Wheat magnesium transporter TaNIPA8-6B and application of an encoding gene thereof in regulating plant stripe rust resistance

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to a wheat magnesium transporter TaNIPA8-6B and application of an encoding gene thereof in regulating plant resistance to stripe rust. The application analyzes the function of the wheat magnesium transporter TaNIPA8-6B through gene editing and overexpression, finds that the wheat magnesium transporter TaNIPA8-6B is induced to express in the reaction of wheat and stripe rust in compatible and incompatible systems, and creates gene editing and overexpression TaNIPA8-6B mutant plants of the gene, and identifies that the gene knockout mutant of the gene shows a disease susceptibility to the incompatible race CYR23 of stripe rust, and the overexpression mutant plant shows a disease resistance to the compatible race CYR32 of stripe rust, and determines that TaNIPA8-6B the gene plays a positive regulation role in the disease resistance of wheat to stripe rust, and provides a high-quality germplasm resource for the resistance of wheat to stripe rust.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of wheat magnesium transporter TaNIPA8-6B and its encoding gene in regulating plant stripe rust resistance. Background Technology

[0002] Common wheat ( Triticum aestivum Wheat, as one of the most widely planted crops in the world, holds a very important position. However, wheat is often threatened by a variety of diseases, among which wheat stripe rust is one of the most serious. This disease is caused by wheat-specific strain of *Striga styracifolium* (Striga styracifolium). Puccinia striiformis Wheat stripe rust is caused by infection with *F. sp. tritici*, a strictly obligate obligate parasitic fungus that primarily spreads over long distances via high-altitude air currents. Large outbreaks can occur when there is a high initial inoculum population, abundant rainfall, high autumn seedling density, and weak varietal resistance. Wheat stripe rust can also reproduce sexually on its alternate host, barberry, leading to frequent variations in virulence and the creation of new pathogenic races, ultimately resulting in the loss of varietal resistance.

[0003] Transgenic breeding is a breeding method that applies genetic engineering to breeding work, introducing genes to cultivate new varieties with specific requirements. Based on transgenic breeding technology, several genes related to wheat stripe rust resistance have been identified in this field, such as wheat stripe rust resistance genes. YrChr1B wheat stripe rust resistance gene YrLumai15 and wheat stripe rust resistance genes TaALD1 However, with the loss of disease resistance in wheat varieties, the discovery of new stripe rust-resistant genes and the creation of new stripe rust-resistant wheat varieties are becoming increasingly important for the prevention and control of wheat stripe rust.

[0004] Magnesium ions (Mg 2+ Magnesium (Mg) is a core nutrient element for plant growth, development, and stress response. It participates in various physiological and biochemical reactions, such as acting as a cofactor for ATP, an enzyme activator, and participating in the balance of transmembrane potential gradients. In higher plants, it participates in the assembly of thylakoid membranes and the stacking of grana, thus maintaining the stability of chloroplast structure. The homeostasis of magnesium ions depends on the dynamic balance of transmembrane transport proteins. In mammals, the NIPA (Non-imprinted in Prader-Willi / Angelman syndrome) family is an evolutionarily conserved group of Mg... 2+ NIPA transport proteins are involved in processes such as neural development, immune regulation, and tumor drug resistance. Functional studies of NIPA homologs in plants are limited, and there are currently no reports on the role of NIPA proteins in plant disease resistance. Summary of the Invention

[0005] The purpose of this invention is to provide the application of wheat magnesium transporter TaNIPA8-6B and its encoding gene in regulating plant resistance to stripe rust. The wheat magnesium transporter TaNIPA8-6B can regulate plant resistance to stripe rust, thereby increasing the biological use of wheat magnesium transporter TaNIPA8-6B.

[0006] This invention provides TaNIPA8-6B The use of a gene or its encoded protein in any one or more of the following:

[0007] Ⅰ: Regulate plant resistance to stripe rust;

[0008] II: Breeding and / or improving plant varieties resistant to stripe rust;

[0009] III: Control of plant stripe rust;

[0010] The TaNIPA8-6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:1.

[0011] Preferably, the application of regulating plant stripe rust resistance includes: positive regulation of the... TaNIPA8-6B The application of genes or their encoded proteins in enhancing plant resistance to stripe rust, and / or negatively regulating the aforementioned TaNIPA8-6B Application of genes or their encoded proteins in reducing plant resistance to stripe rust.

[0012] This invention also provides positive regulation TaNIPA8-6B The application of biological materials expressing genes or their encoded proteins in improving plant resistance to stripe rust, wherein... TaNIPA8-6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:1.

[0013] Preferably, the biomaterial includes any one or more of the following:

[0014] 1) Contains TaNIPA8-6B Expression cassettes of gene ORF sequences;

[0015] 2) Contains TaNIPA8-6B Recombinant expression vectors of gene ORF sequences;

[0016] 3) A recombinant expression vector containing the expression cassette described in 1);

[0017] 4) Contains TaNIPA8-6B Engineered bacteria with ORF sequences of genes;

[0018] 5) Engineered bacteria containing the expression cassette described in 1);

[0019] 6) Engineered bacteria containing the recombinant expression vector described in 2) or 3).

[0020] This invention also provides negative regulation TaNIPA8-6B The application of biological materials expressing genes or their encoded proteins in reducing plant resistance to stripe rust, the aforementioned TaNIPA8-6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:1.

[0021] Preferably, the biomaterial includes any one or more of the following:

[0022] A) Knock out the above TaNIPA8-6B Nucleic acid molecules of genes;

[0023] B) A recombinant expression vector containing the nucleic acid molecules described in A);

[0024] C) Engineered bacteria containing the nucleic acid molecules described in A);

[0025] D) Engineered bacteria containing the recombinant expression vector described in B).

[0026] Preferably, the nucleic acid molecule includes sgRNA1 and sgRNA2; sgRNA1 and sgRNA2 are respectively targeted by... TaNIPA8-6B The primers for the first and second target sites of the gene were synthesized by in vitro transcription; the nucleotide sequences of the forward primer TaNIPA8-6B-Target1-F and the reverse primer TaNIPA8-6B-Target1-R for the first target site are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively; the nucleotide sequences of the forward primer TaNIPA8-6B-Target2-F and the reverse primer TaNIPA8-6B-Target2-R for the second target site are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0027] Preferably, the plant includes wheat.

[0028] This invention also provides a method for breeding wheat varieties resistant to stripe rust, comprising the following steps: increasing the content of magnesium transporter TaNIPA8-6B in recipient wheat or promoting the content of magnesium transporter TaNIPA8-6B in recipient wheat. TaNIPA8-6B Gene expression was used to obtain the stripe rust-resistant wheat variety;

[0029] The TaNIPA8-6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:1.

[0030] This invention also provides a method for breeding wheat mutants with reduced resistance to stripe rust, comprising the following steps: reducing the content of magnesium transporter TaNIPA8-6B in recipient wheat or inhibiting the content of magnesium transporter TaNIPA8-6B in recipient wheat. TaNIPA8-6B Gene expression was used to obtain wheat mutants with reduced resistance to stripe rust.

[0031] The TaNIPA8-6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:1.

[0032] Beneficial effects:

[0033] This invention provides TaNIPA8- The application of genes in one or more of the following three areas: I: regulating plant stripe rust resistance; II: breeding and / or improving plant varieties resistant to stripe rust; III: controlling plant stripe rust; TaNIPA8-6B 6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:1. This invention utilizes gene editing and overexpression techniques to functionally analyze the wheat magnesium transporter TaNIPA8-6B, revealing that it is induced in both affinity and incompatibility systems between wheat and stripe rust fungus. Therefore, this invention provides a gene editing and overexpression method. TaNIPA8-6B Mutant plants of the gene were identified, and the gene knockout mutants were found to be susceptible to the incompatible race CYR23 of stripe rust, while the overexpression mutants were found to be resistant to the compatible race CYR32 of stripe rust. This clarified the genetic makeup of the mutants. Figure 1 Genes play a positive regulatory role in wheat resistance to stripe rust, providing high-quality germplasm resources for wheat resistance to stripe rust. Attached Figure Description

[0034] 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.

[0035] Figure 2 A flowchart illustrating the study of wheat magnesium transporter TaNIPA8-6B in wheat varieties resistant to stripe rust, provided in this embodiment of the invention.

[0036] TaNIPA8-6B The wheat magnesium transporter protein in Example 1 Figure 3 A schematic diagram of gene expression pattern analysis, where data were analyzed using t-tests and ANOVA. "**" and "***" represent... P<0.01 and P<0.001 ;

[0037] Figure 4 The image shows the wheat magnesium transporter TaNIPA8-6B gene editing vector obtained in Example 2;

[0038] TaNIPA8-6B Provided in Example 2 Figure 5 Schematic diagram of PCR detection results for gene-edited plants;

[0039] TaNIPA8-6B Provided in Example 3 Figure 6 A diagram illustrating gene editing detection results;

[0040] TaNIPA8-6B Provided in Example 3 Figure 7 Schematic diagram of phenotypic results of gene-edited plants inoculated with the incompatible race CYR23 of stripe rust;

[0041] TaNIPA8-6B The gene provided in Example 4 Figure 8 Schematic diagram of the overexpression vector;

[0042] Figure 9 This is a schematic diagram of the PCR detection results of the TaNIPA8-6B overexpressing plants provided in Example 4;

[0043] TaNIPA8-6B The target gene in the TaNIPA8-6B overexpressing plant provided in Example 4. Figure 10 The diagram illustrates the expression levels, where data were analyzed using t-tests and ANOVA. "**" and "***" represent... P<0.01 and P<0.001 ;

[0044] TaNIPA8-6B This is a schematic diagram of the phenotypic results of the TaNIPA8-6B overexpressing plants inoculated with the stripe rust affinity race CYR32, as provided in Example 5. Detailed Implementation

[0045] This invention provides TaNIPA8-6B The use of a gene or its encoded protein in any one or more of the following:

[0046] Ⅰ: Regulate plant resistance to stripe rust;

[0047] II: Breeding and / or improving plant varieties resistant to stripe rust;

[0048] III: Control of plant stripe rust;

[0049] The TaNIPA8-6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:1.

[0050] The amino acid sequence shown in SEQ ID NO:1 of this invention is specifically: MGDWIIGALINIVGSVAINFGTNLLKLGHDQREKLYSSNNQSDGKFVPRSVMYFQTWRIGILFFAVGNCLNFMSFAYAAQSLLAALGSIQFVSNIAFAYVVLNKTISVKVMVATTFIVFGNVFLVSFGNHQSPVYTPEQLIAKYSNLVFVLYCMSLVFVVALSQYLYRCGETILSDNAKDTSTHWRTLLPFSYAIVSGAIGSCSVLF AKSLSNMLRLTMSSRYQFHSWFTYSILLLFLCTAGFWMARLNEGLSLFDAILIVPMFQIAWTFFSICTGFVYFQEYQVFDTLRIIMFLLGMTFVFIGISLLAPDENKVADT KDGSNATKDAAIDMNSEAHVSGNCRPRKLPMEETEVDDMDSFSTSVKVKAKCILSRAKSACSMSLGLGEETISASSVLAMPMVSSRTTGFRGIRTDRSKYIPLGSTDWDNL.

[0051] As one implementation method, the TaNIPA8-6B

[0052] As one implementation method, the application of regulating plant stripe rust resistance is as follows: positive regulation of the... TaNIPA8-6B The application of genes or their encoded proteins in enhancing plant resistance to stripe rust, and / or negatively regulating the aforementioned TaNIPA8-6B Application of genes or their encoded proteins in reducing plant resistance to stripe rust. In one embodiment, the plant is a monocotyledonous plant; in another embodiment, the monocotyledonous plant is a grass; in yet another embodiment, the grass is wheat. In one embodiment, the stripe rust is stripe rust induced by a stripe rust fungus; in another embodiment, the stripe rust fungus is the wheat-specific variant of *Striga styli*; in yet another embodiment, the wheat-specific variant of *Striga styli* can be a compatible race and / or an incompatible race of the wheat-specific variant of *Striga styli*. In specific embodiments, this invention uses the incompatible race CYR23 and the compatible race CYR32 of *Striga rust* as examples to identify the phenotypes of gene-edited mutant plants and overexpression mutant plants, but this should not be considered as the entire scope of protection of this invention.

[0053] This invention also provides positive regulation TaNIPA8-6B The application of biological materials expressing genes or their encoded proteins in improving plant resistance to stripe rust, wherein... TaNIPA8-6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:1.

[0054] In one embodiment, the biomaterial includes any one or more of the following:

[0055] 1) Contains TaNIPA8-6B Expression cassettes of gene ORF sequences;

[0056] 2) Contains TaNIPA8-6B Recombinant expression vectors of gene ORF sequences;

[0057] 3) A recombinant expression vector containing the expression cassette described in 1);

[0058] 4) Contains TaNIPA8-6B Engineered bacteria with ORF sequences of genes;

[0059] 5) Engineered bacteria containing the expression cassette described in 1);

[0060] 6) Engineered bacteria containing the recombinant expression vector described in 2) or 3).

[0061] As one embodiment, the nucleotide sequence of the ORF sequence is shown in SEQ ID NO:2. As one embodiment, it is used to amplify the... TaNIPA8-6BThe forward and reverse primers for the gene are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively: forward primer TaNIPA8-6B-cDNA-F: 5'-ATGGGCGACTGGATTATAGGAG-3', and reverse primer TaNIPA8-6B-cDNA-R: 5'-CTATAGATTATCCCAATCAGTGGACC-3'. In one embodiment, the initial vector in the recombinant expression vector is a plasmid vector; in another embodiment, the plasmid vector is pANIC6E. 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 plant is a monocotyledonous plant; in another embodiment, the monocotyledonous plant is a grass; in yet another embodiment, the grass is wheat. The limitations of the stripe rust described in this invention are the same as those in the above-described technical solutions and will not be repeated here.

[0062] This invention also provides negative regulation TaNIPA8-6B The application of biological materials expressing genes or their encoded proteins in reducing plant resistance to stripe rust, the aforementioned TaNIPA8-6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:1.

[0063] In one embodiment, the biomaterial includes any one or more of the following:

[0064] A) Knock out the above TaNIPA8-6B Nucleic acid molecules of genes;

[0065] B) A recombinant expression vector containing the nucleic acid molecules described in A);

[0066] C) Engineered bacteria containing the nucleic acid molecules described in A);

[0067] D) Engineered bacteria containing the recombinant expression vector described in B).

[0068] In one embodiment, the nucleic acid molecule includes sgRNA1 and sgRNA2; sgRNA1 and sgRNA2 are respectively targeted by... TaNIPA8-6BPrimers for the first target (Target1) and the second target (Target2) of the gene are synthesized via in vitro transcription. The nucleotide sequences of the forward primer TaNIPA8-6B-Target1-F and the reverse primer TaNIPA8-6B-Target1-R for the first target are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively; the nucleotide sequences of the forward primer TaNIPA8-6B-Target2-F and the reverse primer TaNIPA8-6B-Target2-R for the second target are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively. This invention does not specifically limit the in vitro transcription process; conventional in vitro transcription procedures in the art can be used.

[0069] In one embodiment, the initial vector in the recombinant expression vector is a Cas9 expression vector. In another 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 employs conventional methods for constructing recombinant expression vectors and engineered bacteria in the art.

[0070] In one embodiment, the plant is a monocotyledonous plant; in another embodiment, the monocotyledonous plant is a grass; in yet another embodiment, the grass is wheat. The definition of stripe rust in this invention is the same as in the above-described technical solutions and will not be repeated here.

[0071] This invention also provides a method for breeding wheat varieties resistant to stripe rust, comprising the following steps: increasing the content of magnesium transporter TaNIPA8-6B in recipient wheat or promoting the content of magnesium transporter TaNIPA8-6B in recipient wheat. TaNIPA8-6B Gene expression was used to obtain the stripe rust-resistant wheat variety; TaNIPA8-6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:1.

[0072] As one implementation method, the TaNIPA8-6B The nucleotide sequence of the gene's ORF is shown in SEQ ID NO:2. As one embodiment, the method for obtaining the stripe rust-resistant wheat variety of the present invention can be: introducing the [specific gene] into recipient wheat. TaNIPA8-6B Gene; as another implementation, the introduction of the gene into recipient wheat TaNIPA8-6B The gene can be transferred into recipient wheat using the recombinant expression vector described above, to increase the content of magnesium transporter TaNIPA8-6B in the recipient wheat or promote the development of magnesium transporter TaNIPA8-6B in the recipient wheat. TaNIPA8-6BThe specific steps for gene expression can be as follows: introducing a 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.

[0073] This invention also provides a method for breeding wheat mutants with reduced resistance to stripe rust, comprising the following steps: reducing the content of magnesium transporter TaNIPA8-6B in recipient wheat or inhibiting the content of magnesium transporter TaNIPA8-6B in recipient wheat. TaNIPA8-6B Gene expression yielded a wheat mutant with reduced resistance to stripe rust; TaNIPA8-6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:1.

[0074] As one implementation method, the TaNIPA8-6B The nucleotide sequence of the gene's ORF is shown in SEQ ID NO:2. As one embodiment, the method for obtaining the wheat mutant with reduced stripe rust resistance according to the present invention can be: introducing the gene with knockout into recipient wheat. TaNIPA8-6B Nucleic acid molecules of the gene; as another implementation, the introduction of the knockout gene into recipient wheat. TaNIPA8-6B The method for obtaining nucleic acid molecules from genes can be to include the gene containing the knockout described in the above technical solution. TaNIPA8-6B The recombinant expression vector of the gene's nucleic acid molecule was transferred into recipient wheat to reduce the content of the magnesium transporter TaNIPA8-6B in the recipient wheat or inhibit its activity. Figure 1 Gene expression can be achieved through the following steps: [The text abruptly shifts to a seemingly unrelated topic about gene expression and gene knockout.] TaNIPA8- The gene's nucleic acid recombinant expression vector was introduced into Agrobacterium to obtain engineered bacteria, and the engineered bacteria were then transformed into wheat embryos to obtain wheat mutants with reduced stripe rust resistance.

[0075] 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.

[0076] The following embodiments are in accordance with TaNIPA8-6B The steps in the analysis and verification of wheat magnesium transporter TaNIPA8-6B regulating wheat resistance to stripe rust, and the breeding or improvement of wheat varieties resistant to stripe rust, are described. Examples 1-4 are obtained according to step S101. TaNIPA8-6B 6B Gene knockout and overexpression materials, and on TaNIPA8-6B Molecular detection was performed on genetically modified wheat; Example 5 is the identification performed according to step S102. TaEF-α Resistance to stripe rust was determined by gene knockout and overexpression, and the regulatory role of wheat magnesium transporter TaNIPA8-6B in the wheat-stripe rust interaction was identified.

[0077] Unless otherwise specified, the biological materials and reagents used in the following examples can be purchased from commercially available sources.

[0078] Example 1

[0079] TaNIPA8-6B The study of expression at different time points during the interaction between wheat and stripe rust fungus followed the steps below:

[0080] At the "two-leaf-one-heart" stage of the wheat variety Fielder, the two leaves were inoculated with the stripe rust-friendly race CYR32 and the incompatible race CYR23 (CYR23 and CYR32 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). The control group was not inoculated with stripe rust.

[0081] Wheat leaves were sampled at 0 hpi (hour post-inoculation), 6 hpi, 12 hpi, 24 hpi, 48 hpi, 72 hpi, and 96 hpi after infection. Before sampling, the leaves were sprayed with water and the 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 ammonia. RNA was extracted using a rapid universal plant RNA extraction kit (purchased from Beijing Huayueyang Biotechnology Co., Ltd.), and reverse transcription was performed using a reverse transcription kit (purchased from Thermo Fisher Scientific). TaNIPA8-6B As an internal reference gene, RT-qPCR was performed using the Thermo Fisher Scientific QuantStudio 1 real-time quantitative PCR system, and analysis was performed using TaNIPA8-6B specific primers. TaEF-α The expression of .

[0082] in Figure 2 Quantitative specific primers and internal reference genes Figure 2 The quantitative primer sequences are as follows:

[0083] Forward primer TaNIPA8-6B-qPCR-F: 5'-GCCACAAAGGACGCGGCAAT-3' (SEQ ID NO:5); Reverse primer TaNIPA8-6B-qPCR-R: 5'-AGCAAGCCGACTTTGCTCTCGA-3' (SEQ ID NO:6).

[0084] Forward primer TaEF-α-F: 5'-TGGTGTCATCAAGCCTGGTATGGT-3' (SEQ ID NO:7); Reverse primer: TaEF-α-R: 5'-ACTCATGGTGCATCTCAACGGACT-3' (SEQ ID NO:8).

[0085] The reaction system and reaction procedure for RT-qPCR are shown in Table 1 and Table 2, respectively.

[0086] Table 1 RT-qPCR reaction system

[0087]

[0088] Table 2 RT-qPCR reaction procedure

[0089]

[0090] After the reaction is complete, use 2 -△△Ct The calculation method is used to analyze the data, and the results are as follows: TaNIPA8-6B As shown.

[0091] Depend on TaNIPA8-6B We can conclude that: TaNIPA8-6B The gene was induced to express in both affinity and incompatibility systems between wheat and stripe rust, with the highest expression level at 6 hpi in the incompatibility system and at 72 hpi in the affinity system.

[0092] Example 2

[0093] BtgZ Methods for constructing gene knockout wheat plants (TaNIPA8-6B-KO)

[0094] The background material used in this embodiment is wild-type wheat Fielder, with gene knockout. Bsa TaNIPA8-6B gene knockout plants were created using Agrobacterium-mediated genetic transformation. The method for preparing TaNIPA8-6B gene knockout plants is as follows:

[0095] (1) Construction of gene editing vector

[0096] The forward and reverse primer sequences for gene editing target design are as follows:

[0097] Target1: TaNIPA8-6B-Target1-F: 5'-GTGGCTTGTTGGAGGA-3' (SEQ ID NO: 9); TaNIPA8-6B-Target1-R: 5'-AACCCAGATTTAATTTATCG-3' (SEQ ID NO: 10);

[0098] Target2: TaNIPA8-6B-Target2-F: 5'-GCGATGGCAAATTCGTCC-3' (SEQ ID NO: 11); TaNIPA8-6B-Target2-R: 5'-CATGCTAACTAACTATAGTTGCTTC-3' (SEQ ID NO: 12).

[0099] The synthesized primers were double-stranded under the following system conditions: 10 μL each of the forward and reverse primers for the gene editing target, 50 μL of 1× TE Buffer and 30 μL of ddH2O. After mixing by pipetting, the mixture was boiled in a water bath for 5 min, cooled to room temperature and stored at -20℃ for later use.

[0100] Using T4 DNA ligase (purchased from Novizan Biosciences), sgRNA2 and Figure 3 I. The intermediate vector sgRNA, which was digested by a single enzyme, 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.

[0101] The ENTRY4-F / R primers are as follows:

[0102] ENTRY4-F: 5'-GCGTTTCTACAAACTCTTCCTG-3' (SEQ ID NO: 13);

[0103] ENTRY4-R: 5'-TGGGTCTAGATATCTCGAGTG-3' (SEQ ID NO: 14).

[0104] Using T4 DNA ligase, sgRNA1 was constructed into... Figure 4 I. The constructed recombinant plasmid was introduced into DH5α competent Escherichia coli cells onto the linearized intermediate vector sgRNA. Positive transformants were screened and verified by monoclonal PCR. After amplification by liquid culture, plasmid DNA was extracted.

[0105] The intermediate vector plasmid was constructed into the final vector Cas9-PCL4 using the LR reaction system shown in Table 3. E. coli was then transformed into Cas9-PCL4. Positive single colonies were detected by PCR using NOS-F / R primers. Plasmids were extracted from the positive single colonies by shaking and sequencing to obtain the TaNIPA8-6B-Cas9 gene editing vector. This gene editing vector consists of the TaU6 promoter and Target1 target sgRNA, the TaU6 promoter and Target2 target sgRNA, the ZmUbi maize ubiquitin promoter, and the Cas9 protein. Figure 4As shown, Target1 and Target2 are two target sgRNAs of the TaNIPA8-6B gene; TaU6 is the promoter that initiates Target1 and Target2; Cas9 is the core element of the gene editing system that encodes the Cas9 protein; and ZmUbi is the maize ubiquitin promoter that initiates Cas9 expression.

[0106] Table 3 LR reaction system

[0107]

[0108] The NOS-F / R primers are as follows:

[0109] NOS-F: 5'-AAGCACATACGTCAGAAACATTAT-3' (SEQ ID NO: 15);

[0110] NOS-R: 5'-TGGGTGAGATTCCTTGAAGTTGAGTA-3' (SEQ ID NO: 16).

[0111] (2) Obtaining genetically modified wheat

[0112] Using Agrobacterium-mediated genetic transformation, the gene-editing vector TaNIPA8-6B-Cas9 was transferred into wheat immature embryos, and transgenic wheat TaNIPA8-6B-KO was obtained. The specific steps are as follows:

[0113] Take 100 μL of Agrobacterium competent cells EHA105 and place them on ice until completely thawed. Add the gene editing vector TaNIPA8-6B-Cas9 to the Agrobacterium competent cells and gently shake to mix thoroughly. Preheat the water bath to 37°C and perform transformation as follows: incubate on ice for 5 min; incubate in liquid nitrogen for 5 min; incubate in a 37°C water bath for 5 min; incubate on ice for 5 min; add 800 μL of antibiotic-free LB liquid medium, gently mix, and incubate at 28°C and 160 rpm for 2-3 h; incubate at 5000 rpm for 5 min and collect the cells. Discard the supernatant in a clean bench, reserving about 100 μL of supernatant. Transfer this supernatant to a plate containing Kan (50 mg / L) and Rif (25 mg / L), spread evenly, and incubate for 2-3 h. Dry the bacterial culture in the air; seal the petri dish with sealing film, invert it in an incubator at 28℃, and incubate in the dark for 2-3 days. Obtain gene knockout plants through an Agrobacterium-mediated genetic transformation system.

[0114] (3) PCR verification

[0115] Twenty-four transgenic wheat lines, L1-L24, from generation T2 of TaNIPA8-6B-KO T2, were validated by PCR. Transgenic positive plants were detected using Cas9-F / R primers, and the PCR products were analyzed by 1.5% agarose gel electrophoresis.

[0116] The Cas9-F / R primers are as follows:

[0117] Cas9-F: 5'-AGATGGATGATGTTCTCCGCTTGCTC-3' (SEQ ID NO: 17);

[0118] Cas9-R: 5'-TTCCTCTACCTGGCTTCCCACTACGA-3' (SEQ ID NO: 18).

[0119] DNA extracted from the transgenic wheat TaNIPA8-6B-KO was tested for positive results. The reaction samples and reagents were added sequentially to the PCR tubes according to the reaction system shown in Table 4.

[0120] Table 4 PCR reaction system for detecting positive results

[0121]

[0122] Add the above samples and reagents to the PCR tube in sequence, centrifuge briefly to mix, and set the PCR reaction program as shown in Table 5.

[0123] Table 5. Procedure for detecting positive PCR reactions

[0124]

[0125] The reaction consisted of 35 denaturation-annealing-extension cycles. After the reaction, 5-10 μL of the PCR product was subjected to agarose gel electrophoresis (1.5% agarose gel, 160V, 20 min). The electrophoresis results were observed using a gel imaging system. TaNIPA8-6B As shown.

[0126] Depend on Figure 5 It can be concluded that the TaNIPA8-6B gene knockout wheat plant TaNIPA8-6B-KO was successfully constructed.

[0127] Example 3

[0128] The phenotypic identification of stripe rust fungi by gene knockout wheat TaNIPA8-6B-KO was performed as follows:

[0129] The PCR detection in Example 3 was used to determine it as Figure 6Gene knockout positive plants were used as plant materials. Two lines, L9 and L17, from the T2 generation were selected (the sequence alignment results of the two lines after PCR detection are shown in the figure). Figure 6 As shown in the image, when the leaves reached the "two leaves and one bud" stage, the leaves were inoculated with the incompatible race CYR23 of stripe rust. After phenotypic identification, the leaves were 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 with stripe rust, sporulation was observed on the leaves. The phenotypic identification results are as follows. TaNIPA8-6B As shown.

[0130] Depend on TaNIPA8-6B It can be found TaNIPA8-6B Gene knockout plants showed a clear susceptibility to stripe rust.

[0131] Example 4

[0132] TaNIPA8-6B Methods for constructing wheat plants with gene overexpression (TaNIPA8-6B-OE)

[0133] The transgenic background material used in this embodiment is wild-type wheat Fielder, overexpressed... TaNIPA8-6B And created using Agrobacterium-mediated genetic transformation. Figure 7 Overexpression in plants. The specific steps are as follows:

[0134] (1) Construction of overexpression vector

[0135] Design overexpression genes Figure 7 The primers, including the forward and reverse primers, are as follows:

[0136] TaNIPA8-6B-6E-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTACCCATACGACGTCCCAGACTACGCTGGCGACTGGATTATAGGA-3' (SEQ ID NO: 19);

[0137] TaNIPA8-6B-6E-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACTAAGCTGTAGTCTGGGACGTCGTATGGGTATAGATTATCCCAATCAGTGGAC-3' (SEQ ID NO: 20).

[0138] Primers were constructed using the TaNIPA8-6B overexpression vector to amplify the full-length gene sequence. The amplified fragment was then incorporated into the expression vector pANIC6E via a homologous recombination gateway reaction, resulting in the TaNIPA8-6B-pANIC6E overexpression vector. The elements of the TaNIPA8-6B-pANIC6E overexpression vector are as follows: GUSPlus As shown.

[0139] Depend on GUSPlus It can be concluded that the overexpression vector consists of the left boundary (LB) of the T-DNA, the right boundary (RB) of the T-DNA, and various components. The left boundary (LB) is the starting point for the transfer of T-DNA from the plasmid to the plant genome during Agrobacterium-mediated plant transformation, marking the beginning of the T-DNA sequence and serving as a key signal for vector integration into the plant genome. The right boundary (RB) of the overexpression vector is the ending portion of the T-DNA. The overexpression vector contains the following components: OsAct1, the rice promoter Actin 1, is used to drive the efficient expression of the selection marker gene Bar in plants; Bar is the selection marker gene, encoding a phosphotransferase, which can induce glyphosate resistance in plants and is used to quickly identify cells that have successfully been transformed with the target gene; the 35ST following the Bar gene is the 35S terminator of cauliflower mosaic virus, used to terminate the transcription process of the Bar gene and add a poly-A tail, which can stabilize the mRNA structure and improve gene expression efficiency; PvUbi1 is the ubiquitin 1 promoter of common bean, used to drive gene expression. GUSPlus Expression; genes TaNIPA8-6B Encoding β-glucosidase, gene expression can be detected through colorimetric reactions, allowing observation of gene expression patterns and locations; the Nopaline synthase terminator (NOST) of Agrobacterium tumefaciens is used to terminate gene expression. TaNIPA8-6B Transcription; the core element, the maize ubiquitin 1 promoter (ZmUbi1), drives the target gene. Figure 8 The expression of the gene, the Acv5 element used for screening target genes, and the Octopine synthase gene terminator (OCST) of Agrobacterium are all part of the gene expression process. Figure 8 Transcription. The right boundary (RB) of the T-DNA is the end of the overexpression vector.

[0140] (2) Obtaining genetically modified wheat

[0141] Using Agrobacterium-mediated genetic transformation, the overexpression vector TaNIPA8-6B-pANIC6E was transferred into wheat embryos, and transgenic wheat TaNIPA8-6B-OE was obtained.

[0142] The specific operation steps are the same as step (2) in Example 3, and the overexpressed wheat material is obtained by Agrobacterium-mediated genetic transformation.

[0143] (3) PCR verification

[0144] This example demonstrates PCR verification of three transgenic wheat lines, L10, L24, and L40, from the TaNIPA8-6B-OE T1 generation. Transgenic positive plants were detected using 6E-F / R primers, and the PCR products were analyzed by 1.5% agarose gel electrophoresis.

[0145] The 6E-F / R primers are as follows:

[0146] 6E-F: 5'-TTAGCCCTGCCTCATACG-3' (SEQ ID NO: 21);

[0147] 6E-R: 5'-CTATCATAGATGTCGCTATAAACC-3' (SEQ ID NO: 22).

[0148] DNA extracted from transgenic wheat TaNIPA8-6B-OE was subjected to positive detection. The specific operating steps were the same as step (3) in Example 3 of this invention, yielding the following result: TaEF-α The PCR verification results of the transgenic wheat TaNIPA8-6B-OE are shown.

[0149] Depend on TaNIPA8-6B It can be concluded that the overexpression vector TaNIPA8-6B-pANIC6E was successfully expressed in transgenic wheat plants.

[0150] (4) RT-qPCR verification

[0151] In this embodiment, the expression level of the TaNIPA8-6B gene in three lines (L10, L24, and L40) of the transgenic wheat TaNIPA8-6B-OE T1 generation was verified by quantitative PCR.

[0152] Wheat leaves from three lines (L10, L24, and L40) of the TaNIPA8-6B-OE T1 generation, which were not inoculated with stripe rust, and Fielder leaves, which were also not inoculated with stripe rust, were sampled and reverse transcribed. Figure 9 As an internal reference gene, RT-qPCR was performed using the Thermo Fisher Scientific QuantStudio 1 real-time quantitative PCR system, and analysis was performed using TaNIPA8-6B specific primers. Figure 9 The expression situation and specific operation steps are the same as in Embodiment 1 of the present invention. After the reaction is completed, 2 -△△Ct The calculation method is used to analyze the data, and the results are as follows: TaNIPA8-6B As shown.

[0153] Depend on Figure 10 We can conclude that:TaNIPA8-6B The expression levels of the gene in the overexpression lines L10, L24 and L40 were significantly increased compared to Fielder, proving that the TaNIPA8-6B-OE material was successfully created.

[0154] Example 5

[0155] This embodiment provides the phenotypic identification of stripe rust fungus by the transgenic wheat TaNIPA8-6B-OE, and the steps are as follows:

[0156] Overexpressing plants of TaNIPA8-6B were created using Agrobacterium-mediated genetic transformation. Positive plants were identified by PCR. Three lines (L10, L23, and L40) from the T1 generation were selected. At the "two leaves and one bud" stage, the two leaves were inoculated with the stripe rust-compatible race CYR32 for phenotypic identification. The plants were then placed in a humidifier in the dark 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 with stripe rust, sporulation on the leaves was observed. Phenotypic results are as follows: ​ As shown, TaNIPA8-6B-OE overexpression plants exhibited significantly reduced sporulation and hypersensitive necrosis response to stripe rust compared to Fielder plants, demonstrating that TaNIPA8-6B overexpression enhances wheat resistance to stripe rust.

[0157] 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. TaNIPA8-6B The application of genes or their encoded proteins in improving wheat stripe rust resistance and / or breeding wheat stripe rust-resistant varieties is characterized by, Increasing the expression of the magnesium transporter TaNIPA8-6B improves wheat resistance to stripe rust. TaNIPA8-6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:

1.

2. Positive regulation TaNIPA8-6B The application of biological materials expressing genes or their encoded proteins in improving wheat resistance to stripe rust, wherein... TaNIPA8-6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:

1.

3. The application according to claim 2, characterized in that, The biomaterials include any one or more of the following: 1) Contains TaNIPA8-6B Expression cassettes of gene ORF sequences; 2) Contains TaNIPA8-6B Recombinant expression vectors of gene ORF sequences; 3) A recombinant expression vector containing the expression cassette described in 1); 4) Contains TaNIPA8-6B Engineered bacteria with ORF sequences of genes; 5) Engineered bacteria containing the expression cassette described in 1); 6) Engineered bacteria containing the recombinant expression vector described in 2) or 3).

4. A method for breeding wheat varieties resistant to stripe rust, characterized in that, This includes the following steps: increasing the content of magnesium transporter TaNIPA8-6B in recipient wheat or promoting the development of magnesium transporter TaNIPA8-6B in recipient wheat. TaNIPA8-6B Gene expression was used to obtain the stripe rust-resistant wheat variety; The TaNIPA8-6B The amino acid sequence of the magnesium transporter TaNIPA8-6B encoded by the gene is shown in SEQ ID NO:1.

Images