Wheat salt-tolerant gene tsgt1 and application thereof
By cloning and transforming the wheat salt tolerance gene TaSGT1, the problem of insufficient research on wheat salt tolerance under abiotic stress was solved, and the salt tolerance and agronomic traits of wheat were improved, exhibiting characteristics such as early maturity, short stature, and drought resistance.
Patent Information
- Application Number
- CN202510228083.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2026-02-24
- Estimated Expiration
- 2045-02-28
AI Technical Summary
There is limited research on the role of the wheat SGT1 gene under abiotic stress in existing technologies, especially its role in improving wheat salt tolerance. Furthermore, soil salinization seriously affects crop yields, making it urgent to develop new salt-tolerant crop varieties.
The wheat salt-tolerant gene TaSGT1 was screened out through bioinformatics analysis. Specific primers were designed to clone its coding region, and overexpression and CRISPR gene editing vectors were constructed. The TaSGT1 gene was transformed by infecting wheat callus tissue with Agrobacterium.
It improved the salt tolerance of wheat and showed characteristics such as early maturity, short stature, and drought resistance, thus promoting the improvement of wheat agronomic traits.
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Figure CN119955811B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to a wheat salt tolerance gene. TaSGT1 And its applications. Background Technology
[0002] Soil salinization severely impacts crop yields, and in recent years, with industrial development and rising global temperatures, soil salinization has become increasingly serious, posing a global social concern. Currently, in addition to mitigating soil salinization, developing new salt-tolerant crop varieties has become a very urgent task.
[0003] Cloning salt-tolerant genes in plants and then using transgenic technology to transfer these genes into high-biomass plants to develop efficient new transgenic plant varieties for cultivation in saline-alkali land is a promising technology with broad application prospects.
[0004] Wheat is one of the world's most important food crops, ranking first among cereal crops in terms of planted area, total output, and total trade value. SGT1 The gene-encoded protein typically possesses five functional regions: the TPR region, VR1 region, CS region, VR2 region, and SGS region, which are highly conserved in eukaryotes. Studies have shown that... TaSGT1 Genes in plants play a central role in disease resistance by activating immune responses through interactions with various molecular chaperones. Currently, this is particularly relevant for wheat. SGT1 There is limited research on the role of genes under abiotic stress, particularly regarding improving wheat salt tolerance. TaSGT1 No related research on genes has been reported yet. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention provides a wheat salt tolerance gene. TaSGT1 And its applications.
[0006] This invention first utilizes bioinformatics analysis and protein-protein interaction screening to identify wheat salt tolerance genes. TaSGT1 Then, based on the sequences of Chinese spring wheat in the database, a design was created. TaSGT1 Gene-specific primers were used to clone cDNA from NaCl-treated salt-tolerant wheat variety Shanrong 3 seedlings. TaSGT1 The coding region was used to construct overexpression and CRISPR gene editing vectors, and finally, wheat transgenic wheat with overexpression and gene knockout was obtained by infecting wheat callus tissue with Agrobacterium.
[0007] The first aspect of the present invention is to provide a wheat salt tolerance gene. TaSGT1 The wheat salt tolerance gene mentioned TaSGT1The nucleotide sequence is selected from any one of the sequences shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, or a nucleotide sequence having 75% or more homology with any of the nucleotide sequences shown above. Furthermore, the present invention also provides the above-mentioned wheat salt tolerance gene. TaSGT1 The encoded protein, wherein the protein sequence is selected from any one of the sequences shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, or a protein sequence having the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues to any of the protein sequences shown above.
[0008] In addition, recombinant plasmids containing the above-mentioned nucleotide sequences or pLGY-OE3 vectors, and wheat salt tolerance genes, are also included. TaSGT1 The gene editing vector pYLC-RISPR / Cas9Pubi-B-SGT1 is also a key technology protected by this invention.
[0009] A second aspect of the present invention is that it provides the aforementioned wheat salt tolerance gene. TaSGT1 Applications, particularly the use of wheat salt tolerance genes. TaSGT1 Application in cultivating plants with at least one of the following characteristics: salt tolerance, drought tolerance, early maturity, and dwarfism.
[0010] Preferably, the plant is wheat.
[0011] The beneficial effects of this invention are as follows:
[0012] This invention is the first to clone a wheat salt tolerance gene. TaSGT1 And wheat salt tolerance genes were introduced via Agrobacterium tumefaciens-mediated transformation. TaSGT1 Gene transfer into wheat, experimental results showed that overexpression TaSGT1 The salt tolerance of transgenic wheat was significantly improved, and the overexpression of transgenic wheat showed characteristic traits such as early maturity, short stature, and drought resistance. Attached Figure Description
[0013] Figure 1 In Embodiment 1 of the present invention TaSGT1 Expression analysis diagram of salt-tolerant wheat Shanrong 3 under NaCl treatment;
[0014] Figure 2 In Embodiment 2 of the present invention TaSGT1 Figure showing the agronomic traits analysis of overexpressed transgenic wheat;
[0015] Figure 3 In Embodiment 3 of the present invention TaSGT1Figure showing the salt tolerance phenotype analysis of overexpression transgenic wheat and gene-edited wheat;
[0016] Figure 4 In Embodiment 4 of the present invention TaSGT1 Figure showing the drought resistance phenotype analysis of overexpression transgenic wheat and gene-edited wheat. Detailed Implementation
[0017] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.
[0018] Example 1 Wheat under salt treatment conditions TaSGT1 Gene expression and analysis
[0019] 1.1 Material treatment: The salt-tolerant wheat seeds of Shanrong No. 3 germinated normally. After one week, the endosperm was removed and the seeds were cultured in Hangload medium for another week. The salt stress group was treated with 200 mM NaCl in liquid medium. Wheat roots were collected at 0 h, 0.5 h, 3 h, 6 h and 24 h after treatment and immediately placed in liquid nitrogen for storage.
[0020] 1.2 Extraction of total RNA from wheat: The Trizol extraction kit manufactured by Invitrogen was used for extraction. The specific operation steps were performed according to the instructions provided with the kit.
[0021] 1.3 Synthesis of first-strand cDNA: The total wheat RNA was reverse transcribed using reverse transcriptase to synthesize the first strand of cDNA.
[0022] 1.4 qRT-PCR amplification reaction
[0023] Using cDNA from wheat roots under control and treatment conditions as templates, the CDS sequence of the target gene was submitted to the Wheatomics website for sequence alignment, thereby obtaining the CDS sequence of the gene with high chromosome similarity. Specific fluorescent quantitative PCR primers were designed based on the differences between the three sequences.
[0024] in:
[0025] SEQ ID No. 1 is the cDNA sequence of the TaSGT1A (gene ID: TraesCS3A03G0585800LC) gene;
[0026] SEQ ID No. 2 is the cDNA sequence of the TaSGT1B gene (gene ID: TraesCS3B03G0666200);
[0027] SEQ ID No. 3 is the cDNA sequence of the TaSGT1D (gene ID: TaesCS3D03G0539200) gene.
[0028] The primer sequences are as follows:
[0029] TaSGT1AF: 5′-ATGGCCGCCGCCGCCGCC-3′;
[0030] TaSGT1AR: 5′-GTTGGCATCAGCTACAGCCTCAGTG-3′;
[0031] TaSGT1BF: 5′-TGAAGCTGAGGACCAGGATGGC-3′;
[0032] TaSGT1BR: 5′-CCGGGAAGTTCAATTGAGACACTC-3′;
[0033] TaSGT1DF: 5′-GAGCTCTACACCCAGGCCATT-3′;
[0034] TaSGT1DR:5′-CCTCGAGTCACCAGATGCATAT-3′;
[0035] TaActinF:5′-GTTCCAATCTATGAGGGATACACGC-3′;
[0036] TaActinR:5′-GAACCTCCACTGAGAACAACATTACC-3′.
[0037] 1.5 Reaction system and reaction procedure: The system described in Table 1 below was used for amplification. The reaction procedure was as follows: 94℃ for 30 s; 94℃ for 15 s for 40 cycles, 55℃ for 15 s, and 72℃ for 25 s.
[0038] Table 1 qPCR amplification system
[0039] Components Volume (μL) 2×SYBR qPCR mixture 5 R-TaSGT1F (2 μM) 1 R-TaSGT1R (2 μM) 1 cDNA 1 <![CDATA[ddH2O]]> 2
[0040] The qRT-PCR amplification results obtained using the method in Example 1 are shown in the appendix. Figure 1 .
[0041] Appendix Figure 1 middle, Figure 1 A represents the expression patterns of the three partially homologous TaSGT1 genes under NaCl treatment. Figure 1 B is TaSGT1BGene expression patterns under treatment with the plant hormone abscisic acid (ABA). In the figure, relative expression is the relative expression level (the same applies below).
[0042] Example 2 TaSGT1 Agronomic trait analysis of transgenic lines
[0043] 2.1 TaSGT1 Construction of transgenic wheat vector
[0044] Using homologous recombination, TaSGT1 The copy gene coding sequence on chromosome B was ligated to the pLGY-OE3 plant overexpression vector, and the target fragment was obtained by PCR amplification. Gene-specific primers containing the homologous arm of the pLGY-OE3 vector and the complete CDS were designed using CE Design. BamHI and AvrII restriction sites and protective bases were added to the 5' ends of the upstream and downstream primers.
[0045] The primer sequences are:
[0046] P-SGT1F:5′-tgttatacttctgcagccctaggATGGCCGCCGCCGCCGCC-3′(BamHI);
[0047] P-SGT1R:5′-gaaagctctgagctcggatccTTAATACTCCCACTTCTTGAGCTCC-3′ (AvrⅡ).
[0048] Amplification using this primer pair TaSGT1 The cDNA sequence was obtained, and then the vector pLGY-OE3 was double-digested with restriction endonucleases BamHI and AvrII, respectively. The completely digested vector was separated by electrophoresis on a 1% agarose gel, recovered from the gel, and then compared with the amplified cDNA sequence. TaSGT1 Gene fragments were linked to construct the plant expression vector pLGY-OE3 / TaSGT1 .
[0049] according to TaSGT1 Gene sequences, involving three TaSGT1 Homologous recombination of two gene gRNA sequences with partial homologous genes TaSGT1 The CRISPR gene knockout vector pYLC-RISPR / Cas9Pubi-B- SGT1 .
[0050] 2.2 TaSGT1 Identification of genetically modified wheat
[0051] TaSGT1Identification of overexpression in wheat: RNA was extracted from positive lines and reverse transcribed into cDNA. Using cDNA as a template, qRT-PCR was performed with primers R-TaSGT1F and R-TaSGT1R, following the same system and conditions as in Example 1. The expression level of the transformed gene was detected in different wheat lines. TaSGT1 The expression levels are shown in Figure 2 A, Figure 2 In A, WT is wild-type, OE1 and OE4 are overexpressing transgenic lines, and KO12 and KO20 are gene knockout lines. The same applies to the following.
[0052] Since the overexpression vector pLGY-OE3 exhibits glyphosate resistance in eukaryotes, the overexpressing plants can be identified using a Bar reagent detection kit for glyphosate. Positive lines should show two bands, while negative lines should show one band.
[0053] Figure 2 As can be seen from A, the target gene has been efficiently expressed in the overexpression line. TaSGT1 Identification of gene-edited wheat: PCR amplification TaSGT1 Gene sequence and sequencing were used to identify the editing status of target sites in gene-edited lines. The gene-editing sequencing identification results are as follows: Figure 2 As shown in B, Figure 2 In B, 1D, 3D, 7D, 8D, etc., all refer to the number of missing bases.
[0054] The results showed that different gene-editing lines exhibited editing of the target sequence, with the editing type being the deletion of multiple base sequences.
[0055] 2.3 TaSGT1 Phenotypic identification of agronomic traits in transgenic wheat
[0056] Wheat seeds were disinfected for 15 minutes with a 7.5% sodium hypochlorite solution (including 7.5% sodium hypochlorite and 0.01% Triton-X 100), rinsed 5-6 times with sterile water, and soaked in distilled water for 24 hours. The seeds were then placed in petri dishes lined with filter paper and transplanted into pots with nutrient soil for outdoor cultivation until maturity. During this period, the flowering period, plant height, number of grains per ear, and thousand-grain weight were monitored.
[0057] The results are attached. Figure 2 C- Figure 2 I, where C: heading phenotype of overexpression and transgenic control, D: TaSGT1 Phenotypic results of gene-edited and wild-type wheat at heading date: E: different wheat plant height phenotypes; F: heading date statistics; G: grain number per spike statistics; H: plant height statistics; I: 1000-grain weight statistics.
[0058] Figure 2 C, 2D, and 2F show overexpression. TaSGT1 Promotes vegetative growth and flowering; Figure 2 E and 2H indicate overexpression. TaSGT1 It can reduce plant height; Figure 2 G indicates knockout. TaSGT1 It can increase the number of grains per ear; Figure 2 I showed knockout TaSGT1 It can increase the weight of a thousand grains.
[0059] Example 3 TaSGT1 Identification of salt tolerance
[0060] Wheat cultivation: Seeds of T2 generation homozygous overexpression transgenic wheat lines and gene-edited homozygous lines were disinfected with 7.5% sodium hypochlorite solution (including 7.5% sodium hypochlorite and 0.01% Triton-X 100) for 15 minutes, then rinsed 5-6 times with sterile water, soaked in distilled water for 24 hours, and then placed in petri dishes lined with filter paper. Five days later, they were transferred to 96-well hydroponic boxes. When they reached the two-leaf-one-heart stage, they were treated with NaCl. The growth conditions were 22±2℃, 16 / 8 h photoperiod, and light intensity of 30-40 μmol·m. -2 ·s -1 .
[0061] NaCl treatment: For germination phenotypic identification, wheat seeds were initially soaked in 100 mM NaCl until obvious phenotypic differences appeared. The length of the wheat taproot was photographed and recorded. For seedling phenotypic identification, when the wheat reached the two-leaf-one-heart stage, it was first treated with 100 mM NaCl for one day, and then treated with 200 mM NaCl. After 3 days, obvious phenotypic differences appeared. After that, it was restored to normal hydroponics. The phenotypic characteristics were photographed in a timely manner, and the fresh weight of the aboveground parts and the proportion of wilted leaf area were recorded.
[0062] Appendix Figure 3 The results showed that the growth of overexpressed transgenic wheat differed from that of wild-type lines. Figure 3In Figure 1, A represents different salt tolerance phenotypes of wheat during germination; B represents the root length statistics of different wheat primary roots in Figure A; C represents wheat phenotypes under normal culture conditions; D represents the salt tolerance phenotypes of different wheat strains treated with 200 mM NaCl for 3 days in 2-week-old seedlings; E represents the phenotype of wheat cultured in normal medium for 1 day in Figure D; F represents the fresh weight of wheat aboveground shoots in Figure E; G represents the fresh weight of wheat roots in Figure E; and H represents the root phenotype of wheat in Figure E.
[0063] from Figure 3 The results of A-3B show that the root length of gene-edited wheat was significantly shorter than that of wild type during the germination period; Figure 3 As observed in C-3G, the aboveground fresh weight of transgenic wheat lines overexpressing during the seedling stage was higher than that of the wild type, and the proportion of wilted leaf area was lower than that of the wild type.
[0064] Example 4 TaSGT1 Drought resistance assessment
[0065] Wheat cultivation: As described in Example 3, when the wheat reached the two-leaf-one-heart stage, it was treated with 20% PEG to simulate drought stress.
[0066] 20% PEG treatment: Significant phenotypic differences were observed three days after 20% PEG treatment. After one day of normal hydroponics, the phenotypic characteristics were photographed and recorded. The aboveground fresh weight and the proportion of wilted leaf area after dry treatment were also statistically analyzed. Results are attached. Figure 4 As shown.
[0067] Appendix Figure 4 In the figure, A: Phenotypes of different wheat lines under normal culture conditions; B: Phenotypes of different wheat lines under 20% PEG treatment; C: Aboveground phenotypes after 3 days of PEG treatment followed by 1 day of rehydration; D: Root phenotypes after 3 days of PEG treatment followed by 1 day of rehydration; E: Aboveground phenotype statistics of wheat in Figure C; F: Rate of wilt area statistics of wheat in Figure C; G: Fresh weight statistics of wheat roots in Figure D; H: Phenotypes of different wheat before drought treatment; I: Phenotypes of different wheat after 3 weeks of drought treatment followed by 1 day of rehydration; J: Statistical analysis of wilted area of wheat in Figure I.
[0068] The above results indicate that the aboveground fresh weight of the overexpression line was higher than that of the wild type, and the proportion of wilted leaf area was lower than that of the wild type. Figure 4B, 4C, 4E, 4F) indicate overexpression. TaSGT1 Genes can significantly improve the drought resistance of wheat.
Claims
1. Wheat salt tolerance genes TaSGT1 Its application in breeding wheat with salt and drought tolerance traits is characterized by, The wheat salt tolerance gene mentioned TaSGT1 The nucleotide sequence is selected from any one of the sequences shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3.