Application of TaHPPD protein in regulation and control of wheat stripe rust resistance

Abstract

The invention belongs to the technical field of genetic engineering and molecular breeding, and particularly relates to application of TaHPPD protein in regulation and control of wheat stripe rust resistance. The TaHPPD protein disclosed by the invention can be used for weakening the accumulation of reactive oxygen species (ROS) and cell necrosis reaction caused by CNL protein NLRMro and reducing ion leakage. A TaHPPD gene in wheat is knocked out through a gene editing technology, a TaHPPD knockout (TaHPPD-KO) wheat material is obtained, and wheat with wheat stripe rust resistance is obtained. The result of the embodiment shows that the TaHPPD-KO wheat plant shows obvious resistance compared with the control Fielder. The TaHPPD protein can be used for regulating and controlling the stripe rust resistance of wheat, and can be used for cultivating stripe rust resistant wheat.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and molecular breeding, and particularly relates to the application of TaHPPD protein in regulating wheat stripe rust resistance. Background Art

[0002] Wheat is one of the most important food crops in the world. Wheat stripe rust caused by Puccinia striatum is a major fungal disease in wheat production. It is widely distributed, spreads quickly, and harms a large area. It is one of the important reasons for wheat production reduction. At present, wheat stripe rust is mainly controlled by chemical pesticides. However, the use of chemical pesticides has aggravated environmental pollution and the resistance of pathogens, making the application of pesticides increasingly restricted. Therefore, it is particularly important to cultivate disease-resistant varieties and explore the resources of disease-resistant genes in wheat. Summary of the invention

[0003] The purpose of the present invention is to provide an application of TaHPPD protein in regulating wheat stripe rust resistance. The present invention finds that TaHPPD protein can negatively regulate wheat stripe rust resistance and can be used to cultivate wheat resistant to stripe rust.

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

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

[0006] As a preferred embodiment, the regulation 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.

[0007] The present invention also provides a target sequence for targeted knockout of the TaHPPD gene, and the target sequence is shown as SEQ ID NO.17.

[0008] The present invention also provides an editing vector for targeted knockout of the TaHPPD gene, wherein the editing vector comprises the target sequence described in the above scheme.

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

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

[0011] The present invention also provides an engineered bacterium for targeted knockout of the TaHPPD gene, wherein the engineered bacterium comprises the editing vector described in the above scheme.

[0012] As a preferred embodiment, the basic bacteria of the engineering bacteria include Agrobacterium.

[0013] The present invention also provides the use of the target sequence described in the above scheme, the editing vector described in the above scheme, or the engineering bacteria described in the above scheme in improving wheat stripe rust resistance or cultivating wheat resistant to stripe rust.

[0014] Beneficial effects:

[0015] The present invention provides an 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. The TaHPPD protein of the present invention can weaken the resistance of CNL proteins to NLR. Moro The accumulation of reactive oxygen species (ROS) and cell necrosis reactions triggered by the invention can reduce ion leakage. The present invention uses gene editing technology to knock out the TaHPPD gene in wheat, obtain TaHPPD knockout (TaHPPD-KO) wheat materials, and obtain wheat with resistance to wheat stripe rust. The results of the embodiment show that in the infection experiment of wheat with stripe rust fungus CYR32, TaHPPD-KO wheat plants showed significant resistance compared with the control Fielder. Specifically, the infection site of the leaves of TaHPPD-KO wheat plants produces a large-area allergic necrosis reaction, and the macroscopic phenotype of reduced summer spores of stripe rust fungi; the microscopic phenotype of increased reactive oxygen area and blocked infection of stripe rust fungi hyphae. The TaHPPD protein can be used to regulate wheat stripe rust resistance and can be used to cultivate wheat resistant to stripe rust. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1Figure 2 is a graph showing the functional verification result of the TaHPPD gene in Example 2; wherein A is a graph showing the phenotype of tobacco inoculated for 48 hours and its DAB staining result; B is a graph showing the phenotype of tobacco inoculated for 72 hours and its trypan blue staining result; C is a graph showing the statistical result of the active oxygen content at 36 hours of inoculation, and D is a graph showing the statistical result of the ion leakage rate at 60 hours and 108 hours of inoculation;

[0018] Figure 2 The Western blot test results in Example 2 are shown in the figure; the left one is the Western blot test result of HA antibody; the right one is the Western blot test result of GFP antibody;

[0019] Figure 3 The result diagram of knocking out TaHPPD in wheat in Example 3 and Comparative Example 1; wherein A is a schematic diagram of the TaHPPD gene editing target; B is a schematic diagram of the TaHPPD gene editing nucleotide sequence; C is the type of expressed protein after TaHPPD editing;

[0020] Figure 4 The results of the infection of TaHPPD-KO and control Fielder wheat by stripe rust in Application Example 1 are shown in Figure 1; A is a typical photo of H2O2 accumulation near the infection point of TaHPPD-KO and Fielder wheat after infection by stripe rust at 72h and 96h after inoculation; B is the statistical result of the H2O2 accumulation area; C is a typical photo of the growth and development of stripe rust in TaHPPD-KO and Fielder wheat at 72h and 96h after inoculation; D is the hyphae area of ​​stripe rust at 72h and 96h after inoculation;

[0021] Figure 5 These are observation pictures of TaHPPD-KO and control Fielder wheat after infection in Application Example 1; A is a comparison of the resistance-susceptibility phenotypes of leaves of TaHPPD-KO plants and Fielder plants; and B is a biomass statistics chart. DETAILED DESCRIPTION

[0022] The present invention provides an application of TaHPPD protein in regulating wheat stripe rust resistance, wherein the TaHPPD protein comprises 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 details are as follows:

[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 an embodiment, the coding sequence of TaHPPD-6A of the present invention is shown as SEQ ID NO.4, the coding sequence of TaHPPD-6B is shown as SEQ ID NO.5, and the coding sequence of TaHPPD-6D is shown as SEQ ID NO.6, which are as follows:

[0027]

[0028]

[0029]

[0030] As an embodiment, the regulation of the present invention includes: increasing the expression of TaHPPD protein to reduce wheat stripe rust resistance, or reducing the expression of TaHPPD protein to increase wheat stripe rust resistance. In a specific embodiment, the method for increasing the expression of TaHPPD protein can be to construct an expression vector and transfer it into wheat; the method for reducing the expression of TaHPPD protein can be to use an editing vector to knock out the wheat TaHPPD gene.

[0031] The present invention also provides a target sequence for targeted knockout of the TaHPPD gene, and the target sequence is shown as SEQ ID NO.17.

[0032] The present invention also provides an editing vector for targeted knockout of the TaHPPD gene, the editing vector comprising the target sequence described in the above scheme. As an embodiment, the editing vector of the present invention further comprises an sgRNA sequence, and the sgRNA sequence is shown in SEQ ID NO. 18. As an embodiment, the target sequence of the present invention is located between the promoter and the sgRNA sequence of the editing vector.

[0033] The present invention also provides an engineering bacterium for targeted knockout of the TaHPPD gene, the engineering bacterium comprising the editing vector described in the above scheme. As an embodiment, the basic bacteria of the engineering bacterium of the present invention comprises Agrobacterium, and in a specific embodiment, the basic bacteria of the engineering bacterium can be Agrobacterium EHA105.

[0034] The present invention also provides the use of the target sequence described in the above scheme, the editing vector described in the above scheme, or the engineering bacteria described in the above scheme in improving wheat stripe rust resistance or cultivating wheat resistant to stripe rust.

[0035] As an 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 can be wheat immature embryos.

[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 in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] Obtaining TaHPPD gene

[0039] The Chinese Spring wheat in the two-leaf stage was inoculated with stripe rust CYR32; the stripe rust CYR32 was disclosed in the literature [Population structure and toxicity characteristics of wheat stripe rust on Aegilops tauschii in Guanzhong, Shaanxi, Wei Guorong et al., Journal of Northwest Agriculture, Vol. 30, No. 8, pp. 1233-1242, 2021]. 48 hours after inoculation, about 100 mg of fresh leaves were cut with clean scissors and placed in a 2 mL RNA-specific centrifuge tube containing sterilized steel balls. The centrifuge tube was marked with the sampled plant number, and then quickly put into liquid nitrogen for rapid freezing. The leaves were crushed using a plant tissue crusher to obtain wheat samples. The leaves were kept at a low temperature during the crushing process to avoid tissue melting and liquid nitrogen expansion causing the centrifuge tube to explode.

[0040] Total RNA was extracted from wheat samples using the Huayueyang Rapid Universal Plant RNA Extraction Kit 3.0 (CAT: 0416-50GK). Since RNA is unstable, the entire operation should be kept as low temperature and as fast as possible.

[0041] RNA was reverse transcribed into cDNA using the IIQ RT SuperMix for qPCR (CAT: R223) was used to reverse transcribe RNA into cDNA.

[0042] Using the cDNA as a template, PCR amplification of TaHPPD-6A and TaHPPD-6D was performed using the primers shown in SEQ ID NO.7 and SEQ ID NO.8, and the amplified products were subjected to agarose gel electrophoresis, and DNA fragments with lengths of 1302 bp and 1311 bp were separated and 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 the primers shown in SEQ ID NO.7 and SEQ ID NO.9, and the amplified products were subjected to agarose gel electrophoresis, and DNA fragments with lengths of 1311 bp were separated and purified and sequenced. The sequencing results are 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] Functional verification of TaHPPD gene

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

[0049] The TaHPPD-6A obtained in Example 1 was connected 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] ① Preparation of pBin-SpeI-EGFP-SpeI-3×HA

[0052] The SpeI recognition sequence and three consecutive HA tag sequences (3×HA) were inserted after the GFP of the pBinGFP2 vector backbone 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 sequence of the 3×HA is shown in SEQ ID NO.11.

[0053]

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

[0055] ②Use Spe I restriction endonuclease to digest the pBin-SpeI-EGFP-SpeI-3×HA vector prepared in the above steps to obtain the linearized vector pBin-SpeI-EGFP-SpeI-3×HA

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

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

[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 vector

[0062] The GFP of the vector pBin-SpeI-EGFP-SpeI-3×HA was removed by Spe I, and the amplified NLR was cloned using one-step cloning technology. Moro The gene was connected to the Spe I site of the vector to obtain pBin-SpeI-NLR Moro -SpeI-3×HA, denoted as NLR Moro -3HA.

[0063] 3) Vector transformation

[0064] The pBinGFP2 vector (denoted as EGFP vector) and the TaHPPD-EGFP vector and NLR prepared in the above steps were Moro -3HA vectors were connected and transformed into Escherichia coli DH5α (transformation method refers to the instructions of Weidi Biotechnology DH5α Chemically Competent Cell, CAT#: DL1001). Use vector universal primers 35S-F and M13-R (SEQ ID NO.15 and SEQ ID NO.16) to detect the colony transfer plasmid and extract the plasmid according to Magen Plasmid Rapid Extraction Kit (CAT: P1001).

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

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

[0067] pBinGFP2 vector and TaHPPD-EGFP vector obtained in the above steps, NLR Moro-3HA vectors were transformed into Agrobacterium EHA105 (pSoup), and the transformation steps were referred to the instructions (Shanghai Weidi Biotechnology Co., Ltd., CAT#: AC1012S). After transformation, single clones were picked and primers 35S-F and M13-R (SEQ ID NO.15 and SEQ ID NO.16) were used to detect the colony transfer plasmid.

[0068] In the clean bench, positive clones were picked up and placed in 10 mL of sterilized LB liquid medium containing Rif and Kan dual resistance, and cultured at 28°C for 3 days at a constant temperature shaker of 220 rpm; the final concentration of Rif in the sterilized LB liquid medium was 20 μg / mL, and the final concentration of Kan was 50 μg / mL. Then, Agrobacterium activation treatment was performed to obtain activated Agrobacterium liquid, which was recorded as EGFP Agrobacterium liquid, TaHPPD-EGFP Agrobacterium liquid and NLR, respectively. Moro -3HA Agrobacterium liquid.

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

[0070] 4) Infection with Nicotiana benthamiana

[0071] Select 5-week-old Nicotiana benthamiana in good growth condition, and use a needle to gently pierce a small hole on the back of the leaf. Be careful not to pierce the leaf. Use a syringe to absorb the mixed activated Agrobacterium solution, gently press the syringe tip against the pierced hole, and slowly inject the solution until the solution spreads in the gaps between the leaves, trying to avoid the veins. When injecting different solutions into a leaf, prevent the injection areas of different solutions from overlapping. Mark the injection area with a marker and return the tobacco to the tobacco room for continued cultivation. When injecting two solutions at the same location, one solution can be injected first, and then the other solution can be injected at the same location 24 hours later. The injection method is as follows: Figure 1 As shown in A and B in the figure, 100 μL of bacterial solution was injected each time. At the same time, EGFP Agrobacterium solution was injected in areas 2 and 3, and TaHPPD-EGFP Agrobacterium solution was injected in areas 4 and 5. After 24 hours, NLR was injected in areas 1, 2, and 4. Moro -3HA Agrobacterium liquid.

[0072] 4) Result detection

[0073] 48 hours after infection, infected leaves were taken, and the macroscopic phenotype was photographed with a camera, and then DAB (3,3'-Diaminobenzidine tetrahydrochloride) was used to stain the H2O2 at the infected site; 72 hours after infection, infected leaves were taken, and the macroscopic phenotype was photographed with a camera, and then trypan blue staining solution was used to stain the cell necrosis at the infected site.

[0074] DAB staining operation method: a. Immerse the leaves in 1mg / mL DAB (pH 3.6) staining solution, and use a vacuum pump to pump for 10 minutes to accelerate the combination of the dye solution; b. Soak in a dark place for 12 hours, and after sufficient staining, use anhydrous ethanol boiling water bath for 10 minutes; c. Soak in anhydrous ethanol until the leaves are bleached and decolorized, and place them in 75% ethanol or clean water to take pictures. The results are shown in Figure 1 A in.

[0075] Trypan blue staining method: a. Immerse the leaves in trypan blue staining solution and boil them in water for 10 minutes until the leaves turn blue; b. Soak for 12 hours and rinse off the excess trypan blue staining solution with clean water; c. Add chloral hydrate aqueous solution to decolorize until the tissue is bleached, changing the chloral hydrate aqueous solution several times during the process, and place it in clean water to take pictures. The results are shown in Figure 1 B in.

[0076] In order to quantitatively detect the content of H2O2, the hydrogen peroxide content detection kit (CAT: BC3595) of Solarbio was used to quantitatively detect the content of H2O2. The specific method is referred to the instruction manual, and the results are referred to Figure 1 C and Table 1.

[0077] Table 1 Data table of H2O2 content in leaves

[0078]

[0079] When the protoplast structure of a plant is affected, its permeability will change, causing organic matter and salts to seep out of the cells. The degree of cell damage can be reflected by the measurement of conductivity, and the degree of plant cell death can be quantified. The detection method is as follows: a. Use a hole puncher to punch holes in 6 leaf discs at the sampling position 60h and 108h after the bacterial solution is injected. Immerse them completely in 4mL sterilized deionized water for 5h, and take 6 biological replicates for each combination; b. Use a conductivity meter to measure the conductivity C1 in the sample solution, boil it in a water bath for 5min, let it cool to room temperature, and use a conductivity meter to measure the conductivity C2 of the sample solution again; c. Calculate the results according to the formula "electrolyte permeation rate = C1 / C2×100%", see the results. Figure 1 D and Table 2.

[0080] Table 2 Electrolyte permeation rate in leaves

[0081]

[0082] In order to verify whether the protein is expressed in tobacco leaves, Western blot was performed using GFP tag and HA tag. The method is as follows: a. Cut the leaves infected with Agrobacterium at the appropriate time, wrap them with tin foil and freeze them quickly with liquid nitrogen; b. Prepare a sterilized mortar in advance and freeze them in liquid nitrogen, take out the leaves and pour them into the mortar containing liquid nitrogen, and fully grind the leaves into powder at low temperature (the main vein of the leaves can be removed); c. Vortex the ground leaves and 1 mL of cell lysate in a 2 mL centrifuge tube to mix them evenly. The cell lysate is Western and IP cell lysate (Biyuntian), and 10 μL of 100 mM phenylmethylsulfonyl fluoride (Phenylmethylsulfonyl fluoride) is added to the lysate. fluoride, PMSF) protease inhibitor. Note that PMSF should not be added in advance; d. Incubate with rotation at 4℃ for 30min; e. Centrifuge at 4℃, 12000rpm for 15min to centrifuge the precipitate as thoroughly as possible; f. Transfer the supernatant to a new centrifuge tube with a pipette on ice, discard the precipitate, and repeat the centrifugation once; g. Take an appropriate amount of supernatant and add SDS-PAGE protein loading buffer (5×) (Biyuntian) in proportion, boil in water bath for 10min, store at -20℃ or perform SDS-polyacrylamide (SDS-PAGE) gel electrophoresis using protein marker as control.

[0083] After electrophoresis, the gel is used to determine the position of the target protein based on the protein marker, and the protein at the corresponding position is cut out for Western blot protein detection. The results are shown in Figure 2 .

[0084] according to Figure 1 It can be seen that transient overexpression of TaHPPD and NLR in Nicotiana benthamiana Moro Protein, NLR Moro Protein can induce cell necrosis. TaHPPD (carrying GFP tag) and NLR Moro After co-expression, the degree of cell necrosis was reduced, while NLR Moro Co-expression with GFP tag did not reduce the extent of cell necrosis, thus, TaHPPD can inhibit the expression of NLR Moro Protein-induced cell necrosis ( Figure 1 B and D), and this phenomenon is due to TaHPPD inhibiting NLR Moro Induced by the accumulation of reactive oxygen species ( Figure 1 A and C). Western blot showed that all proteins were expressed normally ( Figure 2 ). In summary, transient co-expression of NLR in Nicotiana benthamiana Moroand TaHPPD, TaHPPD can attenuate the Moro The accumulation of reactive oxygen species (ROS) and cell necrosis were induced, and ion leakage was greater than that of NLR Moro Reduce.

[0085] Example 3

[0086] Construction of TaHPPD-KO wheat plants

[0087] The selected gene target sequence SEQ ID NO.17 was sent to Zhongji Hi-Tech (Beijing) Biotechnology Co., Ltd. to construct a gene editing vector, and 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, wherein the NGG sequence at the 3' end was not included in the target detection (N represents any nucleotide).

[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 correct, and the gene editing vector was obtained. The gene editing vector was transformed into Agrobacterium EHA105, and the transformation method was the same as in Example 2. The positive plaques were selected and sent to the National Key Laboratory of Crop Stress Resistance and High-efficiency Production of Northwest Agriculture and Forestry University to transform Fielder wheat immature embryos to create transgenic materials. The TaHPPD gene editing target sequence is as follows Figure 3 As shown in Target 1 in A. DNA was extracted from the transformed wheat and SEQ ID NO. 21 and SEQ ID NO. 22 were used to detect positive transgenic lines.

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

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

[0095] The positive plants were tested by PCR using SEQ ID NO.23 and SEQ ID NO.24, and the amplified DNA fragment was about 265 bp long. The PCR product was sent to Hi-Tom (http: / / www.hi-tom.net / hi-tom / ) for high-throughput sequencing to detect the gene editing situation, and the gene-edited material was obtained, which was recorded as TaHPPD-KO.

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

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

[0098] Gene editing results can be found in Figure 3 B and C, where B is a schematic diagram of the TaHPPD gene editing nucleotide sequence, Target1 is the editing site in Example 3, and Target2 is the editing site in Comparative Example 1; C is the type of protein expressed after TaHPPD editing; Fielder in B and C represents the wild-type plant, 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, and the amino acids are inconsistent with the wild type, which indicates that the gene editing is successful. According to Figure 3 It can be seen that the three homologous genes of TaHPPD were all edited and the protein expression was abnormal ( Figure 3 Middle C).

[0099] Comparative Example 1

[0100] A method for constructing TaHPPD-KO wheat plants similar to Example 2, wherein the gene target sequence is as shown in SEQ ID NO. 25 (see Figure 3 The gene target sequence + sgRNA sequence was detected using SEQ ID NO. 26 and SEQ ID NO. 20, wherein the gene target sequence did not include the NGG sequence at the 3' end (N represents any nucleotide) during detection, and a gene editing vector was obtained to transform Agrobacterium EHA105 to create a transgenic material.

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

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

[0103] After sequencing, it was found that when SEQ ID NO.25 was used as the gene target sequence, there was an off-target phenomenon, which was specifically manifested in that the sequencing results were consistent with the Fielder sequence and no editing occurred (see Figure 3 The results showed that the target sequence (SEQ ID NO. 25) failed to play an editing role and failed to successfully construct TaHPPD-KO wheat plants.

[0104] Application Example 1

[0105] Verification of resistance to stripe rust in TaHPPD-KO wheat plants

[0106] (1) Select fresh spores of stripe rust fungus CYR32 within one week and use an appropriate amount of isohexadecane (IHD) to make a 3×10 4 The spore suspension of 1000 g / mL was evenly sprayed on the wheat leaves using an air pump. Fielder wheat was used as the control group, denoted as Fielder. The gene-edited material and its control Fielder used the same batch of spore suspension to ensure that the inoculation amount of the two was consistent.

[0107] After inoculation with stripe rust fungus CYR32, the production of active oxygen and hyphae infection at the infection site of wheat leaves were detected 72h and 96h after inoculation.

[0108] ① The active oxygen in wheat leaves was detected by DAB staining as follows:

[0109] In order to verify the resistance changes caused by gene editing TaHPPD, leaf samples were cut 6 hours before the detection time (i.e., 72 hours and 96 hours of inoculation mentioned above), and the morphological lower end of the leaf was immersed in 1 mg / mL DAB (pH 3.6) staining solution, and the other parts were exposed to the liquid surface and stained under strong light for 6 hours at room temperature. When the leaf veins appear reddish brown, the morphological upper end incision has reddish brown exudation, and black spots are distributed on the surface of the leaf, it proves that the staining is over. Cut the leaves into leaf segments of about 2 cm in length, place them in a new 2 mL centrifuge tube, add a fixing transparent liquid to completely cover the leaf segments, fix and decolorize the leaf segments, and replace the new fixing transparent liquid several times until the leaves no longer fade; the fixing transparent liquid is anhydrous ethanol: glacial acetic acid (1:1, v:v). Discard the fixing transparent liquid, add a chloral hydrate aqueous solution to completely cover the leaf segments, repeat the decolorization several times until the leaf segments are transparent, and replace with a 50% glycerol aqueous solution before microscopic observation for storage. Microscopic observation and statistical results are shown in Figure 4 A and B in.

[0110] Table 3 Average H2O2 area at a single infection point in wheat leaves

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

[0112] ② WGA-Alexa488 staining was used to detect the mycelium infection status. The method was as follows: a. Cut the leaf samples, add fixing transparent liquid [anhydrous ethanol: glacial acetic acid = 1:1, (V:V)] to completely cover the leaf segments, fix and decolorize the leaf segments, and replace the new fixing transparent liquid several times during the period until the leaves no longer fade. b. Discard the fixing transparent solution, add chloral hydrate aqueous solution until the leaf segments are completely covered, and repeat the decolorization several times until the leaf segments are transparent; c. Rinse with 50% ethanol twice, 5 minutes each time, and then rinse with distilled water 3 times; d. Add 1M KOH solution, covering the leaves, and boil in boiling water for about 5 minutes until the leaves become soft (the boiling time depends on the condition of the leaves. Always observe the condition of the leaves. If the boiling time is too long, the leaves will disintegrate and the hyphae structure in the leaves will be destroyed); e. Add 50mM Tris-HCl (pH7.5) to soak the leaves 3 times, 5 minutes each time; f. Use WGA-Alexa488 buffer to stain in the dark for 10 minutes (if the staining time is too long, the staining time can be extended); g. After the staining is completed, add 50mM Tris-HCl (pH7.5) to soak the leaves 3 times, 5 minutes each time, and the leaves are stored in the last Tris-HCl at 4°C for use. Microscope observation statistical results correspond to Figure 4 Middle C and D.

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

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

[0115] In order to observe the resistance changes caused by knocking out TaHPPD, the present invention statistically observed the host cell defense response and fungal growth of TaHPPD-KO wheat after stripe rust infection. The accumulation of active oxygen in TaHPPD-KO wheat was statistically observed by DAB staining. The results showed that the accumulation of active oxygen in TaHPPD-KO wheat was significantly increased compared with Fielder after 72h and 96h of stripe rust infection ( Figure 4 At the same time, the mycelium infection area of ​​TaHPPD-KO wheat was significantly reduced by WGA staining and statistical observation. Figure 4 C and D).

[0116] (2) The present invention further identified the phenotype of gene-edited wheat. After 14 days of inoculation, the phenotypic changes of TaHPPD-KO compared with Fielder were identified. The results are shown in Figure 5 A in the sample and take samples to detect the mycelium content in the leaves.

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

[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: T-VectorpMD TM 19(Simple)(Takara), add 1μL T-Vector pMD TM 19 (Simple) (Takara), 0.3 pmol of the above amplified product (TaEF or PsEF), and add ddH2O to 5 μL. Add 5 μL Solution I, mix thoroughly, react 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 connected to transform E. coli DH5α (the transformation method refers to the instructions of Weidi Biotechnology DH5α Chemically Competent Cell, CAT#: DL1001), the plasmids were extracted and the concentration was measured, and the plasmid copy number was calculated based on the concentration:

[0124]

[0125] The concentration was diluted to 10 using deionized water according to the calculated plasmid copy number. 3 , 10 4 , 10 5 , 10 6 , 10 7 and 108 / μL concentration gradient, using the diluted plasmid concentration gradient using quantitative primers TaEF (SEQ ID NO.27 and SEQ ID NO.28) and PsEF (SEQ ID NO.29 and SEQ ID NO.30) for qRT-PCR, with the logarithm of the plasmid copy number (log) as the horizontal axis, the corresponding C T The values ​​were used as the ordinate for linear fitting to obtain the logarithmic value of the copy number and C T The linear curve equation of the value.

[0126] The qRT-PCR program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, extension at 72°C for 20 s, 40 cycles; 60-95°C with an increase of 1°C each time (melting curve) and holding for 10 s.

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

[0128] according to Figure 5 It can be seen that compared with Fielder, TaHPPD-KO wheat produced a large number of hypersensitive reactions and fewer summer spores ( Figure 5 A); The biomass detection results showed that the content of stripe rust in TaHPPD-KO wheat leaves was significantly reduced ( Figure 5 In summary, compared with the control Fielder, TaHPPD-KO wheat plants showed significant resistance, which was specifically manifested by a large area of ​​hypersensitive necrosis in the infected parts of the leaves of TaHPPD-KO wheat plants, a macroscopic phenotype of reduced summer spores of stripe rust, and a microscopic phenotype of increased active oxygen area and blocked stripe rust hyphae infection.

[0129] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of TaHPPD protein in regulating wheat stripe rust resistance, the TaHPPD protein comprising one or more of TaHPPD-6A, TaHPPD-6B and TaHPPD-6D; the amino acid sequence of the TaHPPD-6A is shown in SEQ ID NO.1, the amino acid sequence of the TaHPPD-6B is shown in SEQ ID NO.2, and the amino acid sequence of the TaHPPD-6D is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that: The coding sequence of the TaHPPD-6A is shown in SEQ ID NO.4, the coding sequence of the TaHPPD-6B is shown in SEQ ID NO.5, and the coding sequence of the TaHPPD-6D is shown in SEQ ID NO.

6.

3. The use according to claim 1, characterized in that: The regulation includes: increasing the expression amount of TaHPPD protein to reduce wheat stripe rust resistance, or decreasing the expression amount of TaHPPD protein to increase wheat stripe rust resistance.

4. A target sequence for targeted knockout of TaHPPD gene, characterized in that: The target sequence is shown in SEQ ID NO.

17.

5. An editing vector for targeted knockout of TaHPPD gene, characterized in that: The editing vector comprises the target sequence of claim 4.

6. The editing vector according to claim 5, characterized in that The editing vector also contains an sgRNA sequence, and the sgRNA sequence is shown in SEQ ID NO.

18.

7. The editing vector according to claim 6, characterized in that The target sequence is located between the promoter and the sgRNA sequence of the editing vector.

8. An engineered bacterium for targeted knockout of the TaHPPD gene, characterized in that: The engineered bacteria comprises the editing vector described in any one of claims 5 to 7.

9. The engineered bacteria according to claim 8, characterized in that The basic bacteria of the engineering bacteria include Agrobacterium.

10. Use of the target sequence according to claim 4, the editing vector according to any one of claims 5 to 7, or the engineered bacteria according to claim 8 or 9 in improving wheat stripe rust resistance or cultivating wheat resistant to stripe rust.

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