M20007), and MBP-OsSPT38 was detected using anti-MBP antibody (Abimat Pharmaceutical Technology (Shanghai) Co., Ltd., Catalog No. M20051). The specific experimental steps were completed with reference to the experimental methods section of "Luo C, Cai XT, Du J, et al. PARAQUAT TOLERANCE3 is an E3 ligase that switches off activated oxidative response by targeting histone-modifying PROTEIN METHYLTRANSFERASE4b. PLoS genetics, 2016, 12(9): e1006332".

[0083] SUMOylation detection experiment in Escherichia coli system: The pCDFDuet-1-OsFd1 (containing a FLAG tag) fusion vector was constructed using the same method as above and transferred into the reconstructed SUMOylation system in the Escherichia coli BL21 (DE3) strain (the system was obtained from South China Normal University. For details about the pCDFDuet-1 vector and SUMOylation system, see “Huang Junwen, Feng Qiyi, Zheng Kaiyong, Huang Junjie, Wang Linbo, Lai Ruiqiang, Lai Jianbin, Yang Chengwei. (2002) An efficient in vitro detection system for plant protein SUMOylation. Acta Botanica Sinica 57, 490-499”). FLAG-OsFd1 was expressed and the protein samples were subjected to western blotting analysis using an anti-FLAG antibody (Abbimate Pharmaceutical Technology (Shanghai) Co., Ltd., Cat. No. M20008). To investigate the function of OsSPT38, the CDS of HA-tagged OsSPT38 or OsSPT38-D was inserted into a separate cloning site of the pCDFDuet-1-OsFd1 fusion vector using the same method as above to construct pCDFDuet-1-OsFd1-OsSPT38 or pCDFDuet-1-OsFd1-OsSPT38D. Subsequent experimental steps were the same as above, and protein samples were analyzed by Western blotting using anti-FLAG (Abbimate Pharmaceuticals (Shanghai) Co., Ltd., Catalog No. M20008) or anti-HA (Abbimate Pharmaceuticals (Shanghai) Co., Ltd., Catalog No. M20003) antibodies. The specific experimental steps were completed with reference to the experimental methods section in “Okada S, Nagabuchi M, Takamura Y, et al. Reconstitution of Arabidopsis thaliana SUMO pathways in E. coli: functional evaluation of SUMO machinery proteins and mapping of SUMOylation sites by mass spectrometry. Plant and cell physiology, 2009, 50(6): 1049-1061”.

[0084] Tobacco system SUMOylation assay: The same method as above was used to construct BGV008-OsFd1 and BGV008-OsFd1 using the BGV008 vector (Weimi Biotechnology (Jiangsu) Co., Ltd.). K125， 134R(containing a GFP tag) fusion vector, and the pCAMBIA1300 vector (SEQ ID NO: 31) was used to construct the pCAMBIA1300-OsSPT38 and pCAMBIA1300-OsSPT38D (containing a FLAG tag) fusion vectors. Through GV3101 Agrobacterium-mediated transfection, the transfection was co-transformed into tobacco (N. benthamiana) leaves. After 48 hours of induction under dark conditions, total protein from tobacco leaves was extracted using radioimmunoprecipitation assay lysis buffer (Shanghai Biyuntian Biotechnology Co., Ltd., Cat. No. P0013B), and protein samples were subjected to Western blotting analysis using GFP (Abimat Pharmaceutical Technology (Shanghai) Co., Ltd., Cat. No. M20004) and FLAG (Abimat Pharmaceutical Technology (Shanghai) Co., Ltd., Cat. No. M20008) antibodies. The specific experimental steps were completed with reference to the experimental methods section in “Joo H, Lim CW, Lee SC. Pepper SUMO E3 ligase CaDSIZ1 enhances drought tolerance by stabilizing the transcription factor CaDRHB1. New Phytologist, 2022, 235(6): 2313-2330”.

[0085] Example 4 Identification of OsSPT38 as a novel SUMO E3 ligase

[0086] OsSPT38 is annotated as an RBR-type E3 ubiquitin ligase (UniProt database, accession number for the OsSPT38 gene is Q2QLR6). Since E3 ubiquitin ligase function depends on an E2 conjugating enzyme, we screened a yeast two-hybrid library (see methods below) for E2 conjugating enzymes that interact with OsSPT38. Among the 30 E2 conjugating enzymes identified in rice, only OsSCE3 interacted with both OsSPT38 and OsSPT38D (Figures 4A-B). Yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and split-luciferase complementation imaging (LCI) experiments (described in Example 3) further confirmed that OsSCE3 indeed interacted with OsSPT38 (Figures 4C-E). OsSCE3 is a SUMO conjugating enzyme in rice (Joo, J., Choi, DH, Lee, YH, Seo, HS, and Song, SI (2019). The rice SUMO conjugating enzymes OsSCE1 and OsSCE3 have opposing effects on drought stress. J Plant Physiol. 240, 152993.), indicating that OsSPT38 may be a SUMO ligase.

[0087] Furthermore, combined with SUMOylation experiments conducted in Escherichia coli and tobacco (N. benthamiana) (see Example 3 for details), it was confirmed that OsSPT38 was involved in mediating SUMOylation modification of substrate proteins.

[0088] Furthermore, domain analysis of OsSPT38 revealed that it contains four domains: RING, IBR, RING, and RNaseH. Further comparative analysis of OsSPT38 with SUMO ligases from Arabidopsis and rice revealed that OsSPT38 contains a C3HC4 RING domain, but lacks the Siz / PIAS RING (SP-RING) domain of classic SUMO E3 ligases (Figure 5). These results suggest that OsSPT38 is a novel SUMO E3 ligase.

[0089] Yeast two-hybrid library screening experiment: The CDS sequence of OsSPT38 or OsSPT38D was inserted into the pGBKT7 vector (Shanghai Ouyi Biomedical Technology Co., Ltd.) by the ClonExpress recombination method (ClonExpressⅡOne Step Cloning Kit, product number C112, produced by Nanjing Novozymes Biotechnology Co., Ltd.) to construct pGBKT7-OsSPT38 and pGBKT7-OsSPT38D fusion vectors, and transformed into the Y2H Gold yeast strain (provided by Shanghai Ouyi Biomedical Technology Co., Ltd.). The total cDNA of the homogenized rice seedlings (Zhonghua 11 background) was inserted into the pGADT7 vector to construct the library plasmid and transformed into the Y187 yeast strain (yeast library construction was completed by Shanghai Ouyi Biomedical Technology Co., Ltd.). According to the "Matchmaker TM Proteins interacting with OsSPT38 or OsSPT38D were screened using the method described in the Gold Yeast Two-Hybrid System User Manual (PT4084-1; TaKaRa).

[0090] Example 5 OsSPT38-mediated SUMOylation promotes substrate protein stabilization

[0091] To investigate the effects of SUMOylation on substrate proteins, we first conducted cell-free degradation experiments (see below for methods). The results showed that OsFd1 degradation was slower in cells treated with protein extracts from spt38D seedlings compared to cells treated with protein extracts from LG31 seedlings (Figures 8A-B), indicating improved OsFd1 protein stability and that SUMOylation of OsFd1 mitigates its degradation under salt and drought stress. OsFd1 protein levels were measured in LG31 and spt38D seedlings treated with 150 mM NaCl and 25% PEG4000 for the indicated time periods. The spt38D mutant exhibited significantly higher levels of OsFd1 compared to wild-type LG31 (Figure 8C). Combined with Example 4, this demonstrates that SUMOylation can mitigate OsFd1 degradation under salt and drought stress, confirming that OsSPT38-mediated SUMOylation enhances OsFd1 protein stability and plays a crucial role in regulating plant stress tolerance.

[0092] Cell-free degradation assay: Crude protein was extracted from two-week-old LG31 or spt38D seedlings using extraction buffer (recipe below) and analyzed using OneDrop TMThe crude protein extracts were adjusted to the same concentration using a spectrophotometer (Wuyi Technology Co., Ltd., model: OD1000). 500 ng of purified GST-Fd1 (see the method section of Example 3 for details) and 50 μg of crude protein extract were co-incubated at a series of indicated time points and Western blot analysis was performed using an anti-GST antibody [Abimat Pharmaceutical Technology (Shanghai) Co., Ltd., catalog number M20007]. The specific experimental steps were completed with reference to the experimental method section in "Wang F, Zhu D, Huang X, et al. Biochemical Insights on Degradation of Arabidopsis DELLA Proteins Gained from a Cell-Free Assay System. The Plant Cell, 2009, 21(8): 2378-2390. DOI: 10.2307 / 40537007".

[0093] Extraction buffer formula: 25 mM Tris-HCl (pH 7.5) (Sigma, Catalog No. T1503), 10 mM MgCl2 (Sinopharm Chemical Reagent Co., Ltd., Catalog No. 10012818), 5 mM DTT (Sangon Biotechnology Co., Ltd., Catalog No. A620058), 0.1% Triton X-100 (Shanghai MacLean Biochemical Technology Co., Ltd., Catalog No. T6328), 10 mM ATP (BBI Life Sciences Co., Ltd., Catalog No. A600020), 10 mM NaCl (Sinopharm Chemical Reagent Co., Ltd., Catalog No. 10019318).

[0094] OsFd1 protein level detection experiment: Two-week-old LG31 or spt38D seedlings were treated with 25% PEG 4000 (Lotte, Korea, Cat. No. PEG-4000) or 150 mM NaCl (Sinopharm Chemical Reagent Co., Ltd., Cat. No. 10019318) and sampled at the designated time points and then snap-frozen in liquid nitrogen. Total protein of the aboveground part of the seedlings was extracted using radioimmunoprecipitation assay lysis buffer (Shanghai Beyotime Biotechnology Co., Ltd., Cat. No. P0013B), and Western blotting analysis was performed using anti-ferredoxin 1 antibody (Agrisera, Cat. No. AS20 4434) to determine the OsFd1 level.

[0095] Example 6 OsSPT38 positively regulates the drought and salt tolerance phenotype of rice

[0096] To further identify the function of OsSPT38 at the genetic level, we also obtained knockout mutants (ko-10, ko-12) and OsSPT38 overexpression (OE) in the LG31 background. W-2, OE W -28) and OsSPT38D overexpression (OE M -5, OE M -27) material drought and salt tolerance phenotype experiment (method is the same as Example 2). The experiment found that compared with the wild type LG31, whether it was treated with 25% PEG4000 or 150mM NaCl, the survival rate of the knockout mutants (ko-10, ko-12) after returning to normal nutrient solution was significantly lower than that of the wild type LG31, while the OsSPT38 overexpression (OE W -2, OE W -28) and OsSPT38D overexpression (OE M -5, OE M -27) strain had a significantly higher survival rate than the wild type (Figure 9A-D). The knockout mutants (ko-10, ko-12) were confirmed by first-generation sequencing. ko-10 had editing at both target 1 and target 2, ultimately resulting in a 1bp base deletion, and ko-12 had a 139bp base deletion between target 1 and target 2 (Figure 9E). At the same time, homozygous OsSPT38 overexpression (OE W -2, OE W -28) and OsSPT38D overexpression (OE M -5, OE M -27) strains, qRT-PCR results showed that compared with wild type LG31, OE W -2.OE W -28, OE M -5.OE M The expression levels of the OsSPT38 gene in the -27 line were all increased ( Figure 9F ).

[0097] Knockout mutants (ko-10, ko-12) were constructed by Weimi Biotechnology (Jiangsu) Co., Ltd. The steps involved using the company's high-throughput CRISPR-Cas9 target design program to design targets in the CDS region of OsSPT38. Targets with high target scores, low off-target rates, and suitable locations were selected as the final targets. The target sequences were constructed into the BGK03 vector and then transformed into LG31 via Agrobacterium-mediated genetic transformation. T0-generation positive seedlings were subsequently obtained through resistance screening and target sequencing. The specific methods for obtaining mutants are described in the reference (Lu, Y., Ye, X., Guo, R., Huang, J., Wang, W., Tang, J., Tan, L., Zhu, J.K., Chu, C., and Qian, Y. (2017). Genome-wide Targeted Mutagenesis in Rice Using the CRISPR / Cas9 System. Mol Plant 10, 1242-1245). The knockout mutants obtained were sequenced and identified as homozygous mutants and then used for subsequent phenotypic experiments.

[0098] OsSPT38 overexpression (OE W -2, OE W -28) and OsSPT38D overexpression (OE M -5, OE M -27) Brief implementation steps for obtaining materials: The CDS sequences of OsSPT38 and OsSPT38D were constructed into the BGV008 vector by the ClonExpress recombination method (the vector construction method was the same as in Example 3, and the BGV008 vector was provided by Weimi Biotechnology (Jiangsu) Co., Ltd.), and then genetically transformed into LG31 by Agrobacterium-mediated genetic transformation. Subsequently, T0 generation positive seedlings were obtained through resistance screening and PCR identification, and the homozygous strains were further propagated and identified for subsequent phenotypic experiments.

[0099] Example 7 Drought and salt tolerance phenotype of Arabidopsis thaliana overexpressing the OsSPT38 gene

[0100] The recombinant protein was expressed by Gateway recombination method (Invitrogen, BP Clonase TM Enzyme Mix, Cat. No. 56481 and LR Clonase TMEnzyme Mix, Catalog No. 56484) was used to insert the CDS sequence of OsSPT38 into the pCB2004 vector (the pCB2004 vector was constructed in our laboratory, for details, see Lei, Z.-Y., Zhao, P., Cao, M.-J., Cui, R., Chen, X., Xiong, L.-Z., Zhang, Q.-F., Oliver, DJ, and Xiang, C.-B. (2007). High-throughput Binary Vectors for Plant Gene Function Analysis. J. Integr. Plant Biol. 49(4), 556-567) to construct the pCB2004OsSPT38 recombinant expression vector, which was then transformed into wild-type Arabidopsis thaliana by Agrobacterium-mediated genetic transformation, and a homozygous overexpression strain was obtained by further glufosinate-ammonium resistance screening. The resulting wild-type (WT) and homozygous OsSPT38-overexpressing (OE-1, OE-2) lines were grown for 10 days on MS (formula below), MS medium containing 200 mM mannitol (Sinopharm Chemical Reagent Co., Ltd., Catalog No. 63008818), or MS medium containing 150 mM NaCl (Sinopharm Chemical Reagent Co., Ltd., Catalog No. 10019318). The results, as shown in Figure 10, show that compared to the WT Arabidopsis line, the OsSPT38-overexpressing Arabidopsis line showed no significant difference in growth on normal MS medium, but exhibited a significant growth advantage in medium containing 200 mM mannitol, with significantly increased biomass. Similarly, the survival rate was higher in medium containing 150 mM NaCl. These experimental results demonstrate the conserved nature of the drought and salt tolerance mediated by the OsSPT38 gene, providing strong evidence for its application in diverse crop breeding applications.

[0101] The Arabidopsis thaliana growth experiment was carried out in an artificial climate chamber with a growth condition of 16 hours of light / 8 hours of darkness and a light intensity of 300 μmol / m 2 / s, the temperature is 22 degrees, and the humidity is set to 70%.

[0102] MS culture medium formula (taking the preparation of 1 L as an example, the final pH is adjusted to 5.8):

[0103] Among them, 10×MS max The formula is as follows (taking 1L as an example):

[0104] 100×MS min The formula is as follows (taking 1L as an example):

[0105] The formula of 100× iron salt is as follows (taking 1L as an example): FeSO4·7H2O 2.78g Na2EDTA·2H2O 3.73g

[0106] Example 8 OsSPT38 promotes SUMOylation of substrate proteins in maize homologous protein Zm00001eb032750

[0107] To further confirm the conserved function of OsSPT38 homologs in other plants as SUMO E3 ligases, promoting the SUMOylation of substrate proteins, we conducted SUMOylation experiments in Escherichia coli using the maize (Zea mays L.) homolog of OsSPT38, Zm00001eb032750. Following the same method as in Example 3, the CDS of Zm00001eb032750 (from the Ensembl Plants database, gene number Zm00001eb032750) carrying an HA tag was inserted into the other cloning site of the pCDFDuet-1-OsFd1 fusion vector to construct the pCDFDuet-1-OsFd1-Zm00001eb032750 expression vector. Furthermore, a SUMOylation experiment was conducted in an Escherichia coli system (the method was the same as in Example 3). The results showed that the addition of Zm00001eb032750 could significantly enhance the SUMOylation modification level of the substrate protein OsFd1 ( FIG. 12 ).

[0108] The above experimental results show that the homologous genes of OsSPT38 in other plants act as SUMO E3 ligases and have a conserved function in promoting the SUMOylation modification of substrate proteins (such as the stress resistance and photosynthetic protein Fd1), providing strong evidence for the application of the homologous genes of OsSPT38 in other plants to enhance the drought and salt tolerance of plants.

[0109] Example 9 Evolutionary Analysis of the OsSPT38 Gene and Identification of Its Homologous Genes

[0110] Evolutionary analysis of the protein encoded by the OsSPT38 gene revealed that homologous genes exist in rice, maize, wheat, soybean, sorghum, millet, rye, barley, oats, rapeseed, cotton, potato, grape, pepper, tobacco, alfalfa, and Arabidopsis thaliana, with amino acid sequence similarities ranging from 52% to 91%, indicating high conservation (Figure 11A). Notably, the glycine residue (Gly) at position 212 of the OsSPT38 protein is highly conserved among homologous genes in monocots, while the dicot homologs contain a basic lysine residue (Lys), with no acidic residues found at this position. This suggests that mutations at this position from neutral or basic amino acids (e.g., Gly or Lys) to acidic amino acids (e.g., Asp) enhance the function of the protein encoded by the homologous gene. The use of the OsSPT38 gene in drought and salt tolerance in plants is a newly discovered finding. For example, the OsSPT38 gene in rice (Oryza sativa L.), also known as LOC_Os12g43560 or Os12g0631200, encodes a protein with the amino acid sequence shown in SEQ ID NO: 2. Its gain-of-function mutant, OsSPT38D, encodes a protein with the amino acid sequence shown in SEQ ID NO: 4. The OsSPT38 homolog in maize (Zea mays L.) is Zm00001eb032750, encoding a protein with the amino acid sequence shown in SEQ ID NO: 5, which shares 88.9% similarity with OsSPT38. The wheat (Triticum aestivum L.) OsSPT38 homologous genes are TraesCS5A02G015900, TraesCS5D02G022000, and TraesCS5B02G014200, encoding proteins with amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, showing 81.7-82.5% similarity to OsSPT38. The soybean (Glycine max L.) OsSPT38 homologous genes are GLYMA_15G126500 and GLYMA_09G020000, encoding proteins with amino acid sequences of SEQ ID NOs: 9 and 10, respectively, showing 59.5-70.5% similarity to OsSPT38. The amino acid sequence of the protein encoded by the OsSPT38 homologous gene SORBI_3008G185500 in sorghum (Sorghum bicolor) is SEQ ID NO: 11, which is 88.0% similar to OsSPT38. The amino acid sequence of the protein encoded by the OsSPT38 homologous gene SETIT_021799mg in millet (Setaria italica) is SEQ ID NO: 12, which is 90.9% similar to OsSPT38.The amino acid sequence of the protein encoded by the SECCEUnv1G0529500 gene in rye (Secale cereale L.) is SEQ ID NO: 13, which is 82.9% similar to OsSPT38. The amino acid sequence of the protein encoded by the HORVU.MOREX.r3.5HG0422080 gene in barley (Hordeum vulgare L.) is SEQ ID NO: 14, which is 82.9% similar to OsSPT38. The OsSPT38 homologous genes AVESA.00001b.r3.4Ag0000409, AVESA.00001b.r3.4Dg0000762, and AVESA.00001b.r3.7Cg0003091 in oat (Avena sativa L.) encode proteins with amino acid sequences of SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, showing 85.8-86.9% similarity to OsSPT38. The OsSPT38 homologous genes BnaA01g10230D and BnaA03g44140D in rapeseed (Brassica napus) encode proteins with amino acid sequences of SEQ ID NO:18 and SEQ ID NO:19, respectively, showing 52.1-61.6% similarity to OsSPT38. The cotton (Gossypium hirsutum L.) OsSPT38 homologous genes B456_009G150700 and B456_010G169300 encode proteins with amino acid sequences of SEQ ID NO:20 and SEQ ID NO:21, respectively, showing 58.6-73.2% similarity to OsSPT38. The potato (Solanum tuberosum L.) OsSPT38 homologous gene PGSC0003DMG400021214 encodes proteins with amino acid sequences of SEQ ID NO:22, showing 69.6% similarity to OsSPT38. The grape (Vitis vinifera) OsSPT38 homologous gene Vitvi10g01351 encodes proteins with amino acid sequences of SEQ ID NO:23, showing 77.7% similarity to OsSPT38. The amino acid sequence of the protein encoded by the OsSPT38 homologous gene T459_33471 in pepper (Capsicum annuum L.) is SEQ ID NO: 24, which is 68.8% similar to OsSPT38. The amino acid sequence of the protein encoded by the OsSPT38 homologous gene ARI10_0 in tobacco (Nicotiana attenuata) is SEQ ID NO: 25, which is 68.8% similar to OsSPT38.The OsSPT38 homologous gene MTR_2g028870 in alfalfa (Medicago truncatula) encodes a protein with an amino acid sequence of SEQ ID NO: 26, which is 72.2% similar to OsSPT38. The OsSPT38 homologous gene AT4G19670 in Arabidopsis thaliana (Arabidopsis thaliana L.) encodes a protein with an amino acid sequence of SEQ ID NO: 27, which is 56.4% similar to OsSPT38. Phylogenetic analysis is shown in Figure 11B.

[0111] Example 10 OsSPT38 improves drought resistance and yield of rice

[0112] To explore the function of OsSPT38 in improving rice drought resistance and yield, we conducted a field drought experiment. The field drought experiment was completed in Sanya, Hainan Province from November 2023 to April 2024. LG31, knockout mutant (ko-12) and overexpression material (OE W -2, OE W -28) seeds were soaked and germinated, then sown in the nursery of the rice field, and the seedlings were transplanted at the 3-leaf stage (about 25 days after sowing). Three independent plots were set up for each material, and 12 rows were planted in each plot, with 10 plants in each row, 20 cm between rows, and 17 cm between plants. The drought treatment group was cut off from water after the seedlings turned green, and the normal control group was managed according to the normal rice cultivation mode. After the rice matured, the side rows were removed, and 20 individual plants were randomly selected from each strain to calculate the plant height, effective tiller number, fruit setting rate, number of grains per ear, and yield per plant. In addition, 30 plants were randomly selected from each plot of each strain for mixed harvesting and yield measurement to obtain the yield per unit area. The experimental results are shown in Figure 13. In the normal control group, compared with the wild-type LG31, the effective tiller number, fruit setting rate, number of grains per ear, and plant height of the knockout mutant (ko-12) were reduced, and the final yield per plant was reduced by 45.9% and the yield per unit area was reduced by 46.4%; while the overexpression material (OE W -2, OE W -28) had no significant difference in fruit setting rate, but the effective tiller number, grain number per ear and plant height increased, and the final yield per plant increased by 19.3%-20.8% and the yield per unit area increased by 20.1%-23.3%. In the drought treatment group, compared with the wild type LG31, the effective tiller number, fruit setting rate and 1000-grain weight of the knockout mutant (ko-12) were significantly reduced, and the final yield per plant decreased by 53.9% and the yield per unit area decreased by 48.8%; while the overexpression material (OE W -2, OE WWhile there was no significant difference in seed set rate between the two strains (-28), there were significant increases in effective tiller number, grain number per panicle, and plant height. Ultimately, yield per plant increased by 46.9%-50.2% and yield per unit area increased by 33.0%-35.2%. These field trial results demonstrate that OsSPT38 positively regulates drought resistance and yield traits in rice, demonstrating its significant application value.

[0113] Sequence Listing

Claims

1. OsSPT38D protein, whose amino acid sequence is shown in SEQ ID NO: 4, which has an amino acid mutation 212D compared to the amino acid sequence of the OsSPT38 protein shown in SEQ ID NO:

2.

2. An OsSPT38D gene encoding the OsSPT38D protein according to claim 1.

3. The OsSPT38D gene according to claim 2, wherein the nucleotide coding sequence is selected from any one of the following: 1) the sequence shown in SEQ ID NO: 3; 2) sequences that have conservative substitutions, deletions, or additions of one or more bases compared to the sequence shown in SEQ ID NO: 3 and have the same function as the sequence shown in SEQ ID NO: 3; 3) A sequence that can hybridize to the sequence shown in SEQ ID NO: 3 under highly stringent conditions.

4. The OsSPT38D gene according to claim 3, characterized in that The sequence having the same function as the sequence shown in SEQ ID NO: 3 has at least 50% identity with the sequence shown in SEQ ID NO: 3, preferably at least 52%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

5. Use of OsSPT38 and / or OsSPT38D proteins or their encoding genes for improving plant stress resistance and / or yield, preferably the stress resistance is salt tolerance and / or drought resistance, wherein the amino acid sequence of the OsSPT38 protein is shown in SEQ ID NO: 2, the OsSPT38D protein is as defined in claim 1, and the gene encoding the OsSPT38D protein is as defined in claim 3 or 4.

6. A recombinant expression vector or host cell comprising the OsSPT38D gene according to claim 3 or 4, wherein the host cell is preferably an Escherichia coli cell or an Agrobacterium cell.

7. Use of the recombinant expression vector or host cell according to claim 6 in improving stress resistance and / or yield of a plant, wherein the stress resistance is preferably salt tolerance and / or drought resistance.

8. A method for improving plant stress resistance and / or plant yield, wherein the stress resistance is preferably salt tolerance and / or drought tolerance, the method comprising any one of the following or a combination thereof: 1) Using gene editing methods, the amino acid 212 of the OsSPT38 protein in plants was mutated to Asp (212D), and plant mutants carrying the 212D mutation were obtained. 2) introducing the 212D mutation into the recipient plant by crossing a plant carrying the OsSPT38D protein with a recipient plant, 3) overexpressing the gene encoding the OsSPT38 protein or the OsSPT8D protein in plants, and 4) Transfecting the gene encoding OsSPT38 protein or OsSPT38D protein into recipient plants.

9. The method according to claims 5, 7, and 8, wherein the plant is a dicotyledonous or monocotyledonous plant, preferably rice, corn, wheat, soybean, sorghum, millet, rye, barley, oat, rapeseed, cotton, potato, grape, pepper, tobacco, alfalfa, and Arabidopsis, most preferably rice and Arabidopsis.

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