Sumo1-wri1 fusion protein and its encoding gene and application in cultivating heat-resistant high-yield plants

Abstract

The application belongs to the technical field of genetic engineering and plant breeding, and particularly relates to a SUMO1-WRI1 fusion protein, a coding gene thereof and application of the SUMO1-WRI1 fusion protein in cultivating heat-resistant high-yield plants. The application constructs a SUMO1-WRI1 fusion protein, which is obtained by directly connecting and fusing SUMO1 and WRI1. WRI1 can improve crop seed quality and yield under high-temperature stress, and the fusion of SUMO1 can significantly enhance the thermal stability and transcriptional regulation activity of WRI1 by means of the structural characteristics of SUMO1, so as to greatly improve the yield and quality improvement effect of WRI1 under high-temperature stress. The technology can effectively improve the seed shriveling problem caused by high-temperature stress without affecting the normal development phenotype of crops, realize the significant improvement of crop seed fullness and yield under heat stress, and further significantly improve the quality and yield of crop seeds, thereby providing an efficient molecular breeding tool for cultivating new heat-resistant high-yield crop varieties.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and plant breeding technology, specifically relating to a SUMO1-WRI1 fusion protein and its encoding gene and its application in cultivating heat-resistant and high-yielding plants. Background Technology

[0002] Extreme heat stress caused by global warming has become a core abiotic stress factor restricting crop yield stability. Heat stress disrupts protein structural stability, carbon metabolism balance, and reproductive development processes, leading to insufficient seed filling, abnormal morphology, and decreased seed plumpness, ultimately resulting in significant yield reduction. During the reproductive growth stage of crops, seed development is particularly sensitive to temperature fluctuations, and yield losses under heat stress are often irreversible. Therefore, identifying key molecular targets for regulating seed heat tolerance development has significant theoretical and applied value.

[0003] WRINKLED1 (WRI1), a transcription factor of the AP2 / EREBP family, is a core regulator of fatty acid synthesis and carbon source allocation in plants. By specifically binding to the AW-box element of target gene promoters, it regulates the expression of key genes in glycolysis and fatty acid synthesis pathways, directly affecting seed oil accumulation and grain filling. Previous studies have confirmed that overexpression of the WRI1 gene can effectively improve crop seed development quality and maintain stable grain filling efficiency under stress conditions, indicating that this gene plays a positive regulatory role in seed development. However, high temperatures easily induce conformational changes or degradation of the WRI1 protein, leading to a significant inhibition of its transcriptional regulatory activity. This makes it difficult to completely resist the adverse effects of heat stress on seed development; therefore, there is still considerable room for optimization of the functional stability of this protein.

[0004] Currently, crop heat tolerance breeding still has significant limitations: First, most crop heat tolerance breeding work focuses on enhancing the overall stress resistance of plants, lacking specific technical means to protect seed development under heat stress during the reproductive period; second, although the traditional WRI1 overexpression strategy can improve seed filling to some extent, the function of this protein is easily inhibited under high temperature conditions, which cannot meet the needs of crop yield protection under extreme heat stress conditions; third, there is a relative lack of relevant methods to optimize the heat tolerance function of WRI1 through molecular modification technology, and a mature and widely applicable solution has not yet been formed.

[0005] Ubiquitin-like proteins (SUMOs) are a class of highly conserved small-molecule regulatory proteins that can alter the spatial conformation of target proteins, enhance their resistance to degradation, or regulate their subcellular localization by forming binding complexes with target proteins, thereby participating extensively in the optimization and regulation of protein function during plant stress responses. Currently, no research reports have been published on a technical strategy of directly fusing SUMO1 with WRI1 to enhance its functional stability under heat stress. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, this invention provides a SUMO1-WRI1 fusion protein, its encoding gene, and a recombinant vector. By directly tandemly fusing SUMO1 and WRI1, the structural characteristics of SUMO1 are used to enhance the stability and transcriptional regulatory activity of WRI1 protein under heat stress, thereby significantly improving the seed plumpness and yield of crops under heat stress. This provides an efficient molecular breeding tool for cultivating new heat-resistant and high-yielding crop varieties.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides the application of SUMO1-WRI1 fusion protein in the cultivation of heat-resistant and high-yielding plants, wherein the amino acid sequence of the SUMO1-WRI1 fusion protein is shown in SEQ ID No. 8.

[0008] WRI1, as a positive regulator of seed development, has its function limited by protein stability at high temperatures. By directly tandemly fusing SUMO1 molecules with WRI1 through genetic engineering, the stability and transcriptional activity of WRI1 under heat stress can be specifically enhanced, thereby significantly improving seed plumpness and yield. GRSX1 of the same molecular weight cannot achieve this optimization effect.

[0009] Preferably, the gene sequence encoding the SUMO1-WRI1 fusion protein is shown in SEQ ID No. 7.

[0010] More preferably, using Arabidopsis cDNA as a template, the full-length SUMO1 gene shown in SEQ ID No. 3 is amplified by PCR using specific primers; using the WRI1 gene sequence shown in SEQ ID No. 1 as a template, the full-length WRI1 gene is amplified by PCR using specific primers, and then the SUMO1 gene is directly tandemly spliced ​​with the WRI1 gene using overlap extension PCR technology to obtain the encoding gene of the SUMO1-WRI1 fusion protein.

[0011] Furthermore, the specific primers for amplifying the full-length SUMO1 gene are shown in SEQ ID No. 12 and SEQ ID No. 13; and the specific primers for amplifying the full-length WRI1 gene are shown in SEQ ID No. 14 and SEQ ID No. 15.

[0012] Preferably, the plants include Arabidopsis thaliana, wheat, corn, soybean, and rapeseed.

[0013] The second aspect of this invention also provides a method for cultivating heat-resistant and high-yielding plants, comprising: constructing a recombinant expression vector containing a gene encoding a SUMO1-WRI1 fusion protein; introducing the recombinant expression vector into host cells by gene introduction and screening to obtain positive host cells; transforming target plants with positive host cells and harvesting T0 generation seeds; subjecting the T0 generation seeds to resistance screening and molecular identification and then continuously self-pollinating to obtain genetically stable homozygous transgenic lines, thereby obtaining heat-resistant and high-yielding transgenic plants.

[0014] Preferably, the gene delivery method includes Agrobacterium-mediated transformation or gene gun method.

[0015] Preferably, the method for constructing the recombinant expression vector is as follows: the SUMO1-WRI1 fusion protein encoding gene and the plant expression vector pCAMBIA1300 are subjected to double enzyme digestion, the target fragment and the vector backbone are recovered, ligated by DNA ligase, transformed into competent E. coli cells, and the recombinant expression vector is obtained by resistance screening and sequencing verification.

[0016] Preferably, the target plant is transformed using an inflorescence infection method with positive host cells, wherein the host cells are Agrobacterium competent cells.

[0017] Preferably, the target plants include Arabidopsis thaliana, wheat, corn, soybean, and rapeseed.

[0018] Compared with the prior art, the beneficial effects of the present invention are: This invention constructs a SUMO1-WRI1 fusion protein, obtained by direct tandem fusion of SUMO1 and WRI1. WRI1 can improve crop seed quality and yield under high-temperature stress, while the fusion of SUMO1, through its own structural characteristics, significantly enhances the thermostability and transcriptional regulatory activity of WRI1, thereby greatly improving its yield and quality-enhancing effect under high-temperature stress. This invention also provides the encoding gene of this fusion protein, the recombinant vector, and its applications. This technology can effectively improve the problem of seed shriveling caused by high-temperature stress without affecting the normal developmental phenotype of crops, achieving a significant increase in seed plumpness and yield under heat stress, thereby significantly improving crop seed quality and yield, and providing an efficient molecular breeding tool for cultivating new heat-resistant and high-yielding crop varieties. Specifically, this invention has the following advantages: (1) High specificity of protein function optimization: Direct fusion of SUMO1 and WRI1 can significantly improve the stability and transcriptional activity of WRI1 under heat stress, while fusion of GRSX1 with the same molecular weight with WRI1 has no such effect, proving that SUMO1 has specificity for WRI1 function optimization and excluding the non-specific effects of molecular weight increase.

[0019] (2) Significant protection effect on seed development: Under heat stress, the SUMO1-WRI1-OE line had the least degree of seed wrinkling, and the seed width decreased by only 4.0% compared with normal temperature, which was significantly lower than the wild type of 18.6% and the WRI1-OE line of 11.4%; the thousand-seed weight increased by 55.6% compared with the wild type and by 16.7% compared with the WRI1-OE line; the total fatty acid content increased by 49.4% compared with the wild type and by 9.6% compared with the WRI1-OE line, and the grain filling was significantly better than the existing WRI1 overexpression technology.

[0020] (3) Outstanding yield guarantee: Under high temperature stress, the SUMO1-WRI1-OE line maintained the highest level of seed weight and oil content, effectively solving the problem of excessive growth and poor seed setting caused by heat stress, and achieving stable yield under adverse conditions.

[0021] (4) Wide range of applications: The method of this invention can be applied to the genetic improvement of various crops such as soybeans, wheat, corn, and rapeseed, and to cultivate heat-resistant and high-yielding new varieties. It is especially suitable for agricultural production in arid and semi-arid regions under the background of global warming, and has important economic value and ecological significance. Attached Figure Description

[0022] Figure 1 To validate the expression of the fusion protein in Arabidopsis thaliana; Figure 2 This is a method of heat stress treatment; Figure 3 Phenotypes of plants with different genotypes at 22℃ and 35℃; Figure 4Phenotypic analysis of seeds from different plant genotypes under normal and heat stress treatments; (A) Morphological comparison of mature seeds of WT, WRI1-OE, SUMO1-WRI1-OE, and GRXS1-WRI1-OE plants under normal and heat stress treatments, scale bar = 500 μm; (B) Length of mature seeds of WT, WRI1-OE, SUMO1-WRI1-OE, and GRSX1-WRI1-OE plants under normal and heat stress treatments. Data are expressed as mean ± standard deviation (SD) (N=3). According to Tukey's HSD test (P<0.05), there was no significant difference in gene expression for the same letter; (C) Width of mature seeds of WT, WRI1-OE, SUMO1-WRI1-OE, and GRSX1-WRI1-OE plants under normal and heat stress treatments. Data are expressed as mean ± standard deviation (SD) (N=3). According to Tukey's HSD test (P<0.05), there was no significant difference in gene expression for the same letter; (D) Seed weight comparison among WT, WRI1-OE, SUMO1-WRI1-OE and GRSX1-WRI1-OE plants under normal and heat stress treatments. Data are expressed as mean ± standard deviation (SD) (N=3). According to Tukey's HSD test (P<0.05), there was no significant difference in gene expression among those labeled with the same letter; (E) Fatty acid content among WT, WRI1-OE, SUMO1-WRI1-OE and GRSX1-WRI1-OE plants under normal and heat stress treatments. Data are expressed as mean ± standard deviation (SD) (N=3). According to Tukey's HSD test (P<0.05), there was no significant difference in gene expression among those labeled with the same letter. Detailed Implementation

[0023] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0025] Based on the positive regulatory role of WRI1 in seed development, this invention uses genetic engineering technology to directly tandem the SUMO1 protein coding sequence with the WRI1 gene to construct a fusion gene, which is then introduced into plants. The structural protection of SUMO1 enhances the stability of the WRI1 protein under heat stress. Simultaneously, using the GRSX1-WRI1 fusion protein as a negative control, systematic phenotypic identification and physiological index detection verify the specificity of SUMO1 in optimizing WRI1 function, ultimately achieving precise improvement in seed development quality under heat stress. Glutoredoxin (GRSX1), as a functional protein with a similar molecular weight to SUMO1, is often used as a negative control for fusion proteins to eliminate the non-specific effects of simply increasing molecular weight on the target protein's function.

[0026] WRI1 gene sequence (SEQ ID No. 1):

[0027] WRI1 protein sequence (SEQ ID No.2): MKKRLTTSTCSSSPSSSVSSSTTTSSPIQSEAPRPKRAKRAKKSSPSGDKSHNPTSPASTRRSSIYRGVTRHRWTGRFEAHLWDKSSWNSIQNKKGKQVYLGAYDSEEAAAHTYDLAALKYWGPDTILNFPAETYTKELEEMQRVTKEEYLASLRRQSSGFSRGVSKYRGVARHHHNGRWEARIGRVFGNKYLYLGTYNTQEEAAAAYDMAAIEYRGANAVTNFDISNYIDRLKKKGVFPFPVNQANHQEGILVEAKQEVETREAKEEPREEVKQQYVEEPPQEEEEKEEEKAEQQEAEIVGYSEEAAVVNCCIDSSTIMEMDRCGDNNELAWNFCMMDTGFSPFLTDQNLANENPIEYPELFNELAFEDNIDFMFDDGKHECLNLENLDCCVVGRESPPSSSSPLSCLSTDSASSTTTTTTSVSCNYLFQGLFVGSE。

[0028] SUMO1 gene sequence (SEQ ID No.3): ATGTCTGCAAACCAGGAGGAAGACAAGAAGCCAGGAGACGGAGGAGCTCACATCAATCTCAAAGTCAAGGGACAGGATGGAAACGAGGTTTTCTTTAGGATCAAGAGAAGCACTCAGCTCAAGAAGCTGATGAATGCTTACTGTGACCGGCAATCTGTGGACATGAACTCCATTGCTTTCTTGTTTGATGGGCGTCGTCTTCGTGCTGAGCAAACTCCCGATGAGCTTGACATGGAGGATGGTGATGAGATCGATGCGATGCTCCATCAGACTGGTGGCAGCGGTGGTGGTGCTACGGCC (removing the stop codon TGA).

[0029] SUMO1 protein sequence (SEQ ID No.4): MSANQEEDKKPGDGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELDMEDGDEIDAMLHQTGGSGGGATA。

[0030] GRXS1 gene sequence (SEQ ID No.5): ATGGAGAAGATATCAAATTTGTTAGAAGACAAGCCCGTGGTGATATTCAGCAAGACGTCCTGCTGTATGAGTCACTCGATCAAGTCGCTTATATCTGGTTACGGTGCGAATTCAACAGTGTATGAGCTAGACGAAATGTCTAATGGACCAGAGATCGAACGAGCACTTGTAGAGCTTGGGTGCAAACCGACTGTGCCAGCTGTCTTTATAGGGCAAGAGCTCGTAGGTGGTGCAAATCAACTTATGTCTCTTCAAGTCAGGAACCAACTAGCTTCGTTGCTCCGAAGAGCTGGAGCCATATGGATT (removing the stop codon TAA).

[0031] GRXS1 protein sequence (SEQ ID No.6): MEKISNLLEDKPVVIFSKTSCCMSHSIKSLISGYGANSTVYELDEMSNGPEIERALVELGCKPTVPAVFIGQELVGGANQLMSLQVRNQLASLLRRAGAIWI.

[0032] SUMO1-WRI1 gene sequence (SEQ ID No.7):

[0033] SUMO1-WRI1 protein sequence (SEQ ID No.8): MSANQEEDKKPGDGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELDMEDGDEIDAMLHQTGGSGGGATAMKKRLTTSTCSSSPSSSVSSSTTTSSPIQSEAPRPKRAKRAKKSSPSGDKSHNPTSPASTRRSSIYRGVTRHRWTGRFEAHLWDKSSWNSIQNKKGKQVYLGAYDSEEAAAHTYDLAALKYWGPDTILNFPAETYTKELEEMQRVTKEEYLASLRRQSSGFSRGVSKYRGVARHHHNGRWEARIGRVFGNKYLYLGTYNTQEEAAAAYDMAAIEYRGANAVTNFDISNYIDRLKKKGVFPFPVNQANHQEGILVEAKQEVETREAKEEPREEVKQQYVEEPPQEEEEKEEEKAEQQEAEIVGYSEEAAVVNCCIDSSTIMEMDRCGDNNELAWNFCMMDTGFSPFLTDQNLANENPIEYPELFNELAFEDNIDFMFDDGKHECLNLENLDCCVVGRESPPSSSSPLSCLSTDSASSTTTTTTSVSCNYLFQGLFVGSE.

[0034] GRXS1-WRI1 gene sequence (SEQ ID No.9):

[0035] GRXS1-WRI1 protein sequence (SEQ ID No. 10): .

[0036] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0037] 1. Construction and identification of recombinant vectors (1) Experimental materials Plant expression vector pCAMBIA1300, Escherichia coli DH5α competent cells, and Agrobacterium tumefaciens EHA105 competent cells were purchased from Tiangen Biotech (Beijing) Co., Ltd.; PCR amplification enzymes, restriction endonucleases (BamHI, SacI), and T4 DNA ligase were purchased from NEB; primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0038] (2) Experimental methods ① Gene amplification and fusion gene construction Using Arabidopsis cDNA as a template, the full-length SUMO1 gene was amplified by PCR with specific primers; the full-length WRI1 gene was amplified using the provided WRI1 gene sequence as a template; and the full-length GFP gene was amplified using a plant expression vector containing the GFP gene (plant binary expression vector pCAMBIA1300-GFP) as a template. Stepwise overlap extension PCR was employed, first tandemly splicing the SUMO1 and WRI1 genes to obtain the SUMO1-WRI1 fusion gene, and then tandemly splicing it with the GFP gene to obtain the full-length SUMO1-WRI1-GFP fusion gene, ensuring continuous and correct reading frames throughout the process.

[0039] First round of PCR (amplifying 3 independent linear fragments respectively): Amplifying the T1 fragment (SUMO1): Primers are T1-F and T1-R; Amplifying the T2 fragment (WRI1): Primers are T2-F and T2-R; Amplifying the T3 fragment (GFP): Primers are GFP-F and GFP-R; Uniform reaction system (50 μL): 2×PCRMasterMix 25 μL, forward and reverse primers 2 μL each, template DNA 1 μL, ddH2O 20 μL; Reaction program: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 40 s (T1) / 150 s (T2) / 40 s (T3), 35 cycles; 72℃ final extension for 2 min; The products were verified by 1.2% agarose gel electrophoresis, and the target fragments were purified by gel recovery.

[0040] Second round PCR (assembling the SUMO1-WRI1 intermediate fusion fragment): Template: T1 (SUMO1) recovery product and T2 (WRI1) recovery product are mixed in equimolar amounts; Primers: T1-F and T2-R; Reaction system: Same as the first round (50 μL); Reaction program: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 62℃ annealing for 30 s, 72℃ extension for 2 min, 35 cycles; 72℃ final extension for 5 min; The product was verified by 1.2% agarose gel electrophoresis, and the SUMO1-WRI1 fusion fragment was purified by gel recovery.

[0041] Third round of PCR (splicing the full-length SUMO1-WRI1-GFP fusion gene): Template: The SUMO1-WRI1 fusion fragment recovery product and the T3 (GFP) recovery product were mixed in equimolar amounts; Primers: T1-F and GFP-R; Reaction system: Same as the first round (50 μL); Reaction program: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 62℃ annealing for 30 s, 72℃ extension for 2.5 min, 35 cycles; 72℃ final extension for 5 min; The product was verified by 1.2% agarose gel electrophoresis, and the full-length fusion gene was purified by gel recovery.

[0042] In addition, the construction process of the GRXS1-WRI1-GFP fusion gene is exactly the same as above, only the corresponding primers are replaced: Round 1: Amplification of T4 fragment (GRXS1, primers T3-F / T3-R), T2 fragment (WRI1, primers T4-F / T2-R), and T3 fragment (GFP, primers GFP-F / GFP-R). Second round: Template = T4 + T2, primer = T3-F / T2-R, to obtain the GRXS1-WRI1 fusion fragment. Third round: Template = GRXS1-WRI1+T3, primer = T3-F / GFP-R, to obtain the full-length GRXS1-WRI1-GFP fusion gene.

[0043] GFP sequence (SEQ ID No.11): AGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTTATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAAGTA。

[0044] The primer sequences are as follows: T1-F (upstream of SUMO1): 5'-GGATCCATGTCTGCAAACCAGGAGGAAG-3' (SEQ ID No.12); T1-R (downstream of SUMO1, containing 15bp overlapping region at the 5' end of WRI1): 5'-TTAGCGCTTCTTTCATGGCCGTAGCACCACC-3' (SEQ ID No.13); T2-F (upstream of WRI1, complementary to the 3' end overlap region of SUMO1): 5'-GGTGGTGCTACGGCCATGAAGAAGCGCTTA-3' (SEQ ID No. 14); T2-R (WRI1 downstream): 5'-GCTGAGGAGATGGAGAAAAGTTCAGTTCTGAA-3' (SEQ ID No. 15); GFP-F (GFP upstream primer): 5'-TTCTCCATCTCCTCAGCATGGTGAGCAAGGGCGAGGA-3' (SEQ ID No. 16); GFP-R (GFP downstream primer): 5'-CGAAAACAAGCACTGAGACTAGTTTTCTCCATCTCCTCAGCT-3' (SEQ ID No. 17).

[0045] T3-F (GRXS1 upstream primer): 5'-GGATCCATGGAGAAGATATCAAATTTG-3' (SEQ ID No. 18); T3-R (GRXS1 downstream primer): 5'-TTAGCGCTTCTTTCATCCATGGCTCCAGCTCTTC-3' (SEQ ID No. 19).

[0046] T4-F (WRI1 upstream primer): 5'-GAAGAGCTGGAGCCATGGATGAAGAAGCGCTTA-3' (SEQ ID No. 20).

[0047] ②Carrier construction: The fusion gene and the plant expression vector pCAMBIA1300 were double-digested with BamHI and Spe I, respectively. The digestion reaction system (30 μL) consisted of 10 μL of vector / PCR product, 3 μL of 10× digestion buffer, 1 μL each of restriction endonucleases BamHI and Spe I, and 15 μL of enzyme-free ultrapure water. The mixture was incubated at 37°C for 4 h. The digested products were separated by agarose gel electrophoresis and purified. They were then mixed with the target fragment at a molar ratio of 3:1 to the vector backbone, and T4 DNA ligase and its buffer were added. The mixture was incubated overnight at 16°C to complete ligation. The ligation product was then transformed into E. coli DH5α competent cells. Positive clones were screened using hygromycin-resistant medium, and the recombinant plant expression vector was successfully constructed by double digestion and sequencing verification. The recombinant expression vector pCAMBIA1300-SUMO1-WRI1-GFP was obtained.

[0048] Simultaneously, using the same method described above, the GRXS1 gene was directly tandemly linked with the WRI1 gene to construct the recombinant expression vector pCAMBIA1300-GRSX1-WRI1-GFP (negative control). Additionally, the full-length WRI1 gene was directly inserted into the BamHI and SacI restriction sites of the pCAMBIA1300 vector to construct the recombinant expression vector pCAMBIA1300-WRI1-GFP (positive control).

[0049] ③ Identification: Single resistant colonies were selected, plasmids were extracted, and PCR identification and double enzyme digestion verification were performed. Positive clones were sent to a sequencing company for sequencing.

[0050] (3) Sequencing results Sequencing confirmed that the SUMO1-WRI1-GFP and GRSX1-WRI1-GFP fusion gene sequences were correct, with complete reading frames and no base mutations or deletions. Double enzyme digestion confirmed that the target fragment was successfully inserted into the vector, and the recombinant expression vectors pCAMBIA1300-SUMO1-WRI1-GFP, pCAMBIA1300-GRSX1-WRI1-GFP, and pCAMBIA1300-WRI1-GFP were successfully constructed.

[0051] (4) Validation of fusion protein The recombinant vector was transformed into BL21 competent cells, and after IPTG induction, specific bands appeared in SDS-PAGE electrophoresis.

[0052] 2. Obtaining homozygous transgenic Arabidopsis lines (1) Experimental materials The seeds of Arabidopsis thaliana Columbia ecotype (WT) were purchased from ABRC; the recombinant Agrobacterium strain was prepared according to Part 1.

[0053] (2) Experimental methods ① The above recombinant vector and empty vector were introduced into Agrobacterium tumefaciens EHA105 competent cells by freeze-thaw method. Positive Agrobacterium strains were obtained by double screening with 50 μg / mL kanamycin and 25 μg / mL rifampin.

[0054] ② Transformation of Arabidopsis thaliana using inflorescence infection method: Preparation of OD 600 Agrobacterium tumefaciens solution at 0.8 was used to infect Arabidopsis thaliana inflorescences during the bolting stage. The inflorescences were immersed in the bacterial solution for 30 seconds, then cultured in the dark for 24 hours. After normal culture, the inflorescences were harvested until the seeds matured.

[0055] ③ Resistance screening: T0 generation seeds were sown on 1 / 2 MS medium containing 50 μg / mL hygromycin, vernalized at 4℃ for 3 days, and then cultured at 22℃ for 1 week. Resistant seedlings were selected and transplanted into nutrient soil (humus and vermiculite were thoroughly mixed at a mass ratio of 3:1, and then an appropriate amount of water was added and mixed). About 4 weeks after seedling, positive plants were rapidly screened by GFP fluorescence.

[0056] ④ Identification of homozygous lines: T1 generation plants were identified by PCR, positive plants were selected for self-pollination and T2 generation seeds were harvested; T2 generation seeds were sown, and if the ratio of resistant plants to non-resistant plants was 3:1, the T1 generation plants were determined to be single-copy plants; T2 generation seeds of single-copy plants were sown, and lines consisting entirely of resistant plants were screened to obtain T3 generation homozygous lines, which were finally obtained as SUMO1-WRI1-GFP-OE, GRSX1-WRI1-GFP-OE, and WRI1-GFP-OE homozygous lines.

[0057] (3) Experimental results Western blot analysis showed that ( Figure 1 The three transgenic lines, SUMO1-WRI1-GFP-OE, GRSX1-WRI1-GFP-OE, and WRI1-GFP-OE, all obtained genetically stable T3 generation homozygous lines with 100% homozygosity of the target gene, which can be used for subsequent functional verification. The GFP antibody can detect the target band at the corresponding positions (100bp for WRI1-GFP, and 100-130bp for SUMO1-WRI-GFP and GRXS1-WRI1-GFP).

[0058] 3. Functional identification of transgenic plants under heat stress (1) Experimental materials T3 generation homozygous transgenic lines, empty vector control lines, and wild-type Arabidopsis thaliana.

[0059] (2) Experimental methods Heat stress treatment (e.g.) Figure 2 (as shown) ① Treatment start time: After disinfecting the seeds of each homozygous line and wild type (WT), sow them and culture them at 22℃ for 4 weeks (16h light / 8h dark) until flowering. Mark the siliques pollinated on the same day. When the siliques develop to 8 DAP (Days After Pollination), start the heat stress treatment.

[0060] ② Stress treatment parameters: Marker plants of different genotypes were transferred to a high-temperature climate chamber, with the temperature set at 35℃ (16 h day) / 25℃ (8 h night), and the light intensity and humidity kept consistent with the conventional culture conditions, and the stress was continued for 12 days.

[0061] ③ Recovery culture: After the heat stress treatment, the plants were transferred back to the normal culture conditions (22℃, 16 h light / 8 h dark) and cultured until the siliques were fully mature, to ensure that the seeds could successfully complete the grain filling and dehydration maturation process.

[0062] ④ Control group setup: Plants from each line sown at the same time as the experimental group were set up as the control group. They were kept under normal culture conditions (22℃, 16 h light / 8 h dark) throughout the process, without heat stress treatment, and the other culture conditions were kept the same as the experimental group for subsequent comparative analysis.

[0063] Phenotypic and yield determination: Plant height and number of pods were measured regularly during the stress period; after seed harvest, seed morphology was observed using a stereomicroscope and the shriveling rate was counted; the weight of 1,000 seeds was measured using an electronic balance (3 replicates); the yield per plant was calculated and variance analysis was performed using SPSS software (P<0.05 was considered significant).

[0064] (3) Experimental results Phenotypic analysis: according to Figure 3 It can be seen that under heat stress, plant height and the number of pods increased for all genotypes. However, under the same treatment, there were no developmental phenotypic differences between different genotypes. Regarding seed indicators (Table 1 and...), ... Figure 4 In the control group (22℃), there were no significant differences in seed morphology and plumpness among the different strains. However, in the heat stress group (35℃), the degree of seed wrinkling varied significantly among the different strains. The degree of wrinkling was quantified by the decrease in seed width: the SUMO1-WRI1-OE strain had the plumpest seeds, with a length of 657.24 mm and a width of 383.94 mm, with the width decreasing by only 4.0% compared to normal temperature; the WRI1-OE strain had a length of 564.62 mm and a width of 347.73 mm, with the width decreasing by 11.4% compared to normal temperature; the GRXS1-WRI1-OE strain had a length of 601.00 mm and a width of 323.92 mm, with the width decreasing by 11.5% compared to normal temperature; and the wild-type seeds had the most severe wrinkling, with a length of 551.73 mm and a width of 293.84 mm, with the width decreasing by 18.6% compared to normal temperature.

[0065] Regarding yield and quality indicators, the performance of each strain was as follows: In the heat stress group, the SUMO1-WRI1-OE strain had a thousand-grain weight of 19.6 mg / 1000 grains and a total fatty acid content of 366 μg / mgDW; the WRI1-OE strain had a thousand-grain weight of 16.8 mg / 1000 grains and a total fatty acid content of 334 μg / mgDW; the GRXS1-WRI1-OE strain had a thousand-grain weight of 12.2 mg / 1000 grains and a total fatty acid content of 241 μg / mgDW; and the wild type had a thousand-grain weight of 12.6 mg / 1000 grains and a total fatty acid content of 245 μg / mgDW. It is evident that the thousand-grain weight was 55.6% higher than the wild type and 16.7% higher than the WRI1-OE strain; the total fatty acid content was 49.4% higher than the wild type and 9.6% higher than the WRI1-OE strain, and the grain filling was significantly improved.

[0066] Under heat stress, there were no significant differences in vegetative and reproductive growth phenotypes among different genotypes, indicating that SUMO1-WRI1 fusion does not affect the basic heat tolerance development of plants; combined with seed indicators ( Figure 4 The SUMO1-WRI1-OE strain consistently maintained higher seed plumpness, thousand-seed weight, and total fatty acid content under both normal temperature and heat stress, demonstrating that this fusion can effectively increase seed oil yield and improve seed quality while ensuring normal heat-resistant growth of the plant.

[0067] Table 1: Seed Indicators of Each Genotype The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. The application of SUMO1-WRI1 fusion protein in the cultivation of heat-resistant and high-yielding plants, characterized in that, The amino acid sequence of the SUMO1-WRI1 fusion protein is shown in SEQ ID No.

8.

2. The application according to claim 1, characterized in that, The gene sequence encoding the SUMO1-WRI1 fusion protein is shown in SEQ ID No.

7.

3. The application according to claim 2, characterized in that, Using Arabidopsis cDNA as a template, the full-length SUMO1 gene shown in SEQ ID No. 3 was amplified by PCR with specific primers; using the WRI1 gene sequence shown in SEQ ID No. 1 as a template, the full-length WRI1 gene was amplified by PCR with specific primers. Then, the SUMO1 gene was directly tandemly spliced ​​with the WRI1 gene using overlap extension PCR technology to obtain the encoding gene of the SUMO1-WRI1 fusion protein.

4. The application according to claim 3, characterized in that, The specific primers for amplifying the full-length SUMO1 gene are shown in SEQ ID No. 12 and SEQ ID No. 13; the specific primers for amplifying the full-length WRI1 gene are shown in SEQ ID No. 14 and SEQ ID No.

15.

5. The application according to claim 1, characterized in that, The plants mentioned include Arabidopsis thaliana, wheat, corn, soybean, and rapeseed.

6. A method for cultivating heat-resistant and high-yielding plants, characterized in that, include: A recombinant expression vector containing the SUMO1-WRI1 fusion protein encoding gene was constructed. The recombinant expression vector was introduced into host cells by gene introduction and positive host cells were obtained by screening. The positive host cells were then used to transform the target plant and T0 generation seeds were harvested. The T0 generation seeds were subjected to resistance screening and molecular identification and then continuously self-crossed to obtain genetically stable homozygous transgenic lines, which are heat-resistant and high-yielding transgenic plants.

7. The method for cultivating heat-resistant and high-yielding plants according to claim 6, characterized in that, The gene delivery methods include Agrobacterium-mediated transformation or gene gun transformation.

8. The method for cultivating heat-resistant and high-yielding plants according to claim 6, characterized in that, The method for constructing the recombinant expression vector is as follows: the SUMO1-WRI1 fusion protein encoding gene and the plant expression vector pCAMBIA1300 are subjected to double enzyme digestion, the target fragment and the vector backbone are recovered, ligated by DNA ligase, transformed into competent E. coli cells, and the recombinant expression vector is obtained through resistance screening and sequencing verification.

9. A method for cultivating heat-resistant and high-yielding plants according to claim 6, characterized in that, The target plant was transformed using the inflorescence infection method with positive host cells, wherein the host cells were Agrobacterium competent cells.

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