Wheat GPI-anchored protein gene TaLLG3 and its application in resistance to stripe rust

Abstract

This invention belongs to the field of crop genetic engineering technology, specifically relating to the wheat GPI-anchored protein gene. TaLLG3 And its application in resistance to stripe rust. This invention utilizes overexpression and gene editing techniques to target the wheat GPI-anchored protein gene. TaLLG3 The function of the wheat GPI-anchored protein gene was analyzed and verified, and it was found that overexpression of the wheat GPI-anchored protein gene... TaLLG3 It can enhance wheat's resistance to stripe rust pathogen; and target wheat GPI-anchored protein genes. TaLLG3 After gene editing, its resistance to stripe rust pathogen was significantly weakened. This indicates that the wheat GPI-anchored protein gene... TaLLG3 It plays a positive regulatory role in the immune response to wheat stripe rust, providing genetic resources for breeding wheat varieties resistant to stripe rust.

Description

Technical Field

[0001] This invention belongs to the field of crop genetic engineering technology, specifically relating to the wheat GPI-anchored protein gene. TaLLG3 And its application in combating stripe rust. Background Technology

[0002] Wheat stripe rust is caused by the fungus *Strombus styracifolius* (…). Pucciniastriiformisf.sp.tritici This fungal disease is caused by *Striga styracifolium*, named for the bright yellow, striped rust spots that appear on the leaves when the disease occurs. *Striga styracifolium* is an obligate parasitic fungus that relies on living wheat tissue to complete its complex life cycle, which includes multiple stages such as urediniospores and teliospores. Urediospores are bright yellow, oval-shaped, and have fine spines on their surface, allowing them to be dispersed over long distances by air currents; teliospores are dark brown and primarily serve to overwinter. This pathogen exhibits a strong ability to vary, and multiple physiological races have been identified, leading to a high risk of loss of resistance in varietals.

[0003] During wheat cultivation, the wheat-specific strain of *Stripetra rust* (…) Puccinia striiformis f. sp. tritici, Pst Wheat stripe rust, caused by [unspecified fungal infection], is one of the most serious fungal diseases affecting wheat production. Research and practical experience show that, in addition to reasonable control measures and the rational use of pesticides, breeding and promoting disease-resistant wheat varieties is the most economical, effective, and environmentally friendly control measure. However, disease-resistant varieties are easily affected by new variant races and lose their resistance to stripe rust. Therefore, the discovery of genes related to wheat stripe rust resistance, research on resistance mechanisms, and the breeding of disease-resistant varieties are crucial for the control of wheat stripe rust. Summary of the Invention

[0004] The purpose of this invention is to provide wheat GPI-anchored protein genes. TaLLG3 Its application in resistance to stripe rust is positively regulating the wheat GPI-anchored protein gene. TaLLG3 It can effectively improve crop resistance to stripe rust and provide genetic resources and technical support for the breeding of stripe rust-resistant crop varieties.

[0005] This invention provides a wheat GPI-anchored protein gene. TaLLG3 The application of wheat GPI-anchored protein TaLLG3, or its encoded protein, in regulating crop resistance to stripe rust and / or breeding stripe rust-resistant crop varieties; the amino acid sequence of said wheat GPI-anchored protein TaLLG3 is shown in SEQ ID NO:1.

[0006] Preferably, the application includes: positively regulating the wheat GPI-anchored protein gene. TaLLG3 Or the application of wheat GPI-anchored protein TaLLG3 in improving crop resistance to stripe rust, and / or negative regulation of the wheat GPI-anchored protein gene. TaLLG3Or the application of wheat GPI-anchored protein TaLLG3 in reducing crop resistance to stripe rust.

[0007] This invention also provides the gene that positively regulates wheat GPI-anchored protein as described in the above technical solution. TaLLG3 Alternatively, the application of biomaterials expressing the wheat GPI-anchored protein TaLLG3 in improving crop resistance to stripe rust, wherein the wheat GPI-anchored protein gene... TaLLG3 The protein encodes wheat GPI-anchored protein TaLLG3, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] Preferably, the biomaterial includes any one or more of the following:

[0009] 1) Contains wheat GPI-anchored protein gene TaLLG3 Recombinant expression vectors;

[0010] 2) Contains wheat GPI-anchored protein gene TaLLG3 Engineered bacteria;

[0011] 3) Engineered bacteria containing the recombinant expression vector described in 1).

[0012] Preferably, the initial strain of the engineered bacteria is Agrobacterium.

[0013] This invention also provides the negative regulation of wheat GPI-anchored protein gene as described in the above technical solution. TaLLG3 Alternatively, the application of biomaterials expressing the wheat GPI-anchored protein TaLLG3 in reducing crop resistance to stripe rust, wherein the wheat GPI-anchored protein gene... TaLLG3 The protein encodes wheat GPI-anchored protein TaLLG3, the amino acid sequence of which is shown in SEQ ID NO:1.

[0014] Preferably, the biomaterial includes any one or more of the following:

[0015] A) Knock out the wheat GPI-anchored protein gene TaLLG3 Nucleic acid molecules;

[0016] B) A recombinant expression vector containing the nucleic acid molecules described in A);

[0017] C) Engineered bacteria containing the nucleic acid molecules described in A);

[0018] D) Engineered bacteria containing the recombinant expression vector described in B).

[0019] Preferably, the nucleic acid molecule includes gRNA1 and gRNA2, as shown in SEQ ID NO:15 and SEQ ID NO:16, respectively.

[0020] Preferably, the crop includes wheat.

[0021] The present invention also provides a method for breeding wheat varieties, wherein the wheat varieties include stripe rust-resistant wheat varieties or wheat mutants with reduced stripe rust resistance;

[0022] The method for breeding the stripe rust-resistant wheat variety includes the following steps: increasing the expression level of wheat GPI-anchored protein TaLLG3 in recipient wheat or promoting the expression of wheat GPI-anchored protein gene in recipient wheat. TaLLG3 The expression was used to obtain the stripe rust-resistant wheat variety;

[0023] The method for cultivating wheat mutants with reduced stripe rust resistance includes the following steps: reducing the expression level of wheat GPI-anchored protein TaLLG3 in recipient wheat or inhibiting the wheat GPI-anchored protein gene in recipient wheat. TaLLG3 The expression of [the substance] was used to obtain the wheat mutant with reduced resistance to stripe rust;

[0024] The wheat GPI-anchored protein gene TaLLG3 The protein encodes wheat GPI-anchored protein TaLLG3, the amino acid sequence of which is shown in SEQ ID NO:1.

[0025] Beneficial effects:

[0026] This invention provides a wheat GPI-anchored protein gene. TaLLG3 The application of the wheat GPI-anchored protein TaLLG3, or its encoded protein, in regulating crop resistance to stripe rust and / or breeding stripe rust-resistant crop varieties. This invention utilizes overexpression and gene editing techniques to target the wheat GPI-anchored protein gene. TaLLG3 The function of the wheat GPI-anchoring protein TaLLG3 was analyzed and validated, and it was found that overexpression of the wheat GPI-anchoring protein gene... TaLLG3 It can enhance wheat's resistance to stripe rust pathogen; and target the wheat GPI-anchored protein gene. TaLLG3 After gene editing, its resistance to stripe rust pathogen was significantly weakened. This indicates that the wheat GPI-anchored protein gene... TaLLG3 It plays a positive regulatory role in the immune response to wheat stripe rust, providing genetic resources for breeding wheat varieties resistant to stripe rust.

[0027] Based on this, the present invention creates an overexpression of the GPI-anchored protein gene. TaLLG3A transgenic wheat variety resistant to stripe rust exhibits significant resistance to the stripe rust fungus. Therefore, the GPI-anchored protein gene of this wheat can be utilized. TaLLG3 The creation of stripe rust-resistant strains provides excellent wheat material for the breeding of stripe rust-resistant varieties. Compared with traditional disease-resistant breeding techniques, plant disease-resistant genetic engineering technology can overcome reproductive isolation and incompatibility between species, achieving targeted improvement of target traits in a shorter period of time, and providing crops with more comprehensive, continuous, and broad-spectrum protection. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0029] Figure 1 The wheat GPI-anchored protein gene in Example 2 TaLLG3 A schematic diagram of expression profile analysis, where, compared to 0 h infection time, express P <0.05, express P <0.01;

[0030] Figure 2 Example 3: Wheat GPI-anchored protein gene TaLLG3 Phenotypic identification results of wheat plants L2, L6, L10, and Fielder inoculated with rust fungus pathotype CYR32 14 days after expression, showing the results compared to the Fielder wild type. express P <0.01, *express P <0.001;

[0031] Figure 3 The wheat GPI-anchored protein gene stably inherited in Example 4 TaLLG3 Phenotypic identification results of wheat plants TaLLG3-ko#L3, L4, L5, L10 knocked out and Fielder plants inoculated with stripe rust physiological race CYR23 14 days later. Compared with the Fielder wild type, express P <0.001;

[0032] Figure 4 The wheat GPI-anchored protein gene in Example 5 TaLLG3 The diagram shows the bursting of reactive oxygen species around the infection point of the knockout plant after it was infected with stripe rust, as well as the area statistics. In this diagram, SV represents the substomatal vesicles, and H2O2 represents reactive oxygen species. express P <0.05, express P <0.001;

[0033] Figure 5 The wheat GPI-anchored protein gene in Example 5 TaLLG3 A statistical chart showing the area of ​​stripe rust mycelial infection and the length of mycelial hyphae in knocked-out plants. express P <0.05, express P <0.01, express P <0.001. Detailed Implementation

[0034] This invention provides a wheat GPI-anchored protein gene. TaLLG3 The application of wheat GPI-anchored protein TaLLG3, or its encoded protein, in regulating crop resistance to stripe rust and / or breeding stripe rust-resistant crop varieties; the amino acid sequence of said wheat GPI-anchored protein TaLLG3 is shown in SEQ ID NO:1.

[0035] SEQ ID NO:1 is shown below.

[0036] 5'-MALTRLFISHFPTAVLAGLASASASPFLSDSLFQGSTGSTGSLLQTKNDCPMSFETQNYTILTNKCKRPQYPPTECCDAFKEFACPFAAYINNQSTNCADTMFSYIDFHGYPKGLFADECLKGKEGVSCEGIPAVDTGVPSGGRQVQGVSRPLVVLLCGLGALLFP-3'.

[0037] As one embodiment, the wheat GPI-anchored protein gene of the present invention TaLLG3 The nucleotide sequence is shown in SEQ ID NO:2.

[0038] SEQ ID NO:2 is shown below.

[0039] 5’-ATGGCGCTGACCCGGCTGTTCATCTCCCACTTCCCCACCGCCGTCCTGGCCGGACTCGCCTCCGCCTCCGCGTCGCCCTTCCTGTCTGACAGCTTATTCCAGGGCAGCACCGGATCGACGGGGAGGAGCTTGCTGCAGACCAAGAATGACTGCCCTATGAGCTTCGAGACCCAGAACTACACGATCCTCACAAACAAGTGCAAAAGGCCACAATACCCTCCTACCGAATGTTGTGATGCTTTCAAGGAATTTGCGTGCCCATTTGCCGCGTACATCAACAACCAGAGCACTAACTGTGCAGACACAATGTTCAGCTACATCGACTTCCATGGCTACCCAAAAGGCCTGTTCGCCGACGAGTGCCTAAAAGGAAAGGAAGGGGTTTCTTGCGAAGGCATCCCAGCGGTAGACACCGGCGTGCCCAGTGGCGGGCGACAAGTTCAAGGGGTTTCGCGTCCTTTGGTTGTGCTCCTGTGTGGACTAGGAGCATTGTTGTTCCCTTGA-3’。

[0040] During pathogen infection, plants release a large number of signaling molecules, such as pathogen / microbe-associated molecular patterns (P / MAMPs) and damage-associated molecular patterns (DAMPs). Plant plasma membrane-localized pattern recognition receptors (PRRs) often sense these signaling molecules, activating immunity and conferring host resistance to pathogens. PRRs are plasma membrane-localized receptor kinases (RKs) or receptor-like proteins (RLPs). PRRs typically sense pathogen / microbe-associated molecular patterns (PAMPs / MAMPs) and damage-associated molecular patterns, which are host-derived molecules released during pathogen attack or cell damage. Sensing DAMPs, in addition to the non-self-surveillance enabled by PAMP recognition, allows plant cells to indirectly monitor a wider variety of pathogens and expand responses triggered solely by PAMP sensing. Wheat GPI (glycosylphosphatidylinositol) anchored proteins are a class of functional proteins anchored to the cell membrane surface via GPI structures and play an important role in plant immune responses. GPI-anchored proteins can act as membrane-localized receptors or co-receptors, recognizing conserved molecular patterns in fungi (such as chitin) and activating PTI (PAMP-Triggered Immunity) immune responses.

[0041] As one implementation, the application includes: positively regulating the wheat GPI-anchored protein gene. TaLLG3 Or the application of wheat GPI-anchored protein TaLLG3 in improving crop resistance to stripe rust, and / or negative regulation of the wheat GPI-anchored protein gene. TaLLG3 Alternatively, the application of wheat GPI-anchored protein TaLLG3 in reducing crop resistance to stripe rust. As another embodiment, improving crop resistance to stripe rust can be achieved by enhancing crop resistance to stripe rust; as another embodiment, improving crop resistance to stripe rust can be achieved by enhancing crop resistance to *Striga styracifolium*, particularly enhancing crop resistance to the wheat-specific form of *Striga styracifolium*. As another embodiment, reducing crop resistance to stripe rust can be achieved by weakening the pathogenicity and / or growth and development ability of *Striga styracifolium*, further by shortening the hyphal length of *Striga styracifolium* and / or the infection area of ​​*Striga styracifolium* on wheat. As one embodiment, the crop includes gramineous crops; as another embodiment, the gramineous crop is wheat. As one embodiment, the stripe rust is *Striga styracifolium*-induced stripe rust; as another embodiment, the *Striga styracifolium* is the wheat-specific form of *Striga styracifolium*.

[0042] As one implementation method, the positive regulation method of the present invention is overexpression and / or hyperexpression; the negative regulation method is knockout and / or knockdown.

[0043] This invention also provides the gene that positively regulates wheat GPI-anchored protein as described in the above technical solution. TaLLG3 Alternatively, the application of biomaterials expressing the wheat GPI-anchored protein TaLLG3 in improving crop resistance to stripe rust, wherein the wheat GPI-anchored protein gene... TaLLG3 The amino acid sequence of the wheat GPI-anchored protein TaLLG3 is shown in SEQ ID NO:1.

[0044] In one embodiment, the biomaterial includes any one or more of the following:

[0045] 1) Contains wheat GPI-anchored protein gene TaLLG3 Recombinant expression vectors;

[0046] 2) Contains wheat GPI-anchored protein gene TaLLG3 Engineered bacteria;

[0047] 3) Engineered bacteria containing the recombinant expression vector described in 1).

[0048] As one implementation method, the wheat GPI-anchored protein gene TaLLG3 The nucleotide sequence is shown in SEQ ID NO:2. As one embodiment, it is used to amplify the wheat GPI-anchored protein gene. TaLLG3 The forward and reverse primers are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. In one embodiment, the initial vector in the recombinant expression vector is a plasmid vector, or it can be a pANIC6E plasmid vector. This invention does not specifically limit the construction method of the recombinant expression vector; conventional overexpression vector construction methods in the art can be used, such as the homologous recombination Gateway. In one embodiment, the initial strain in the engineered bacteria is Agrobacterium. This invention does not specifically limit the construction method of the engineered bacteria; conventional engineered bacteria construction methods in the art can be used. In one embodiment, the organism is a gramineous crop; in another embodiment, the gramineous plant is wheat. The limitation of the stripe rust disease described in this invention is the same as in the above technical solutions and will not be repeated.

[0049] This invention also provides the negative regulation of wheat GPI-anchored protein gene as described in the above technical solution. TaLLG3 Alternatively, the application of biomaterials expressing the wheat GPI-anchored protein TaLLG3 in reducing crop resistance to stripe rust, wherein the wheat GPI-anchored protein gene... TaLLG3The amino acid sequence of the wheat GPI-anchored protein TaLLG3 is shown in SEQ ID NO:1.

[0050] In one embodiment, the biomaterial includes any one or more of the following:

[0051] A) Knock out the wheat GPI-anchored protein gene TaLLG3 Nucleic acid molecules;

[0052] B) A recombinant expression vector containing the nucleic acid molecules described in A);

[0053] C) Engineered bacteria containing the nucleic acid molecules described in A);

[0054] D) Engineered bacteria containing the recombinant expression vector described in B).

[0055] In one embodiment, the nucleic acid molecule includes gRNA1 and gRNA2, as shown in SEQ ID NO:15 and SEQ ID NO:16, respectively. In one embodiment, the initial vector in the recombinant expression vector is a Cas9 expression vector, or pCas9. In one embodiment, the initial strain of the engineered bacteria is Agrobacterium. This invention does not specifically limit the construction method of the recombinant expression vector and the engineered bacteria, and uses conventional methods for constructing recombinant expression vectors and engineered bacteria in the art. In one embodiment, the organism is a gramineous crop; in another embodiment, the gramineous plant is wheat. The definition of stripe rust in this invention is the same as in the above technical solutions and will not be repeated.

[0056] The present invention also provides a method for breeding wheat varieties, wherein the wheat varieties include stripe rust-resistant wheat varieties or wheat mutants with reduced stripe rust resistance;

[0057] The method for breeding the stripe rust-resistant wheat variety includes the following steps: increasing the expression level of wheat GPI-anchored protein TaLLG3 in recipient wheat or promoting the expression of wheat GPI-anchored protein gene in recipient wheat. TaLLG3 The expression was used to obtain the stripe rust-resistant wheat variety;

[0058] The method for cultivating wheat mutants with reduced stripe rust resistance includes the following steps: reducing the expression level of wheat GPI-anchored protein TaLLG3 in recipient wheat or inhibiting the wheat GPI-anchored protein gene in recipient wheat. TaLLG3 The expression of [the substance] was used to obtain the wheat mutant with reduced resistance to stripe rust;

[0059] The wheat GPI-anchored protein gene TaLLG3The amino acid sequence of the wheat GPI-anchored protein TaLLG3 is shown in SEQ ID NO:1.

[0060] As one implementation method, the method for obtaining the stripe rust-resistant wheat variety of the present invention may be: introducing the wheat GPI-anchored protein gene into recipient wheat. TaLLG3 As another implementation, the wheat GPI-anchored protein gene is introduced into recipient wheat. TaLLG3 The recombinant expression vector described in the above technical solution can be transferred into recipient wheat to increase the expression level of wheat GPI-anchored protein TaLLG3 in recipient wheat or promote the expression of wheat GPI-anchored protein gene in recipient wheat. TaLLG3 The specific steps for expressing the recombinant expression vector can be as follows: introducing the recombinant expression vector into Agrobacterium to obtain engineered bacteria, and then transforming the engineered bacteria into wheat embryos to obtain the stripe rust-resistant wheat variety.

[0061] As one implementation method, the method for obtaining the wheat mutant with reduced stripe rust resistance according to the present invention may be: introducing the wheat GPI-anchoring protein gene knocked out into recipient wheat. TaLLG3 Nucleic acid molecules; as another embodiment, the introduction of the wheat GPI-anchored protein gene knocked out into recipient wheat. TaLLG3 The method for nucleic acid molecules can be to include the above-mentioned technical solution containing the wheat GPI-anchored protein gene knocked out. TaLLG3 The recombinant expression vector of the nucleic acid molecule was transferred into recipient wheat to reduce the expression level of wheat GPI-anchored protein TaLLG3 in recipient wheat or to inhibit the expression of wheat GPI-anchored protein gene in recipient wheat. TaLLG3 The specific steps for expression can be as follows: [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] TaLLG3 The recombinant expression vector of the nucleic acid molecule was introduced into Agrobacterium to obtain engineered bacteria, and the engineered bacteria were transformed into wheat embryos to obtain wheat mutants with reduced stripe rust resistance.

[0062] This invention validates the wheat GPI-anchored protein gene. TaLLG3 Expression profiles of the wheat GPI-anchored protein gene under wheat stripe rust infection were obtained. TaLLG3 Expression induced by infection with wheat stripe rust pathogen. Wheat GPI-anchored protein gene. TaLLG3 It plays a positive regulatory role in the immune response against wheat stripe rust, and overexpression of the wheat GPI-anchored protein gene is involved. TaLLG3 Enhance wheat's resistance to stripe rust pathogen.

[0063] This invention, through the identification of disease incidence in wheat plants overexpressing GPI-anchored protein genes after infection with stripe rust, discovered that the wheat GPI-anchored protein gene... TaLLG3Overexpressing plants showed significantly lower disease incidence and spore biomass compared to wild-type plants, indicating that overexpression of the wheat GPI-anchored protein gene is a significant improvement. TaLLG3 This enhances wheat's resistance to stripe rust. Wheat GPI-anchored protein gene. TaLLG3 The mycelial length and infection area of ​​stripe rust fungus in the knockout plants were significantly higher than those in the wild type, while the area of ​​reactive oxygen species accumulation was significantly lower in the knockout plants, indicating that the wheat GPI-anchored protein gene was knocked out. TaLLG3 The growth and development of stripe rust fungus were promoted and its pathogenicity was significantly enhanced in transgenic wheat, indicating that the wheat GPI-anchored protein gene... TaLLG3 It positively regulates wheat resistance to stripe rust, providing genetic resources for breeding stripe rust-resistant wheat varieties.

[0064] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0065] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials can be purchased through conventional commercial channels; and unless otherwise specified, the percentages in the following embodiments refer to mass percentages.

[0066] Example 1

[0067] Wheat GPI-anchored protein gene TaLLG3 To obtain it, follow these steps:

[0068] Design primers for full-length gene amplification, including forward primers. TaLLG3 -F (5'-ATGGCGCTGACCCGGCTG-3', SEQ ID NO:3) and reverse primer TaLLG3 -R (5'-TCAAGGGAACAACAATGC-3', SEQ ID NO:4). The wheat GPI-anchored protein gene was amplified using cDNA from wheat Fielder plants (provided by Northwest A&F University) as a template. TaLLG3 Sequencing was performed to obtain the wheat GPI-anchored protein gene. TaLLG3 The nucleotide sequence is shown in SEQ ID NO:2, and the corresponding amino acid sequence of wheat GPI-anchored protein TaLLG3 is shown in SEQ ID NO:1.

[0069] Example 2

[0070] Wheat GPI-anchored protein gene TaLLG3 The expression profile analysis steps are as follows:

[0071] Based on Example 1, real-time quantitative PCR technology was used to analyze the wheat GPI-anchored protein gene. TaLLG3 Expression profile under stripe rust infection.

[0072] During the “two-leaf-one-heart” stage of the wheat variety Fielder, the two leaves were inoculated with the stripe rust-friendly race CYR31 and the incompatible race CYR23 (CYR23 and CYR31 strains were bred by the Plant Immunology Research Group of the National Key Laboratory of Crop Stress Resistance and High-Efficiency Production at Northwest A&F University).

[0073] Wheat leaves were sampled at 0, 6, 12, 24, 48, and 72 hours post-infection. Before sampling, the leaves were moistened and spores on the leaf surface were wiped off with a paper towel. The collected samples were placed in 2 mL centrifuge tubes and then into liquid nitrogen. RNA was extracted using a rapid universal plant RNA extraction kit (purchased from Beijing Huayueyang Biotechnology Co., Ltd.). cDNA was obtained by reverse transcription using a reverse transcription kit (purchased from Thermo Fisher Scientific). Using the wheat elongation factor gene as an internal reference, real-time quantitative PCR was performed using Thermo Fisher Scientific's QuantStudio 1 real-time quantitative PCR system, utilizing the wheat GPI-anchored protein gene. TaLLG3 Specific fragment quantitative primer analysis TaLLG3 The expression of .

[0074] Among them, wheat GPI-anchored protein gene TaLLG3 The primer sequences for specific fragment quantification and the primer sequence for the wheat elongation factor gene (internal reference gene) are as follows:

[0075] Internal reference primer: TaEF -F:5'-TGGTGTCATCAAGCCTGGTATGGT-3' (SEQ ID NO:5), TaEF -R: 5'-ACTCATGGTGCATCTCAACGGACT-3' (SEQ ID NO: 6),

[0076] Wheat GPI-anchored protein gene TaLLG3 Specific fragment quantification primers: TaLLG3 -qRT-F: 5'-TGAGTGGTGTGATTCCCTGG-3' (SEQ ID NO:7), TaLLG3 -qRT-R: 5'-CGGATGTGCACTCTCCCAAT-3' (SEQ ID NO: 8).

[0077] The reaction conditions were: 95℃ pre-denaturation for 1 min; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; and finally 72℃ extension for 10 min.

[0078] Wheat GPI-anchored protein gene ​ Expression levels at different infection times in wheat infected with stripe rust are as follows: ​ As shown, CYR23 is an incompatible race of stripe rust fungus, while CYR31 is an compatible race of stripe rust fungus.

[0079] Depend on ​ It can be concluded that: wheat GPI-anchored protein gene ​ It was induced to express in both compatible and incompatible wheat races, with the highest expression level at 12 hours in the incompatible system and at 48 hours in the compatible system.

[0080] Example 3

[0081] Wheat GPI-anchored protein gene ​ Cultivation and disease resistance identification of overexpression plants.

[0082] (1) Wheat GPI-anchored protein gene ​ The wheat GPI-anchored protein gene was constructed in the plant transgenic expression vector pANIC6E. ​ The sequence is shown in SEQ ID NO:2, and the specific steps are as follows:

[0083] use ​ Primer construction for overexpression vector: ​ -6E-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTACCCATACGACGTCCCAGACTACGCTATGGCGCTGACCCGGCTG-3' (SEQ ID NO:9) and ​ -6E-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAAGCGTAGTCTGGGACGTCGTATGGGTATCAAGGGAACAACAATGC-3' (SEQ ID NO:10) Amplified full-length gene sequence;

[0084] The amplified fragment was constructed into the expression vector pANIC6E via the homologous recombination Gateway reaction to obtain the TaLLG3-pANIC6E overexpression vector.

[0085] (2) Obtaining genetically modified wheat

[0086] Obtained through Agrobacterium-mediated genetic transformation ​ -OE transgenic overexpression plants.

[0087] (3) PCR verification was performed ​-OE transgenic positive detection.

[0088] Extracting T1 generation ​ DNA from -OE transgenic overexpressing plants was amplified by PCR using universal detection primers for pANIC6E (6E-F: 5'-TTAGCCCTGCCTTCATACG-3', SEQ ID NO: 11 and 6E-R: 5'-CTATCATAGATGTCGCTATAAACC-3', SEQ ID NO: 12). The PCR products were then detected by 1.5% agarose gel electrophoresis. The results are shown below. ​ As shown.

[0089] Depend on ​ We can conclude that: ​ -OE transgenic overexpression plants were successfully constructed.

[0090] (5) Genetically modified wheat ​ - The phenotypic identification of stripe rust fungi by OE is performed as follows:

[0091] Three strains (L2, L6, and L10) from the T1 generation were selected. When they reached the "two leaves and one bud" stage, the two leaves were inoculated with the stripe rust-compatible race CYR32. The inoculated plants were then placed in a humidity chamber in darkness for 24 hours, followed by a photoperiod growth incubator with 16°C light for 16 hours and 11°C darkness for 8 hours. Fourteen days after inoculation, sporulation on the leaves was observed, and phenotypic identification results are as follows: ​ As shown, it was found ​ Transgenic overexpression plants showed enhanced resistance to stripe rust and reduced spore biomass.

[0092] In addition, DNA was extracted from the stripe rust inoculation site on wheat overexpression material leaves, diluted to 400 ng / μL, and used as a template to analyze the stripe rust internal reference gene. ​ primers ( ​ -F: 5'-TTCGCCGTCCGTGATATGAGACAA-3', SEQ ID NO: 13; ​ -R: 5'-ATGCGTATCATGGTGGTGGAGTGA-3', SEQ ID NO:14) and wheat internal reference gene ( ​ () ​ -F: 5'-TGGTGTCATCAAGCCTGGTATGGT-3', SEQ ID NO: 5; ​ The quantitative primers (-R: 5'-ACTCATGGTGCATCTCAACGGACT-3, SEQ ID NO: 6) were used to detect the relative biomass of stripe rust fungus in wheat leaves by qRT-PCR. The results are as follows: ​As shown.

[0093] It can be seen that wheat GPI-anchored protein genes ​ Transgenic overexpression plants showed enhanced resistance to stripe rust.

[0094] Example 4

[0095] Wheat GPI-anchored protein gene ​ Cultivation and phenotypic identification of knockout plants

[0096] (1) Wheat GPI-anchored protein gene ​ The cultivation steps for knocking out plants are as follows:

[0097] The first step, gene editing target design: based on wheat ​ Based on the genome sequence of the gene and the target design requirements of CRISPR-Cas9 technology, two specific targets for wheat were designed. ​ gRNA of genes;

[0098] ​ -gRNA1: 5'-AAGTTGACCGTGGACCC-3' (SEQ ID NO: 15);

[0099] ​ -gRNA2: 5'-GAGCACTAACTGTGCAG-3' (SEQ ID NO: 16).

[0100] The second step involved constructing gRNA tandemly into the gene editing vector pCas9 via in vitro bridging and transforming it into Agrobacterium strain EHA105. The transformation process is as follows:

[0101] Using T4 DNA ligase to connect gRNA2 and ​ I. The intermediate vector psgRNA (a commercially available vector, which can be found on AddGene, website: https: / / www.addgene.org / browse / sequence / 235209 / ) was ligated overnight at 16°C; the ligation product was transformed into E. coli DH5α competent cells, and positive single colonies were detected using ENTRY4-F / R primers. Plasmids were extracted from the positive single colonies by shaking and sent for sequencing.

[0102] The ENTRY4-F / R primers are as follows:

[0103] ENTRY4-F: 5'-GCGTTTCTACAAACTCTTCCTG-3' (SEQ ID NO: 17);

[0104] ENTRY4-R: 5'-TGGGTCTAGATATCTCGAGTG-3' (SEQ ID NO: 18).

[0105] Using T4 DNA ligase, gRNA1 was constructed into... ​ I. The constructed recombinant plasmid was introduced into DH5α competent Escherichia coli cells onto the linearized intermediate vector psgRNA. Positive transformants were screened and verified by monoclonal PCR. After amplification by liquid culture, plasmid DNA was extracted.

[0106] The intermediate vector plasmid was constructed into the gene editing vector pCas9 (a commercially available vector, searchable on the website of Shanghai Novopro Biotechnology Co., Ltd., https: / / www.novopro.cn / vector / V12286) using the LR reaction. The pCas9 vector was then transformed into *E. coli*. Positive single colonies were detected using NOS-F / R primer PCR. Plasmids were extracted from the positive single colonies by shaking and then sequenced, yielding wheat... ​ Gene editing vector CRSPR-Cas9-gRNAs.

[0107] The NOS-F / R primers are as follows:

[0108] NOS-F: 5'-AAGCACATACGTCAGAAACATTAT-3' (SEQ ID NO: 19);

[0109] NOS-R: 5'-TGGGTGAGATTCCTTGAAGTTGAGTA-3' (SEQ ID NO: 20);

[0110] The third step involved Agrobacterium-mediated genetic transformation of wheat callus: the Agrobacterium-mediated CRSPR-Cas9-gRNAs transformed individuals constructed in the second step were infecting the immature embryos of the recipient variety Fielder, resulting in wheat with targeted editing capabilities. ​ Plants that have been genetically modified were identified, and positive plants were identified through resistance screening and PCR testing.

[0111] The fourth step involved testing and sequencing the progeny of transgenic positive plants to obtain... ​ Mutant plants successfully edited on B copy ​ KO.

[0112] (2) Genetically modified wheat ​ The phenotypic identification of stripe rust fungi by KO is as follows:

[0113] Three strains (L3, L4, L5, and L10) from the T1 generation were selected. When they reached the "two leaves and one bud" stage, the two leaves were inoculated with the incompatible race CYR23 of stripe rust. They were then placed in a humidity chamber in darkness for 24 hours, followed by a photoperiod growth incubator with 16°C light for 16 hours and 11°C darkness for 8 hours. Fourteen days after inoculation, sporulation on the leaves was observed, and phenotypic identification results are as follows: ​ As shown, it was found ​ Transgenic knockout plants showed reduced resistance to stripe rust and increased spore biomass.

[0114] In addition, DNA was extracted from the leaf inoculation site of wheat overexpression material with stripe rust, diluted to 400 ng / μL, and used as a template to detect the stripe rust internal reference gene PstEF. ​ -F: 5'-TTCGCCGTCCGTGATATGAGACAA-3', SEQ ID NO: 13; ​ -R: 5'-ATGCGTATCATGGTGGTGGAGTGA-3', SEQ ID NO:14) and wheat internal reference gene ( ​ () ​ -F: 5'-TGGTGTCATCAAGCCTGGTATGGT-3', SEQ ID NO: 5; ​ The quantitative primers (-R: 5'-ACTCATGGTGCATCTCAACGGACT-3, SEQ ID NO: 6) were used to detect the relative biomass of stripe rust fungus in wheat leaves by qRT-PCR. The results are as follows: ​ As shown.

[0115] It can be seen that wheat GPI-anchored protein genes ​ Transgenic knockout plants showed reduced resistance to stripe rust.

[0116] Example 5

[0117] Wheat GPI-anchored protein gene ​ The steps for identifying disease resistance in knockout plants are as follows:

[0118] Wheat leaves from L3 and L4 lines inoculated with the incompatible race CYR23 of stripe rust were collected at different time points (24 and 48 h). The leaves were cut into small segments and placed morphologically apical-upward in centrifuge tubes containing DAB staining solution. After 4 h under strong light, the leaves were removed and immersed in a decolorizing solution (anhydrous ethanol: glacial acetic acid, volume ratio = 1:1), with the solution changed every 24 h until the leaves became transparent. The decolorized and transparent wheat leaves were then fixed in chloral hydrate solution for 24 h and preserved using 30% glycerol. The area of ​​reactive oxygen species near the infection sites in the leaf tissue was observed and counted using an Olympus fluorescence microscope under bright field. The presence of observed substomatal vesicles was used as the criterion for determining the infection site. The results are as follows: ​ As shown.

[0119] Depend on ​ Conclusion: Wheat GPI-anchored protein gene ​ The area of ​​reactive oxygen species accumulation in knockout plants was significantly lower than that in wild-type plants, indicating that knocking out the wheat GPI-anchored protein gene... ​ It weakens wheat's resistance to stripe rust.

[0120] Decolorized and transparent wheat leaves were stained with wheat germ agglutinin (WGA, which specifically binds to a small glycoprotein on the chitin of fungal cell walls; WGA coupled with fluorescein was used to stain infected leaves, allowing clear observation of hyphal structures under fluorescence). The hyphal structure of *Strombus stripe rust* was then observed using an Olympus fluorescence microscope with the GFP fluorescence channel, and hyphal length and infection area were counted. Hyphae length was defined as the distance from the tip of the longest hyphae to the tip of the substomatal fossa. 30–50 infection points were counted for each tissue sample, and the samples were repeated three times. Statistical significance analysis was performed on the statistical data. Results are as follows: ​ As shown.

[0121] Depend on ​ It can be seen that wheat GPI-anchored protein gene ​ The mycelial length and infection area of ​​stripe rust fungus in the knockout plants were significantly higher than those in the control Fielder, indicating that knocking out the wheat GPI-anchored protein gene... ​ In genetically modified wheat, the growth and development of stripe rust fungus are promoted, and disease resistance is significantly weakened.

[0122] comprehensive ​ The results show that the wheat GPI-anchored protein gene ​ It plays a positive regulatory role in the process of wheat resistance to stripe rust.

[0123] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Upregulation of a wheat GPI-anchored protein gene TaLLG3 or use of a wheat GPI-anchored protein TaLLG3 in increasing wheat stripe rust resistance and / or breeding a wheat variety resistant to stripe rust. The amino acid sequence of the wheat GPI-anchored protein TaLLG3 is shown as SEQ ID NO:

1.

2. A biological material for up-regulating the expression of a wheat GPI-anchored protein gene TaLLG3 or a wheat GPI-anchored protein TaLLG3 in improving the resistance of wheat to stripe rust and / or breeding a wheat variety resistant to stripe rust, the wheat GPI-anchored protein gene TaLLG3 encoding a wheat GPI-anchored protein TaLLG3, the amino acid sequence of the wheat GPI-anchored protein TaLLG3 being shown as SEQ ID NO:

1.

3. Use according to claim 2, characterized in that, The biological material comprises any one or more of the following: 1) recombinant expression vector comprising a wheat GPI-anchored protein gene TaLLG3 ; 2) an engineered bacteria containing a wheat GPI-anchored protein gene TaLLG3 ; 3) an engineered bacterium containing the recombinant expression vector of 1).

4. Use according to claim 3, characterized in that, The initial strain in the engineered bacterium is Agrobacterium.

5. A method of breeding a wheat variety resistant to stripe rust, characterized in that, The method comprises the following steps: increasing the expression amount of a wheat GPI-anchored protein TaLLG3 in a receptor wheat or promoting the expression of a wheat GPI-anchored protein gene TaLLG3 in the receptor wheat, so as to obtain the anti-streak rust wheat variety. TaLLG3 The method comprises the following steps: increasing the expression amount of a wheat GPI-anchored protein TaLLG3 in a receptor wheat or promoting the expression of a wheat GPI-anchored protein gene TaLLG3 in the receptor wheat, so as to obtain the anti-streak rust wheat variety. The wheat GPI-anchored protein gene TaLLG3 A wheat GPI-anchored protein TaLLG3 is encoded, and the amino acid sequence of the wheat GPI-anchored protein TaLLG3 is shown as SEQ ID NO: 1.

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