Application of TaHPPD protein in regulating resistance to stripe rust in wheat

Abstract

The application belongs to the technical field of genetic engineering and molecular breeding, and particularly relates to application of TaHPPD protein in regulation of wheat stripe rust resistance. Moro The TaHPPD protein can weaken active oxygen (ROS) accumulation and cell necrosis reaction triggered by CNL class protein NLR, and reduce ion leakage. The application obtains TaHPPD knockout (TaHPPD-KO) wheat material by knocking out TaHPPD gene in wheat through gene editing technology, and obtains wheat with wheat stripe rust resistance. The results of the embodiment show that, compared with the control Fielder, the TaHPPD-KO wheat plant shows significant resistance. The TaHPPD protein can be used to regulate wheat stripe rust resistance, and can be used for cultivating wheat resistant to stripe rust.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and molecular breeding technology, specifically relating to the application of TaHPPD protein in regulating wheat stripe rust resistance. Background Technology

[0002] Wheat is one of the world's most important food crops. Wheat stripe rust, caused by *Strombus styracifolius*, is a major fungal disease affecting wheat production, characterized by its wide distribution, rapid spread, and large affected area, making it a significant cause of wheat yield reduction. Currently, wheat stripe rust is mainly controlled using chemical pesticides. However, the use of chemical pesticides exacerbates environmental pollution and pathogen resistance, further restricting pesticide application. Therefore, breeding disease-resistant varieties and mining disease-resistant genes in wheat are particularly important. Summary of the Invention

[0003] The purpose of this invention is to provide the application of TaHPPD protein in regulating wheat stripe rust resistance. This invention discovers that TaHPPD protein can negatively regulate wheat stripe rust resistance and can be used to breed stripe rust-resistant wheat.

[0004] This invention provides the application of TaHPPD protein in regulating wheat stripe rust resistance, wherein the TaHPPD protein includes one or more of TaHPPD-6A, TaHPPD-6B and TaHPPD-6D; the amino acid sequence of TaHPPD-6A is shown in SEQ ID NO.1, the amino acid sequence of TaHPPD-6B is shown in SEQ ID NO.2, and the amino acid sequence of TaHPPD-6D is shown in SEQ ID NO.3.

[0005] As a preferred embodiment, the encoding sequence of TaHPPD-6A is shown in SEQ ID NO.4, the encoding sequence of TaHPPD-6B is shown in SEQ ID NO.5, and the encoding sequence of TaHPPD-6D is shown in SEQ ID NO.6.

[0006] As a preferred embodiment, the regulation includes: increasing TaHPPD protein expression to reduce wheat stripe rust resistance, or decreasing TaHPPD protein expression to increase wheat stripe rust resistance.

[0007] The present invention also provides a target sequence for knocking out the TaHPPD gene, the target sequence being shown in SEQ ID NO. 17.

[0008] The present invention also provides an editing vector for targeting and knocking out the TaHPPD gene, wherein the editing vector contains the target sequence described in the above scheme.

[0009] As a preferred embodiment, the editing vector further comprises an sgRNA sequence as shown in SEQ ID NO. 18.

[0010] As a preferred embodiment, the target sequence is located between the promoter of the editing vector and the sgRNA sequence.

[0011] The present invention also provides an engineered bacterium that targets and knocks out the TaHPPD gene, wherein the engineered bacterium comprises the editing vector described in the above scheme.

[0012] As a preferred embodiment, the base bacteria of the engineered bacteria include Agrobacterium.

[0013] This invention also provides the application of the target sequence described in the above scheme, the editing vector described in the above scheme, or the engineered bacteria described in the above scheme in improving wheat stripe rust resistance or cultivating stripe rust-resistant wheat.

[0014] Beneficial effects:

[0015] This invention provides the application of TaHPPD protein in regulating wheat stripe rust resistance. The TaHPPD protein includes one or more of TaHPPD-6A, TaHPPD-6B, and TaHPPD-6D. The amino acid sequence of TaHPPD-6A is shown in SEQ ID NO.1, the amino acid sequence of TaHPPD-6B is shown in SEQ ID NO.2, and the amino acid sequence of TaHPPD-6D is shown in SEQ ID NO.3. The TaHPPD protein of this invention can attenuate the resistance of NLR, a protein of the CNL class. Moro The TaHPPD gene triggers the accumulation of reactive oxygen species (ROS) and cell necrosis, reducing ion leakage. This invention uses gene editing technology to knock out the TaHPPD gene in wheat, obtaining TaHPPD knockout (TaHPPD-KO) wheat material, thus producing wheat with resistance to wheat stripe rust. Results from the examples show that in wheat stripe rust CYR32 infection experiments, TaHPPD-KO wheat plants exhibited significant resistance compared to the control Fielder. Specifically, TaHPPD-KO wheat plants showed a macroscopic phenotype of large-area hypersensitive necrosis at the infection sites on the leaves and a reduction in stripe rust urediniospores; and a microscopic phenotype of increased reactive oxygen species area and inhibited stripe rust mycelial infection. The TaHPPD protein can be used to regulate wheat stripe rust resistance and can be used to breed stripe rust-resistant wheat. Attached Figure Description

[0016] 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.

[0017] Figure 1The diagram shows the results of TaHPPD gene function verification in Example 2; where A is the tobacco phenotype and its DAB staining results after 48 hours of inoculation; B is the tobacco phenotype and its trypan blue staining results after 72 hours of inoculation; C is the statistical results of reactive oxygen species content after 36 hours of inoculation; and D is the statistical results of ion leakage rate after 60 hours and 108 hours of inoculation.

[0018] Figure 2 The images show the Western blot detection results in Example 2; the left image is the Western blot detection result for HA antibody; the right image is the Western blot detection result for GFP antibody.

[0019] Figure 3 The figures show the results of TaHPPD knockout in wheat in Example 3 and Comparative Example 1; where A is a schematic diagram of the TaHPPD gene editing target; B is a schematic diagram of the TaHPPD gene editing nucleotide sequence; and C is the type of protein expressed after TaHPPD editing.

[0020] Figure 4 The images show the results of TaHPPD-KO and Fielder wheat (the control) after infection with stripe rust fungus in Application Example 1. A shows typical photographs of H2O2 accumulation near the infection site in TaHPPD-KO and Fielder wheat at 72h and 96h post-inoculation; B shows the statistical results of the H2O2 accumulation area; C shows typical photographs of the growth and development of stripe rust fungus in TaHPPD-KO and Fielder wheat at 72h and 96h post-inoculation; and D shows the mycelial area of ​​stripe rust fungus at 72h and 96h post-inoculation.

[0021] Figure 5 The images show observations of TaHPPD-KO and the control Fielder wheat after infection in Example 1; where A is a comparison of the resistance and susceptibility phenotypes of leaves from TaHPPD-KO and Fielder plants; and B is a biomass statistical graph. Detailed Implementation

[0022] This invention provides the application of TaHPPD protein in regulating wheat stripe rust resistance. The TaHPPD protein includes one or more of TaHPPD-6A, TaHPPD-6B, and TaHPPD-6D. The amino acid sequence of TaHPPD-6A is shown in SEQ ID NO.1, the amino acid sequence of TaHPPD-6B is shown in SEQ ID NO.2, and the amino acid sequence of TaHPPD-6D is shown in SEQ ID NO.3. Specifically:

[0023] SEQ ID NO.1:MPPTPTTPAATGAAAVTPEHARPRRMVRFNPRSDRFHTLAFHHVEFW CADAASAAGRFAFALGAPLAARSDLSTGNSVHASQLLRSGNLAFLFTAPYANGCDAATASLPSFSADAARQFSADHGLAVRSIALRVADAAEAFRASVDGGARPAFSPVDLGRGFGFAEVELYGDVVLRFVSHPDGRDVPFLPGFEGVSNPDAVDYGLTRFDHVVGNVPELAPAAAYVAGFTGFHEFAEFTTEDVGTAESGLNSMVLANNSEGVLLPLNEPVHGTKRRSQIQTFLEHHGGSGVQHIAVASSDVLRTLREMRARSAMGGFDFLPPPLPKYYEGVRRIAGDVLSEAQIKECQELGVLVDRDDQGVLLQIFTKPVGDRPTLFLEMIQRIGCMEKDERGEEYQKGGCGGFGKGNFSELFKSIEDYEKSLEAKQSAAVQGS;

[0024] SEQ ID NO.2:MPPTPTTPAATGAAAAAAVTPEHARPRRMVRFNPRSDRFHTLAFHHV EFWCADAASAAGRFAFALGAPLAARSDLSTGNSVHASQLLRSGNLAFLFTAPYANGCDAATASLPSFSADAARRFSADHGLAVRSIALRVADAAEAFRASVDGGARPAFSPVDLGRGFGFAEVELYGDVVLRFVSHPDDTDVPFLPGFEGVSNPDAVDYGLTRFDHVVGNVPELAPAAAYVA GFAGFHEFAEFTTEDVGTAESGLNSMVLANNSEGVLLPLNEPVHGTKRRSQIQTFLEHHGGPGVQHIAVASSDVLRTLREMRARSAMGGFDFLPPPLPKYYEGVRRIAGDVLSEAQIKECQELGVLVDRDDQGVLLQIFTKPVGDRPTLFLEMIQRIGCMEKDERGEEYQKGGCGGFGKGNFSELFKSIEDYEKSLEAKQSAAVQAS;

[0025] SEQ ID NO.3:MPPTPTTTPAATGAGAAAAVTPEHARPRRMVRFNPRSDRFHTLSFHHVE FWCADAASAAGRFAFALGAPLAARSDLSTGNSVHASQLLRSGNLAFLFTAPYANGCDAATASLPSFSADAARRFSADHGLAVRSIALRVADAAEAFRASVDGGARPAFSPVDLGRGFGFAEVELYGDVVLRFVSHPDGTDVPFLPGFEGVSNPGAVDYGLTRFDHVVGNVPELASAAAYVAGFTGFHEFAEFTT EDVGTAESGLNSMVLANNSEGVLLPLNEPVHGTKRRSQIQTFLEHHGGPGVQHIAVASSDVLRTLREMRARSAMGGFDFLPPPLPKYYEGVRRIAGDVLSEAQIKECQELGVLVDRDDQGVLLQIFTKPVGDRPTLFLEMIQRIGCMEKDERGEEYQKGGCGGFGKGNFSELFKSIEDYEKSLEAKQSAAVQGS.

[0026] As one implementation, the encoding sequence of TaHPPD-6A is shown in SEQ ID NO.4, the encoding sequence of TaHPPD-6B is shown in SEQ ID NO.5, and the encoding sequence of TaHPPD-6D is shown in SEQ ID NO.6, as detailed below:

[0027]

[0028]

[0029]

[0030] As one implementation method, the regulation described in this invention includes: increasing the expression level of TaHPPD protein to reduce wheat stripe rust resistance, or decreasing the expression level of TaHPPD protein to increase wheat stripe rust resistance. In specific embodiments, increasing the expression level of TaHPPD protein can be achieved by constructing an expression vector and transferring it into wheat; decreasing the expression level of TaHPPD protein can be achieved by using an editing vector to knock out the wheat TaHPPD gene.

[0031] The present invention also provides a target sequence for knocking out the TaHPPD gene, the target sequence being shown in SEQ ID NO. 17.

[0032] This invention also provides an editing vector for targeting and knocking out the TaHPPD gene, the editing vector comprising the target sequence described above. As one embodiment, the editing vector of this invention further comprises an sgRNA sequence, as shown in SEQ ID NO. 18. As another embodiment, the target sequence of this invention is located between the promoter and the sgRNA sequence of the editing vector.

[0033] This invention also provides an engineered bacterium that targets and knocks out the TaHPPD gene, the engineered bacterium comprising the editing vector described above. As one embodiment, the base bacterium of the engineered bacterium includes Agrobacterium, and in a specific embodiment, the base bacterium can be Agrobacterium EHA105.

[0034] This invention also provides the application of the target sequence described in the above scheme, the editing vector described in the above scheme, or the engineered bacteria described in the above scheme in improving wheat stripe rust resistance or cultivating stripe rust-resistant wheat.

[0035] As one embodiment, the present invention also provides a method for knocking out the wheat TaHPPD gene, comprising the following steps: introducing the editing vector described in the above scheme into wheat tissue, and culturing wheat with the TaHPPD gene knocked out. In a specific embodiment, the wheat tissue may be a wheat embryo.

[0036] To further illustrate the present invention, the application of the TaHPPD protein provided by the present invention in regulating wheat stripe rust resistance is 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.

[0037] Example 1

[0038] Obtain the TaHPPD gene

[0039] Chinese spring wheat at the two-leaf stage was inoculated with stripe rust fungus CYR32. CYR32 is disclosed in the literature [Community Structure and Toxicity Characteristics of Stripe Rust Fungus on Jointed Wheat in Guanzhong Region, Shaanxi Province, Wei Guorong et al., Northwest Agricultural Journal, Vol. 30, No. 8, pp. 1233-1242, 2021]. Forty-eight hours after inoculation, approximately 100 mg of fresh leaves were cut with clean scissors and placed into 2 mL RNA centrifuge tubes containing sterile steel beads. The centrifuge tubes were labeled with the plant number from which the samples were taken, and then rapidly immersed in liquid nitrogen for quick freezing. Wheat samples were obtained by disrupting the leaves using a plant tissue disruptor, maintaining the leaves at a low temperature during the disruption process to prevent tissue melting and the expansion of liquid nitrogen that could cause the centrifuge tubes to explode.

[0040] Total RNA was extracted from wheat samples using the Huayueyang Rapid Universal Plant RNA Extraction Kit 3.0 (CAT:0416-50GK). Due to the instability of RNA, the entire process should be carried out at a low temperature and as quickly as possible.

[0041] RNA was reverse transcribed into cDNA using Novizan Biotechnology Co., Ltd. IIQ RT SuperMix for qPCR (CAT:R223) reverse transcribes RNA into cDNA.

[0042] Using the cDNA as a template, PCR amplification of TaHPPD-6A and TaHPPD-6D was performed using primers shown in SEQ ID NO.7 and SEQ ID NO.8. The amplification products were subjected to agarose gel electrophoresis, and DNA fragments of 1302 bp and 1311 bp in length were separated, purified, and sequenced. The sequencing results are shown in SEQ ID NO.4 and SEQ ID NO.6, namely TaHPPD-6A and TaHPPD-6D. Similarly, PCR amplification of TaHPPD-6B was performed using primers shown in SEQ ID NO.7 and SEQ ID NO.9. The amplification product was subjected to agarose gel electrophoresis, and DNA fragment of 1311 bp in length was separated, purified, and sequenced. The sequencing result is shown in SEQ ID NO.5, namely TaHPPD-6B.

[0043] SEQ ID NO.7: 5'-ATGCCGCCCACCCCCACCACC-3';

[0044] SEQ ID NO.8: 5'-CTATGATCCCTGAACTGCAGCAGA-3';

[0045] SEQ ID NO.9: 5'-CTATGATGCCTGAACTGCAGCAGA-3'.

[0046] Example 2

[0047] TaHPPD gene function verification

[0048] 1) Construction of TaHPPD-6A-EGFP vector

[0049] The TaHPPD-6A obtained in Example 1 was ligated to the Spe I restriction site in the pBinGFP2 vector using a one-step cloning technique to obtain the TaHPPD-EGFP vector.

[0050] 2) NLR Moro -3HA vector construction

[0051] ①Preparation of pBin-SpeI-EGFP-SpeI-3×HA

[0052] The pBinGFP2 vector backbone is followed by a SpeI recognition sequence and three consecutive HA tag sequences (3×HA) to obtain pBin-SpeI-EGFP-SpeI-3×HA. The pBin-SpeI-EGFP-SpeI-3×HA vector backbone sequence is shown in SEQ ID NO.10, and the 3×HA sequence is shown in SEQ ID NO.11.

[0053]

[0054] SEQ ID NO. 11: 5'-TACCCATACGATGTCCTGACTATGCCGAGTATCCATATGACGTT CCAGATTACGCTGTCTACCCATACGATGTTCCAGATTACGCT-3'.

[0055] ② The pBin-SpeI-EGFP-SpeI-3×HA vector prepared in the above steps was digested with SpeI restriction endonuclease to obtain the linearized vector pBin-SpeI-EGFP-SpeI-3×HA.

[0056] Preparation system: Add 2 μL of 10×Tango buffer, 1 μL of Spe I restriction endonuclease, 2 μg of pBin-SpeI-EGFP-SpeI-3×HA plasmid, and add ddH2O to a final volume of 20 μL. Mix well and digest at 37℃ for 5 h. Perform agarose gel electrophoresis on the product. Cut off the gel block containing the target band and perform gel recovery (method as per Magen Gel DNA Micro-Recovery Kit, CAT:D2110).

[0057] ③ Using leaf cDNA from wheat material Avs+Yr10 (published in the literature [Zhongyi Wu, Gaohua Zhang et al. Transcriptomic analysis of wheat reveals possible resistance mechanism mediated by Yr10 to stripe rust. Stress Biology (2023) 3:44]) as a template, the NLR gene was amplified using SEQ ID NO.13 and SEQ ID NO.14. Moro (SEQ ID NO.12) The product was subjected to agarose gel electrophoresis, and the gel block containing the target band was cut off and the gel was recovered by the above method.

[0058]

[0059] SEQ ID NO.13: 5'-CATTTACGAACGATAGACTAGTGCCACCATGGAGGTCGTGACCG GGGC-3';

[0060] SEQ ID NO. 14: 5'-GAACATCGTATGGGTAACTAGTTTCCTCTATGGACAGCTCTC-3'.

[0061] ④ Preparation of NLR Moro -3HA carrier

[0062] The GFP in the vector pBin-SpeI-EGFP-SpeI-3×HA was excised using SpeI, and the amplified NLR was then cloned using a one-step cloning technique. Moro The gene was ligated to the Spe I site in the vector, resulting in pBin-SpeI-NLR. Moro -SpeI-3×HA, denoted as NLR Moro -3HA.

[0063] 3) Carrier transformation

[0064] The pBinGFP2 vector (denoted as EGFP vector) and the TaHPPD-EGFP vector prepared in the above steps, NLR Moro -3HA vectors were ligated and transformed into E. coli DH5α (transformation method as per the Vedi Bio DH5α Chemically Competent Cell instruction manual, CAT#:DL1001). Colony transformation plasmid detection was performed using universal primers 35S-F and M13-R (SEQ ID NO.15 and SEQ ID NO.16), and plasmids were extracted using the Magen Plasmid Rapid Mini-Prep Kit (CAT:P1001).

[0065] SEQ ID NO.15: 5'-TGACGCACAATCCCACTATC-3';

[0066] SEQ ID NO. 16: 5'-CAGGAAACAGCTATGAC-3'.

[0067] The pBinGFP2 vector and the TaHPPD-EGFP vector obtained in the above steps, NLR Moro-3HA vector was used to transform Agrobacterium EHA105 (pSoup). The transformation procedure was performed according to the instruction manual (Shanghai Weidi Biotechnology Co., Ltd., CAT#: AC1012S). After transformation, single clones were picked and colony transformation plasmids were detected using primers 35S-F and M13-R (SEQ ID NO.15 and SEQ ID NO.16).

[0068] Positive clones were picked in a clean bench and placed in 10 mL of sterile LB broth containing both Rif and Kan antibiotics. The cultures were incubated at 28°C for 3 days using a shaker at 220 rpm. The final concentrations of Rif and Kan in the sterile LB broth were 20 μg / mL and 50 μg / mL, respectively. Agrobacterium was then activated to obtain activated Agrobacterium solutions, which were subsequently labeled as EGFP Agrobacterium solution, TaHPPD-EGFP Agrobacterium solution, and NLR Agrobacterium solution. Moro -3HA Agrobacterium tumefaciens solution.

[0069] The activation method for Agrobacterium is as follows: a) Centrifuge the cultured Agrobacterium suspension at 4500 rpm for 5 min; b) Discard the supernatant, add 5 mL of sterile 10 mM MgCl2 solution, mix well, and centrifuge at 4500 rpm for 5 min. Repeat this step 3 times; c) Discard the supernatant, add an appropriate amount of sterile As buffer solution, mix well, and measure the OD of the bacterial suspension using a Nanodrop 2000 UV spectrophotometer. 600 , OD 600 Dilute to 0.6 with As buffer and store in the dark for 1 hour to obtain activated Agrobacterium bacterial suspension.

[0070] 4) Infection with Tobacco Benedict

[0071] Select healthy, 5-week-old *Nicotiana benthamiana* leaves. Gently pierce a small hole on the underside of each leaf using a needle, being careful not to perforate the leaf. Draw a syringe into a well-mixed solution of activated *Agrobacterium*, gently press the syringe nozzle against the pierced hole, and slowly inject the solution until it spreads between the leaf blades, avoiding the veins as much as possible. When injecting different solutions into a single leaf, prevent overlapping of injection areas by marking the injection areas with a marker. Return the tobacco to the previous growing area for continued cultivation. When injecting two solutions into the same location, inject one solution first, and then inject the other solution at the same location 24 hours later. The injection method is as follows: Figure 1 As shown in A and B, 100 μL of bacterial solution was injected each time. At the same time, EGFP Agrobacterium solution was injected in regions 2 and 3, and TaHPPD-EGFP Agrobacterium solution was injected in regions 4 and 5. After 24 hours, NLR was injected in regions 1, 2 and 4. Moro -3HA Agrobacterium tumefaciens solution.

[0072] 4) Result detection

[0073] 48 h after infection, infected leaves were taken and photographed for macroscopic phenotype. Then, DAB (3,3'-Diaminobenzidine tetrahydrochloride) was used to stain the infected area for H2O2. 72 h after infection, infected leaves were taken and photographed for macroscopic phenotype. Then, trypan blue staining solution was used to stain the infected area for cell necrosis.

[0074] DAB staining procedure: a) Immerse the leaves in 1 mg / mL DAB (pH 3.6) staining solution and use a vacuum pump to aspirate for 10 minutes to accelerate stain binding; b) Soak in the dark for 12 hours, and after sufficient staining, use anhydrous ethanol in a boiling water bath for 10 minutes; c) Soak in anhydrous ethanol until the leaves bleach and decolorize, then place them in 75% ethanol or distilled water for photography. See attached image for results. Figure 1 A in the middle.

[0075] Trypan blue staining procedure: a) Immerse the leaves in trypan blue staining solution and boil in a water bath for 10 minutes until the leaves turn blue; b) Soak for 12 hours, then rinse with clean water to remove excess trypan blue staining solution; c) Add chloral hydrate solution to decolorize until the tissue is bleached, changing the chloral hydrate solution several times during this process. Take photos while the tissue is in clean water. See attached image for results. Figure 1 B in the middle.

[0076] To quantitatively determine the H2O2 content, the Solarbio Hydrogen Peroxide Content Detection Kit (CAT: BC3595) was used. The specific method was described in the instruction manual. Results are available in [link to results]. Figure 1 C in the table and Table 1.

[0077] Table 1. Data on H2O2 content in leaves

[0078]

[0079] When the structure of a plant protoplast is affected, its permeability changes, causing organic matter and salts to seep out of the cells. Electrolyte conductivity measurements can reflect the degree of cell damage and quantify the extent of plant cell death. The detection method is as follows: a) 60h and 108h after bacterial infusion, six leaf discs are perforated at the sampling location using a perforator. The leaf discs are completely immersed in 4mL of sterile deionized water for 5h. Six biological replicates are taken for each combination; b) The conductivity C1 of the sample solution is measured using a conductivity meter. After boiling in a water bath for 5min and cooling to room temperature, the conductivity C2 of the sample solution is measured again using a conductivity meter; c) The results are calculated using the formula "electrolyte leakage rate = C1 / C2 × 100%". (See attached table for results.) Figure 1 D in the table and Table 2.

[0080] Table 2 Electrolyte extravasation rate in leaves

[0081]

[0082] To verify whether the protein was expressed in tobacco leaves, Western blot analysis was performed using GFP and HA tags. The method was as follows: a) Agrobacterium-infected leaves were cut at an appropriate time, wrapped in aluminum foil, and flash-frozen in liquid nitrogen; b) A sterile mortar was prepared in advance and flash-frozen in liquid nitrogen. The leaves were then removed and placed in the mortar containing liquid nitrogen, and ground thoroughly at low temperature until powdery (the midrib of the leaves can be removed); c) The ground leaves were mixed with 1 mL of cell lysis buffer in a 2 mL centrifuge tube by vortexing. The cell lysis buffer was Western blot and IP cell lysis buffer (Beyotime), and 10 μL of 100 mM phenylmethylsulfonyl fluoride was added to the lysis buffer. d) Add fluoride (PMSF) protease inhibitors, note that PMSF should not be added in advance; e) Incubate at 4℃ for 30 min by rotation; f) Centrifuge at 4℃, 12000 rpm for 15 min, centrifuging as thoroughly as possible to remove the precipitate; g) Transfer the supernatant to a new centrifuge tube on ice, discard the precipitate, and repeat the centrifugation once more; g) Take an appropriate amount of supernatant and add SDS-PAGE protein loading buffer (5×) (Beyotime) according to the ratio, boil in water for 10 min, and store at -20℃ or perform SDS-polyacrylamide (SDS-PAGE) gel electrophoresis with protein marker as a control.

[0083] After electrophoresis, the gel was used to determine the location of the target protein based on protein markers. The protein at the corresponding location was then excised for Western blot protein detection. The results are shown in [link to results]. Figure 2 .

[0084] according to Figure 1 It can be seen that transient overexpression of TaHPPD and NLR in Nicotiana benthamiana is possible. Moro Protein, discovered NLR Moro Proteins can induce cell necrosis. TaHPPD (carrying a GFP tag) and NLR Moro Co-expression reduced the degree of cell necrosis, while NLR Moro Co-expression with the GFP tag did not attenuate the degree of cell necrosis; therefore, TaHPPD could inhibit NLR-mediated cell death. Moro Protein-induced cell necrosis ( Figure 1 (B and D), and this phenomenon is due to TaHPPD inhibiting NLR. Moro The accumulation of induced reactive oxygen species leads to ( Figure 1 (A and C). Western blot analysis showed that all proteins were expressed normally. Figure 2 In summary, it can be concluded that NLR is transiently co-expressed in *Nicotiana benthamiana*. MoroAnd TaHPPD, TaHPPD can weaken the NLR Moro This leads to the accumulation of reactive oxygen species (ROS) and cell necrosis, and ion leakage is more pronounced compared to NLR. Moro To alleviate.

[0085] Example 3

[0086] Constructing TaHPPD-KO wheat plants

[0087] The selected gene target sequence SEQ ID NO.17 was submitted to Zhongji Gaoke (Beijing) Biotechnology Co., Ltd. to construct a gene editing vector. The sgRNA sequence of the gene editing vector is shown in SEQ ID NO.18. SEQ ID NO.19 and SEQ ID NO.20 were used to detect the gene target sequence + sgRNA sequence, where the 3' end NGG sequence (N represents any nucleotide) was not included during target detection.

[0088] SEQ ID NO.17: 5'-CCACGTCGAGTTCTGGTGCGCGG-3';

[0089] SEQ ID NO.18: 5'-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-3';

[0090] SEQ ID NO.19: 5'-CGCACCAGAACTCGACG-3';

[0091] SEQ ID NO. 20: 5'-TTGGCGGCAGGGAGA-3'.

[0092] The detection was successful, and the gene-editing vector was obtained. This vector was then transformed into Agrobacterium EHA105 using the same method as in Example 2. Positive plaques were selected and sent to the National Key Laboratory of Crop Stress Resistance and High-Efficiency Production at Northwest A&F University for transformation into Fielder wheat embryo transgenic materials. The TaHPPD gene-editing target sequence is as follows: Figure 3 As shown in Target1 of section A. DNA was extracted from the transformed wheat and detected in positive transgenic lines using SEQ ID NO.21 and SEQ ID NO.22.

[0093] SEQ ID NO.21: 5'-ACCTGAACGCGGTGGTCGGCA-3';

[0094] SEQ ID NO. 22: 5'-GTCGCCCCCGAGCTGAGACAGG-3'.

[0095] Positive plants were subjected to PCR detection using SEQ ID NO.23 and SEQ ID NO.24. The amplified DNA fragment was approximately 265 bp in length. The PCR product was sent to Hi-Tom (http: / / www.hi-tom.net / hi-tom / ) for high-throughput sequencing to detect gene editing. The resulting gene-edited material was designated TaHPPD-KO.

[0096] SEQ ID NO.23: 5'-GGAGTGAGTACGGTGTGCATGGTCCGCTTCAACCCGCG-3';

[0097] SEQ ID NO. 24: 5'-GAGTTGGATGCTGGATGGCGGCGGAGAAGGAGGGCAG-3'.

[0098] See gene editing results Figure 3 In Figures B and C, B represents a schematic diagram of the TaHPPD gene-edited nucleotide sequence, Target1 is the editing site in Example 3, and Target2 is the editing site in Comparative Example 1; C represents the type of protein expressed after TaHPPD editing; in B and C, Fielder represents wild-type plants, KO1 represents positive plant 1, KO2 represents positive plant 2, and KO3 represents positive plant 3; in C, * indicates premature termination of translation, and red-font amino acids indicate frameshift mutations after gene editing, where the amino acids are inconsistent with the wild type, indicating successful gene editing. Figure 3 It can be seen that all three homologous genes of TaHPPD were edited, and the protein expression was abnormal. Figure 3 (C)

[0099] Comparative Example 1

[0100] A method for constructing TaHPPD-KO wheat plants similar to that in Example 2, the difference being that the gene target sequence is shown in SEQ ID NO.25 (see Example 2). Figure 3 The gene target sequence (Target2) of A was detected using SEQ ID NO.26 and SEQ ID NO.20, along with the sgRNA sequence. The gene target sequence was not included in the 3' end NGG sequence (N represents any nucleotide). The gene editing vector was obtained and transformed into Agrobacterium EHA105 to create transgenic material.

[0101] SEQ ID NO.25: 5'-CCAGGTCCGACCTCTCCACGGGG-3';

[0102] SEQ ID NO. 26: 5'-CCGTGGAGAGGTCGGAC-3'.

[0103] Sequencing revealed that using the sequence SEQ ID NO.25 as the gene target sequence resulted in off-target effects. Specifically, the sequencing results were consistent with the Fielder sequence, indicating no editing occurred (see [link to documentation]). Figure 3 The results showed that the target sequence (SEQ ID NO. 25) failed to exert an editing effect and TaHPPD-KO wheat plants could not be successfully constructed.

[0104] Application Example 1

[0105] TaHPPD-KO wheat plant resistance to stripe rust verification

[0106] (1) Select relatively fresh spores of stripe rust fungus CYR32 within one week, and prepare a solution with an appropriate amount of isohexadecane (IHD) at a concentration of 3×10⁻⁶. 4 A spore suspension of 1 spore per mL was uniformly sprayed onto wheat leaves using an air pump. Fielder wheat was used as a control group, denoted as Fielder. The same batch of spore suspension was used for both the gene-edited material and its control, Fielder, to ensure consistent inoculation levels.

[0107] After inoculation with stripe rust fungus CYR32, the production of reactive oxygen species and mycelial infection at the infection sites on wheat leaves were detected at 72 h and 96 h, respectively.

[0108] ① DAB staining was used to detect reactive oxygen species in wheat leaves. The method is as follows:

[0109] To verify the resistance changes induced by TaHPPD gene editing, leaf samples were cut 6 hours before the detection time (i.e., 72h and 96h before inoculation). The morphologically lower part of the leaf was immersed in 1mg / mL LDAB (pH 3.6) staining solution, while the rest of the leaf was exposed to strong light at room temperature for 6 hours. Staining was considered complete when the veins turned reddish-brown, reddish-brown exudate appeared at the morphologically upper cut, and black spots were distributed on the leaf surface. The leaves were then cut into segments approximately 2cm in length and placed in new 2mL centrifuge tubes. Fixation and clearing solution was added to completely submerge the leaf segments, and the segments were fixed and decolorized. The fixation and clearing solution was changed several times until the leaves no longer faded. The fixation and clearing solution was anhydrous ethanol: glacial acetic acid (1:1, v:v). The fixation and clearing solution was discarded, and chloral hydrate aqueous solution was added to completely submerge the leaf segments. This decolorization process was repeated several times until the leaf segments were transparent. Before microscopic observation, the segments were stored in a 50% glycerol aqueous solution. Microscopic observation and statistical results are shown below. Figure 4 A and B in the example.

[0110] Table 3. Average H2O2 area of ​​a single infection site in wheat leaves.

[0111] <![CDATA[H2O2(μm 2 )]]> TaHPPD-KO Fielder 72h 13123.11 2952.94 96h 17944.00 8408.03

[0112] ② The mycelial infection status was detected by WGA-Alexa488 staining. The method is as follows: a. Cut off the leaf sample and add fixation and clearing solution [anhydrous ethanol: glacial acetic acid = 1:1, (V:V)] to completely submerge the leaf segment. Fix and decolorize the leaf segment, and change the fixation and clearing solution several times until the leaf no longer fades. b. Discard the fixative clearing solution, add chloral hydrate solution until the leaf segment is completely submerged, repeat the decolorization process several times until the leaf segment is transparent; c. Rinse twice with 50% ethanol, 5 minutes each time, then rinse three times with distilled water; d. Add 1M KOH solution, enough to submerge the leaf, and boil for about 5 minutes until the leaf softens (boiling time depends on the condition of the leaf; observe the leaf condition constantly. Over-boiling will cause the leaf to disintegrate and damage the mycelial structure); e. Soak the leaf three times with 50mM Tris-HCl (pH 7.5), 5 minutes each time; f. Stain with WGA-Alexa 488 buffer in the dark for 10 minutes (extend the staining time if the staining solution is too long); g. After staining, soak the leaf three times with 50mM Tris-HCl (pH 7.5), 5 minutes each time, and store the leaf in the last Tris-HCl solution at 4℃ for later use. Microscopic observation and statistical results correspond to... Figure 4 C and D in the middle.

[0113] Table 4. Average mycelial infection area at a single infection site in wheat leaves.

[0114] <![CDATA[Hyphal infection area (μm 2 )]]> TaHPPD-KO Fielder 72h 4377.71 5647.72 96h 16117.01 31452.58

[0115] To observe the changes in resistance induced by TaHPPD knockout, this invention statistically observed the host cell defense response and fungal growth of TaHPPD-KO wheat after stripe rust infection. DAB staining was used to observe and statistically analyze the accumulation of reactive oxygen species (ROS) in TaHPPD-KO wheat. The results showed that at 72 h and 96 h after stripe rust infection, the accumulation of ROS in TaHPPD-KO wheat was significantly increased compared to Fielder wheat. Figure 4 (A and B). Simultaneously, by staining stripe rust mycelia with WGA and observing and statistically analyzing them, the mycelial infection area of ​​TaHPPD-KO wheat was significantly reduced ( Figure 4 (C and D in the middle).

[0116] (2) This invention further identified the phenotype of gene-edited wheat. After 14 days of inoculation, the phenotypic changes of TaHPPD-KO compared to Fielder were identified. The results are shown in [reference needed]. Figure 5 A in the sample was used to determine the mycelial content in the leaves.

[0117] Biomass can intuitively reflect the infection status of pathogens in the host through data. The infection status of pathogens in the host is reflected by the relative DNA content of pathogens and hosts. The specific method is as follows: extract DNA from leaf samples, amplify DNA fragments using TaEF (SEQ ID NO.27 and SEQ ID NO.28) and PsEF (SEQ ID NO.29 and SEQ ID NO.30) respectively, and perform TA ligation.

[0118] SEQ ID NO.27: 5'-TGACCAGATCAACGAGCC-3';

[0119] SEQ ID NO.28: 5'-CTCCAGGAGAGACTCATG-3';

[0120] SEQ ID NO.29: 5'-TTCGCCGTCCGTGATATGAGACAA-3';

[0121] SEQ ID NO. 30: 5'-ATGCGTATCATGGTGGTGGAGTGA-3'.

[0122] The TA connection method is as follows: The T vector uses T-VectorpMD TM 19(Simple)(Takara), add 1μL T-Vector pMD TM 19(Simple)(Takara), 0.3 pmol of the above amplification product (TaEF or PsEF), and add ddH2O to a final volume of 5 μL. Add 5 μL of Solution I, mix thoroughly, and incubate at 16 °C for 30 min or overnight to obtain T-PsEF and T-TaEF vectors.

[0123] The T-PsEF and T-TaEF vectors obtained in the above steps were ligated and transformed into E. coli DH5α (transformation method according to the DH5α Chemically Competent Cell manual of Weidi Biotechnology, CAT#:DL1001). The plasmids were extracted, their concentrations were measured, and the plasmid copy number was calculated based on the concentrations.

[0124]

[0125] The concentration was diluted to 10⁻¹⁰ with deionized water based on the calculated plasmid copy number. 3 10 4 10 5 10 6 10 7 and 108 A concentration gradient of / μL was established, and qRT-PCR was performed using the diluted plasmids with quantitative primers TaEF (SEQ ID NO.27 and SEQ ID NO.28) and PsEF (SEQ ID NO.29 and SEQ ID NO.30). The logarithm of plasmid copy number (log) was plotted on the x-axis, corresponding to the C... T The value is used to perform linear fitting on the ordinate to obtain the copy number logarithm and C. T The linear curve equation for the value.

[0126] The qRT-PCR program is as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 10 s; annealing at 60℃ for 30 s; extension at 72℃ for 20 s; 40 cycles; increase the temperature by 1℃ each time from 60 to 95℃ (melting curve), and maintain for 10 s.

[0127] The DNA concentration in leaf samples was measured and diluted to 20 ng / μL with deionized water. qRT-PCR was performed on the same samples using TaEF (SEQ ID NO. 27 and SEQ ID NO. 28) and PsEF (SEQ ID NO. 29 and SEQ ID NO. 30), respectively. Based on the obtained C... T Substituting the values ​​into the corresponding linear equations, the copy numbers of TaEF and PsEF in the same sample were calculated, and the PsEF / TaEF ratio was obtained. Using the Fielder control group ratio as a reference, the infection of stripe rust mycelia in wheat was compared. The results are shown in [link to results]. Figure 5 B in the middle.

[0128] according to Figure 5 It can be seen that, compared to Fielder, TaHPPD-KO wheat produces a large number of hypersensitivity reactions and fewer urediniospores. Figure 5 (A); Biomass analysis results showed that the content of stripe rust fungus in TaHPPD-KO wheat leaves was significantly reduced ( Figure 5 (B) In summary, compared with the control Fielder, TaHPPD-KO wheat plants exhibited significant resistance, specifically manifested in the macroscopic phenotype of large-area allergic necrosis at the infected sites on the leaves and reduced stripe rust urediniospores; and the microscopic phenotype of increased reactive oxygen species area and inhibited stripe rust mycelial infection.

[0129] 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. An editing vector for targeting and knocking out the TaHPPD gene, characterized in that, The editing vector contains a target sequence and an sgRNA sequence, the target sequence being shown in SEQ ID NO.17; the sgRNA sequence being shown in SEQ ID NO.18; The protein encoded by the TaHPPD gene includes one or more of TaHPPD-6A, TaHPPD-6B, and TaHPPD-6D; the amino acid sequence of TaHPPD-6A is shown in SEQ ID NO.1, the amino acid sequence of TaHPPD-6B is shown in SEQ ID NO.2, and the amino acid sequence of TaHPPD-6D is shown in SEQ ID NO.

3.

2. The editing medium according to claim 1, characterized in that, The target sequence is located between the promoter of the editing vector and the sgRNA sequence.

3. The editing medium according to claim 1, characterized in that, The encoding sequence of TaHPPD-6A is shown in SEQ ID NO.4, the encoding sequence of TaHPPD-6B is shown in SEQ ID NO.5, and the encoding sequence of TaHPPD-6D is shown in SEQ ID NO.

6.

4. An engineered bacterium that targets and knocks out the TaHPPD gene, characterized in that, The engineered bacteria include the editing vector described in any one of claims 1 to 3.

5. The engineered bacteria according to claim 4, characterized in that, The basic bacteria of the engineered bacteria include Agrobacterium.

6. The application of the editing vector according to any one of claims 1 to 3 or the engineered bacteria according to claim 4 or 5 in improving wheat stripe rust resistance or cultivating stripe rust-resistant wheat; wherein the wheat is Fielder wheat; and the pathogen of stripe rust is stripe rust fungus CYR32.

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