Application of wheat TaPP2C32 protein or its encoding gene in regulating wheat stem rust resistance

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to the use of a wheat TaPP2C32 protein or a gene encoding it in regulating wheat stem rust resistance. The present invention provides the use of a wheat TaPP2C32 protein or a gene encoding it in regulating wheat stem rust resistance; the amino acid sequence of the wheat TaPP2C32 protein is shown in SEQ ID NO: 2. The present invention finds that the TaPP2C32 protein encoding can regulate the expression of PR genes and ROS scavenging genes. Compared with the control, plants expressing the wheat TaPP2C32 protein encoding gene can weaken wheat stem rust resistance, while plants expressing the protein overexpressing the gene can enhance wheat stem rust resistance. Therefore, the TaPP2C32 protein encoding gene is involved in regulating wheat stem rust resistance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of wheat TaPP2C32 protein or its encoding gene in regulating wheat stem rust resistance. Background Art

[0002] Wheat stem rust is a wheat disease caused by the wheat-specific form of the fungus Puccinia graminis. It primarily affects wheat stems and leaf sheaths, but can also damage leaves and ears. The uredia are oblong, high-raised, brownish-yellow, and irregularly scattered. When the uredia mature, the epidermis cracks in large areas and turns outward like lips, releasing rust-brown uredia. Later, black teliospores form, and the epidermis cracks, releasing black, rust-like teliospores. Using and planting disease-resistant varieties is the most cost-effective strategy for controlling stem rust. However, the population structure and virulence of wheat stem rust frequently vary, easily leading to a loss of resistance in the variety.

[0003] During the interaction between wheat and stem rust, plants will produce a series of defense mechanisms to resist the invasion of stem rust. Protein phosphatase 2C (PP2C) is a Mg-dependent + or Mn + Monomeric serine / threonine protein phosphatases play an important role in plant stress signaling. However, their role in the interaction between wheat and stem rust remains largely unknown. Therefore, identifying genes associated with wheat stem rust resistance is crucial for understanding the mechanisms of wheat resistance to stem rust and for wheat production and breeding. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of wheat TaPP2C32 protein or its encoding gene in regulating wheat stem rust resistance, enrich wheat stem rust resistance-related genes, improve wheat stem rust resistance, prepare stem rust-resistant transgenic wheat, and lay the foundation for genetic breeding of stem rust-resistant wheat.

[0005] The present invention provides the use of wheat TaPP2C32 protein or its encoding gene in regulating wheat stem rust resistance; the amino acid sequence of the wheat TaPP2C32 protein is shown in SEQ ID NO: 2.

[0006] Preferably, the regulation includes positively regulating the expression level of the wheat TaPP2C32 protein or its encoding gene to improve the wheat's resistance to stem rust; or negatively regulating the expression level of the wheat TaPP2C32 protein or its encoding gene to reduce the wheat's resistance to stem rust.

[0007] The present invention also provides the use of wheat TaPP2C32 protein or its encoding gene in genetic breeding of stem rust-resistant wheat and / or preparation of stem rust-resistant transgenic wheat; the amino acid sequence of the wheat TaPP2C32 protein is shown in SEQ ID NO: 2.

[0008] Preferably, the nucleotide sequence of the wheat TaPP2C32 protein encoding gene is shown in SEQ ID NO: 1.

[0009] The present invention also provides a silent fragment of the wheat TaPP2C32 protein encoding gene, wherein the silent fragment is amplified using wheat cDNA as a template and primers V-TaPP2C32-F and V-TaPP2C32-R;

[0010] The nucleotide sequence of the primer V-TaPP2C32-F is shown in SEQ ID NO: 3;

[0011] The nucleotide sequence of the primer V-TaPP2C32-R is shown in SEQ ID NO: 4.

[0012] The present invention also provides a BSMV gene silencing vector for the wheat TaPP2C32 protein encoding gene, wherein the BSMV gene silencing vector comprises a pCa-γbLIC vector, a pCaBS-α vector and a pCaBS-β vector; the pCa-γbLIC vector contains the silencing fragment described in the above technical solution.

[0013] The present invention also provides a recombinant Agrobacterium for silencing the wheat TaPP2C32 protein encoding gene. The recombinant Agrobacterium comprises a basic Agrobacterium and the BSMV gene silencing vector described in the above technical solution introduced into the basic Agrobacterium.

[0014] The present invention also provides a method for regulating wheat stem rust resistance, comprising the following steps:

[0015] Regulate the expression of the wheat TaPP2C32 protein encoding gene in wheat;

[0016] The amino acid sequence of the wheat TaPP2C32 protein is shown in SEQ ID NO: 2.

[0017] Preferably, the wheat stem rust resistance is improved by increasing the expression level of the wheat TaPP2C32 protein encoding gene in the wheat;

[0018] By silencing the wheat TaPP2C32 protein encoding gene in the wheat, the wheat's resistance to stem rust is reduced.

[0019] Preferably, the method for silencing the wheat TaPP2C32 protein encoding gene in the wheat comprises: using the recombinant Agrobacterium described in the above technical solution to infect wheat.

[0020] Beneficial effects:

[0021] The present invention provides a use of a wheat TaPP2C32 protein or a gene encoding it in regulating wheat stem rust resistance; the amino acid sequence of the wheat TaPP2C32 protein is shown in SEQ ID NO: 2. By silencing the TaPP2C32 protein-encoding gene in wheat, the present invention found that PR genes (TaPR1, TaPR2, and TaPR5) specifically induced by pathogens or exogenous hormones in wheat were significantly downregulated, while ROS scavenging genes (TaCAT and TaSOD) were significantly upregulated. After inoculation with stem rust, spore piles increased and the area of ​​stem rust colonies increased. By overexpressing the TaPP2C32 protein-encoding gene in wheat, the present invention found that PR genes (TaPR1, TaPR2, and TaPR5) specifically induced by pathogens or exogenous hormones in wheat were significantly upregulated, while ROS scavenging genes (TaCAT and TaSOD) were significantly downregulated. After inoculation with stem rust, mycelial growth was inhibited, and the area of ​​stem rust colonies was significantly reduced. Compared with the control variety, silencing the wheat TaPP2C32 protein-encoding gene can weaken the wheat stem rust resistance, and overexpressing the wheat TaPP2C32 protein-encoding gene can enhance the wheat stem rust resistance. The wheat TaPP2C32 protein or its encoding gene has a regulatory effect on the wheat stem rust resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0023] Figure 1 Electrophoresis diagram of TaPP2C32 connected to pCa-γbLIC vector (BSMVγ) (A) and phenotype of tobacco plants injected with different BSMV vectors (B);

[0024] Figure 2 Figure 2 shows the resistance of wheat to stem rust after silencing TaPP2C32. A shows the phenotype of the silenced wheat strain 10 days after inoculation with BSMV virus. B shows the relative expression level of TaPP2C32 detected by qPCR 5 days after inoculation with BSMV virus. C shows the phenotype of the silenced wheat strain 14 days after inoculation with wheat stem rust. D shows the relative expression level of TaPP2C32 during infection with wheat stem rust. n = 3, mean ± standard error, **P < 0.01, ***P < 0.0001. <P≤0.001,****P<0.0001;

[0025] Figure 3The expression of related genes in the wheat TaPP2C32 silenced strain inoculated with wheat stem rust; A to E are the expression changes of TaPR1, TaPR2, TaPR5, TaSOD, and TaCAT, respectively; n = 3, mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.0001 <P≤0.001,****P<0.0001;

[0026] Figure 4 Figure 2 shows the resistance of wheat to stem rust after overexpression of TaPP2C32. A is the electrophoresis of the CDS sequence of TaPP2C32. B is the expression level of TaPP2C32 in the TaPP2C32 overexpressing strain detected by qPCR. C is the phenotype of the TaPP2C32 overexpressing strain 14 days after inoculation with wheat stem rust. D is the relative expression level of TaPP2C32 in the TaPP2C32 overexpressing strain during stem rust infection. n = 3, mean ± standard error, **P < 0.01, ***P < 0.0001 <P≤0.001,****P<0.0001;

[0027] Figure 5 Figure 3 shows the hyphal growth of wheat TaPP2C32 overexpressing strains after inoculation with wheat stem rust. A is the result of laser confocal microscopy observation; B is the hyphal length of stem rust; C is the colony area of ​​stem rust; n = 3, mean ± standard error, ***P < 0.0001.

[0028] Figure 6 The expression of related genes in the wheat TaPP2C32 overexpression strain after inoculation with wheat stem rust; A to E are the expression changes of TaPR1, TaPR2, TaPR5, TaSOD, and TaCAT, respectively; n = 3, mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.0001 <P≤0.001,****P<0.0001。 DETAILED DESCRIPTION

[0029] The present invention provides the use of wheat TaPP2C32 protein or its encoding gene in regulating wheat stem rust resistance; the amino acid sequence of the wheat TaPP2C32 protein is shown in SEQ ID NO: 2.

[0030] As an embodiment, the regulation of the present invention includes positively regulating the expression level of the wheat TaPP2C32 protein or its encoding gene to improve the wheat's resistance to stem rust; or negatively regulating the expression level of the wheat TaPP2C32 protein or its encoding gene to reduce the wheat's resistance to stem rust.

[0031] The present invention also provides the use of wheat TaPP2C32 protein or its encoding gene in genetic breeding of stem rust-resistant wheat and / or preparation of stem rust-resistant transgenic wheat; the amino acid sequence of the wheat TaPP2C32 protein is shown in SEQ ID NO: 2.

[0032] As an embodiment, the wheat stem rust of the present invention is caused by wheat stem rust fungus (Puccinia graminisf.sp.tritici Erikss.&E.Henn.).

[0033] As an embodiment, the nucleotide sequence of the wheat TaPP2C32 protein encoding gene of the present invention is shown as SEQ ID NO: 1.

[0034] The sequence information of SEQ ID NO: 1-2 of the present invention is as follows:

[0035]

[0036] SEQ ID NO:2:MSCSVAIPSSPVFSPSRRPLSCKAASASPESVSVSSPAPST AGSPLRPFGLLRAQIREEASPSPKTSSAAPSVAAGSVLKRRRPAPLMVPVDGAAAAAAAAAAVAAVESDPSNEVEEEGDEFAAYCRRGRGRRRVEMEDRHVAKVALGGDPEVALFAVFDGHGGKNAAEFAAQNMPKFMAEVVRKVDGGDSDEIEGAVKKCYLKTDEEFLKRE ESGGACCVTALLQKGGLTVSNTGDCRAVLSRAGTAEALTSDHRASREDERERIENLGGFVVNNRGTWRVQGSLAVSRGIGDAHLKQWVVADPDTRTLLVDPQCEFLVLASDGLWDKVDNQEAIDIARPLCIGNDKTSRIAACRRLVETAGSRGSTDDISVLIIQLQKFSGSS.

[0037] The present invention also provides a silent fragment of the wheat TaPP2C32 protein encoding gene, wherein the silent fragment is amplified using wheat cDNA as a template and primers V-TaPP2C32-F and V-TaPP2C32-R;

[0038] The nucleotide sequence of the primer V-TaPP2C32-F is shown in SEQ ID NO: 3;

[0039] The nucleotide sequence of the primer V-TaPP2C32-R is shown in SEQ ID NO: 4.

[0040] The present invention also provides a BSMV gene silencing vector for the wheat TaPP2C32 protein encoding gene, wherein the BSMV gene silencing vector comprises a pCa-γbLIC vector, a pCaBS-α vector and a pCaBS-β vector; the pCa-γbLIC vector contains the silencing fragment described in the above technical solution.

[0041] The present invention also provides a recombinant Agrobacterium for silencing the wheat TaPP2C32 protein encoding gene. The recombinant Agrobacterium comprises a basic Agrobacterium and the BSMV gene silencing vector described in the above technical solution introduced into the basic Agrobacterium.

[0042] In one embodiment, the basic Agrobacterium of the present invention includes GV3101. In one embodiment, the mass ratio of the pCa-γbLIC vector, the pCaBS-α vector, and the pCaBS-β vector in the recombinant Agrobacterium of the present invention is 1:1:1.

[0043] The present invention also provides a method for regulating wheat stem rust resistance, comprising the following steps:

[0044] Regulate the expression of the wheat TaPP2C32 protein encoding gene in wheat;

[0045] The amino acid sequence of the wheat TaPP2C32 protein is shown in SEQ ID NO: 2.

[0046] In one embodiment, the present invention improves wheat stem rust resistance by increasing the expression of the wheat TaPP2C32 protein-encoding gene in the wheat; or reduces wheat stem rust resistance by silencing the wheat TaPP2C32 protein-encoding gene in the wheat. In one embodiment, the method of silencing the wheat TaPP2C32 protein-encoding gene in the wheat comprises: infecting wheat with the recombinant Agrobacterium described in the above technical solution. In one embodiment, the present invention infects wheat leaves with the recombinant Agrobacterium described in the above technical solution. The present invention does not have strict requirements on the specific steps of the infection; routine procedures in the art can be used.

[0047] To further illustrate the present invention, the application of the wheat TaPP2C32 protein or its encoding gene provided by the present invention in regulating wheat stem rust resistance is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0048] The biological materials used in the following examples are as follows.

[0049] (1) “Sr6” wheat and “Sr10” wheat: stem rust-resistant single gene lines, published in “Li Tianya, Cao Yuanyin, Li Weihua, et al. Derivation and analysis of stem rust-resistant genes in 52 important wheat varieties from four provinces in northern my country [J]. Henan Agricultural Sciences, 2011, 40(10): 84-87.”

[0050] (2) 21C3: physiological race of wheat stem rust, published in “Li Tianya, Cao Yuanyin, Li Weihua, et al. Derivation and analysis of stem rust resistance genes in 52 important wheat varieties from four provinces in northern my country [J]. Henan Agricultural Sciences, 2011, 40(10): 84-87.”

[0051] (3) Barley stripe mosaic virus (BSMV) VIGS vector system (pCa-γbLIC plasmid, pCaBS-α plasmid and pCaBS-β plasmid), disclosed in “Yuan C, Li C, Yan L, Jackson AO, Liu Z, Han C, Yu J, Li D (2011) A High Throughput Barley Stripe Mosaic Virus Vector for Virus Induced Gene Silencing in Monocots and Dicots. PLoS One, Volume 6, Issue 10, e26468”; wherein, the pCa-γbLIC plasmid contains the DNA sequence corresponding to the γb RNA in the three chains of barley stripe mosaic virus (BSMV), and at the same time, a LIC linker site is introduced downstream of the γb gene in the plasmid for cloning exogenous gene fragments. In the examples of the present invention, the pCa-γbLIC plasmid is referred to as the BSMVγ vector;

[0052] (4) pLGY-02 vector, published in “Liang Fang, Liu Yifei, Cui Zhongchi, et al. Rapid identification of the function of wheat pathogenesis-related protein gene TaPR1 using Agrobacterium-mediated method [J]. Journal of Hebei Agricultural University, 2019, 42(2): 12-17.”

[0053] The culture medium used in the following examples is as follows.

[0054] (1) LB liquid medium: weigh 2 g of sodium chloride, 1 g of yeast extract powder, and 2 g of tryptone, dilute to 200 mL, and sterilize at 120°C for 20 min;

[0055] (2) LB solid medium: weigh 2 g of sodium chloride, 1 g of yeast extract powder, 2 g of tryptone, and 4 g of agar powder, dilute to 200 mL, and sterilize at 120°C for 20 min.

[0056] The PCR primers used in the following examples are shown in Table 1.

[0057] Table 1 PCR primers used in the examples

[0058] Primer Name Primer Sequence (5'-3') V-TaPP2C32-F AAGGAAGTTTAACGGCCGAGGCACTCAC(SEQ ID NO:3) V-TaPP2C32-R AACCACCACCACCGTACCTTATCCCACAAGCCATCAGA(SEQ ID NO:4) OE-TaPP2C32-F GGTACCATGTCCTGCTCCGTCGC(SEQ ID NO:5) OE-TaPP2C32-R ACTAGTAGAAGAACCTGAGAACTTCTGCA(SEQ ID NO:6) q-TaPP2C32-F AGAATTGAGAACCTGGGCGG(SEQ ID NO:7) q-TaPP2C32-R TGGCGAtGTCTATGGCTTCC(SEQ ID NO:8) q-TaPR1-F CAATAACCTCGGCGTCTTCATC(SEQ ID NO:9) q-TaPR1-R ATTTACTCGCTCGGTCCCTC(SEQ ID NO:10) q-TaPR2-F AGGATGTTGCTTCCATGTTTGCCG(SEQ ID NO:11) q-TaPR2-R AAGTAGATGCGCATGCCGTTGATG(SEQ ID NO:12) q-TaPR5-F CAAGCAGTGGTATCAACGCAGAG(SEQ ID NO:13) q-TaPR5-R GTGAAGCCACAGTTGTTCTTGATGTT(SEQ ID NO:14) q-TaCAT-F GCCTGTGTTTTTTATCCGAGA(SEQ ID NO:15) q-TaCAT-R CAGGTGCCTCCAACAGTAACA(SEQ ID NO:16) q-TaSOD-F GGCTCTCCAAGGTCGTGT(SEQ ID NO:17) q-TaSOD-R GGGTTGCCGTTGTTGTAG(SEQ ID NO:18) BSMV-11 GGTAGAACTGATGTGAGAGATGTAGAAG(SEQ ID NO:19) BSMV-32 TGGTCTTCCCTTGGGGGACCGAA(SEQ ID NO:20) V-NbPDS-F AAGGAAGTTTAAATTTGCACCCGCAGAAGA(SEQ ID NO:21) V-NbPDS-R AACCACCACCACCGTCTTCTGTTTCGTGTAGTCACCA(SEQ ID NO:22) q-TaGAPDH-F TGCCTTGCTCGTCTTGCTAA(SEQ ID NO:23) q-TaGAPDH-R CTTGATGGAAGGACCATCAAC(SEQ ID NO:24)

[0059] The main instruments used in the following examples are shown in Table 2.

[0060] Table 2 Main instruments used in the examples

[0061]

[0062] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, commercially available or prepared according to conventional methods in the art, and the specifications are laboratory pure. Unless otherwise specified, the experimental methods and conditions used in the following examples are all conventional experimental methods and conditions in the art, and reference can be made to relevant experimental manuals, known documents or manufacturer specifications. Unless otherwise defined, the meaning of all technical and scientific terms used in the present invention is the same as that generally understood by those of ordinary skill in the art to which the present invention belongs.

[0063] Example 1

[0064] 1. Extraction of Total RNA from Wheat

[0065] Total RNA from wheat was extracted using the Trizol method. The specific steps are as follows: wheat leaves were placed in a mortar and ground into powder in liquid nitrogen. The powder was then placed in a pre-cooled 1.5 mL centrifuge tube and placed on ice. 1 mL of RNAiso Plus (Takara) was quickly added and mixed by inversion until the sample was completely dissolved in the RNAiso Plus. 200 μL of chloroform was added, the tube was shaken for 15 seconds, and centrifuged at 12,000 rpm for 5 minutes. The supernatant (approximately 530 μL) was transferred to a new 1.5 mL centrifuge tube. An equal volume of isopropanol was added and mixed by inversion by hand. The tube was allowed to stand at 4°C for 20 minutes, and centrifuged at 12,000 rpm for 20 minutes. The supernatant was discarded and the white precipitate was retained. 350 μL of 75% ethanol was added and pipetted. The tube was centrifuged at 12,000 rpm for 5 minutes, and the ethanol was aspirated. The tube was opened and placed in a clean bench and aired for 5 minutes. 20 μL of sterile water was added and mixed to obtain an RNA solution, which was stored in a -20°C refrigerator.

[0066] 2. Synthesis of Wheat cDNA

[0067] The wheat total RNA obtained in step 1 was reverse transcribed using the Novozymes RNA Reverse Transcription Kit (Cat. No. R423-01) according to the kit instructions to synthesize the first-strand cDNA, which served as a template for gene cloning. The resulting wheat cDNA was stored in a refrigerator at -20°C.

[0068] Example 2

[0069] Construction of TaPP2C32 silencing vector

[0070] 1. Cloning of target genes

[0071] (1) Using the wheat cDNA obtained in Example 1 as a template, the primers V-TaPP2C32-F and V-TaPP2C32-R (Tm: 58°C) in Table 1 were used to amplify the TaPP2C32 silencing target fragment V-TaPP2C32 (250 bp). The PCR reaction system and procedure were as follows:

[0072] PCR reaction system: 10× KOD buffer 5 μL, 2 mM dNTPs 5 μL, 25 mM MgSO 4 2 μL, 10 pmol / μL V-TaPP2C32-F 1.5 μL, 10 pmol / μL V-TaPP2C32-R 1.5 μL, cDNA ≤ 100 ng (RNA equiv.) / 50 μL 2 μL, KOD Plus (1.0 U / μL) 1 μL, DMSO (Solybol, Cat. No. D8370) 1 μL, and ddH 2 O to 50 μL; KOD Plus was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.

[0073] PCR reaction program: 94°C for 2 min; 94°C for 15 s, 58°C for 30 s, 68°C for 15 s, 35 cycles; stored at 4°C.

[0074] (2) PCR products were detected by 1.0% agarose gel electrophoresis (200 V, 15 min). The target band was recovered using a DNA purification kit (Takara, Cat. No. 9761) according to the kit instructions to obtain the TaPP2C32 silencing target fragment V-TaPP2C32, which was then stored at -20°C.

[0075] 2. Digest the BSMVγ vector with ApaⅠ endonuclease (Takara) using the following digestion system and conditions: 3 μL of 10× Buffer, 1 μL of ApaⅠ, 5 μL of BSMVγ vector, and 21 μL of ddH2O. Incubate the digestion reaction system in a 37°C metal bath for 1 hour.

[0076] After the reaction, the digested product was subjected to 1.0% agarose gel electrophoresis and recovered using a DNA purification kit (Takara, Cat. No. 9761) according to the kit instructions to obtain the purified BSMVγ linear vector, which was then stored in a -20°C refrigerator.

[0077] 3. Use Vazyme's T4 DNA Polymerase to connect the purified BSMVγ linear vector with the TaPP2C32 silencing target fragment V-TaPP2C32 as follows.

[0078] (1) Treatment of purified BSMVγ linear vector

[0079] Prepare a reaction system according to the ratio of 10×T4 DNApol Buffer 2μL, 0.1% BSA 1μL, dTTP (100nM) 1μL, T4 DNA Polymerase 0.5μL, purified BSMVγ linear vector 200ng and ddH2O to 20μL; add the above system to a PCR tube, incubate at 25°C for 90min; then at 72°C for 20min; cool the resulting reaction solution on ice for 5-10min.

[0080] (2) Processing of TaPP2C32 silencing target fragment

[0081] Prepare a reaction system with 2 μL of 10× T4 DNApol Buffer, 1 μL of 0.1% BSA, 1 μL of dATP (100 nM), 0.5 μL of T4 DNA Polymerase, 100 ng of the purified TaPP2C3 silencing target fragment, and ddH2O to a volume of 20 μL. Add this system to a PCR tube and incubate at 25°C for 90 minutes and then at 72°C for 20 minutes. Chill the resulting reaction solution on ice for 5–10 minutes.

[0082] (3) Take 10 μL of the BSMVγ linear vector treated in step (1) and 20 μL of the TaPP2C32 silencing target fragment treated in step (2), mix thoroughly, and gradually heat to 66°C for 2 minutes, then place at room temperature for 10 minutes to obtain a ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells by heat shock method and screened with kanamycin. Pick 6 to 8 monoclonal clones and use 2×Taq MasterMix (Dye Plus) of Nanjing Novozymes Biotechnology Co., Ltd., primers BSMV-11 and BSMV-32 in Table 1, and perform bacterial liquid PCR verification according to the following reaction system and reaction procedure.

[0083] PCR reaction system: 2× Taq Master Mix (Dye Plus) 10 μL, 10 μM BSMV-111 μL, 10 μM BSMV-321 μL, bacterial solution 1 μL, and ddH2O to 20 μL;

[0084] The PCR reaction program was as follows: 98°C for 30 s; 35 cycles of 98°C for 30 s, 58°C for 30 s, and 72°C for 10 s; and 72°C for 5 min. After the reaction, the amplified product was sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing verification.

[0085] (4) The positive colonies that were sequenced correctly were extracted with a rapid plasmid extraction kit (Takara, catalog number: 9760) and detected by 1.0% agarose gel electrophoresis (200V, 15min) to obtain the TaPP2C32 silencing vector BSMVγ:TaPP2C32 ( Figure 1 Middle A).

[0086] Comparative Example 1

[0087] Construction of NbPDS silencing vector

[0088] NbPDS is the tobacco phytoene dehydrogenase PDS gene, and its GenBank accession number is ABE99707.1. Using Nicotiana benthamiana leaf cDNA and primers V-NbPDS-F and V-NbPDS-R in Table 1, the NbPDS silencing target fragment V-NbPDS was amplified according to the method of Example 2 to construct the NbPDS silencing vector BSMVγ:NbPDS; wherein the nucleotide sequence of the NbPDS silencing target fragment V-NbPDS is shown in SEQ ID NO: 25, specifically: 5'-ATTTGCACCCGCAGAAGAGTGGATAAATCGTAGTGACTCAGAAATTATT GATGCTACAATGAAGGAACTAGCGAAGCTTTTCCCTGATGAAATTTCGGCAGATCAGAGCAAAGCAAAAATATTGAAGTATCATGTTGTCAAAACCCCAAGGTCTGTTTATAAAACTGTGCCAGGTTGTGAACCCTGTCGGCCCTTGCAAAGATCCCCTATAGAGGGTTTTTATTTAGCTGGTGACTACACGAAACAGAAG-3'.

[0089] Example 3

[0090] Construction of TaPP2C32 overexpression vector

[0091] 1. Cloning of target genes

[0092] (1) Using the wheat cDNA obtained in Example 1 as a template, the full-length TaPP2C32 gene coding sequence (CDS) OE-TaPP2C32 (1158 bp) was amplified using the primers OE-TaPP2C32-F and OE-TaPP2C32-R (Tm: 58°C) in Table 1. The PCR reaction system and procedure were as follows:

[0093] The PCR reaction system consisted of 5 μL of 10× KOD buffer, 5 μL of 2 mM dNTPs, 2 μL of 25 mM MgSO₄, 1.5 μL of 10 pmol / μL OE-TaPP2C32-F, 1.5 μL of 10 pmol / μL OE-TaPP2C32-R, 2 μL of cDNA ≤ 100 ng (RNA equiv.) / 50 μL, 1 μL of KOD Plus (1.0 U / μL), 1 μL of DMSO (Solybol, Cat. No. D8370), and ddH₂O to a volume of 50 μL. KODPlus was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.

[0094] PCR reaction program: 94°C for 2 min; 94°C for 15 s, 58°C for 30 s, and 68°C for 60 s, 35 cycles; stored at 4°C.

[0095] (2) PCR products were detected by 1.0% agarose gel electrophoresis (200 V, 15 min). The target band was recovered using a DNA purification and recovery kit (Takara, Cat. No. 9761) according to the kit instructions to obtain the full-length TaPP2C32 gene CDS OE-TaPP2C32 (overexpressed gene fragment) and stored at -20°C.

[0096] 2. Use Kpn I and Spe I endonucleases (Takara) to double-digest the pLGY-02 vector and the overexpressed gene fragment OE-TaPP2C32 obtained in step 1, respectively, and purify and recover them. The enzyme digestion system and conditions are as follows:

[0097] 10× Buffer 3μL, KpnⅠ 0.5μL, Spe I 0.5μL, pLGY-02 vector / OE-TaPP2C32 fragment 5μL, and ddH2O 21μL; the prepared enzyme digestion reaction system was placed in a 37°C metal bath for 1 hour;

[0098] After the reaction, the digestion products were analyzed by 1.0% agarose gel electrophoresis. DNA purification and recovery kit (Takara, Cat. No. 9761) was used according to the kit instructions to obtain the pLGY-02 linear vector and OE-TaPP2C32 fragment after double digestion and purification, respectively, and stored in a -20°C refrigerator.

[0099] 3. Use Vazyme's T4 DNA Ligase to ligate the double-digested and purified pLGY-02 linear vector and double-digested and purified OE-TaPP2C32 fragment to obtain a ligation product. The ligation system and conditions are as follows: 1.5 μL of double-digested and purified pLGY-02 linear vector, 6.5 μL of double-digested and purified OE-TaPP2C32 fragment, 1 μL of T4 DNA Ligase Buffer, and 1 μL of T4 DNA Ligase; 22°C for 3 hours; 70°C for 10 minutes.

[0100] 4. Transform the ligation product into E. coli DH5α competent cells using the heat shock method and select with kanamycin. Pick 6 to 8 single clones and perform bacterial PCR verification using 2× Taq Master Mix (DyePlus) from Nanjing Novozymes Biotechnology Co., Ltd. according to the following reaction system and reaction procedure. PCR reaction system: 2× Taq Master Mix (Dye Plus) 10μL, 10μM BSMV-111μL, 10μM BSMMV-321μL, bacterial solution 1μL, and ddH2O to 20μL; PCR reaction procedure: 98℃ for 30s; 98℃ for 30s, 58℃ for 30s, 72℃ for 10s, 35 cycles; 72℃ for 5min. After the reaction, the amplified product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing verification.

[0101] 5. The positive colonies that were sequenced correctly were extracted with a rapid plasmid extraction kit (Takara, Cat. No. 9760) and detected by 1.0% agarose gel electrophoresis (200V, 15min) to obtain the TaPP2C32 overexpression vector pLGY-02:TaPP2C32 ( Figure 4 Middle A).

[0102] Example 4

[0103] Transformation of Agrobacterium competent cells

[0104] Agrobacterium GV3101 competent cells were transformed with the plasmids BSMVγ, BSMVγ:TaPP2C32, BSMVγ:NbPDS, pCaBS-α, pCaBS-β, pLGY-02, and pLGY-02:TaPP2C32, respectively. The Agrobacterium transformation method was as follows: GV3101 competent cells were removed from a -80°C freezer, thawed on ice, added with 5 μL of plasmid, gently pipetted to mix, and incubated on ice for 20 min. The cells were then snap-frozen in liquid nitrogen for 1 min, then 37°C for 5 min, and incubated on ice for 5 min. In a laminar flow hood, 400 μL of LB liquid medium was added and shaken at 200 rpm at 30°C for 2.5 h. The cells were centrifuged at 5000 rpm for 1 min, most of the supernatant was aspirated, and the remaining liquid was used to resuspend the cells. The cells were then plated onto LB solid medium containing kanamycin. The cells were sealed and incubated upside down at 30°C for 48 h.

[0105] The obtained recombinant Agrobacterium were named GV3101 / BSMVγ, GV3101 / BSMVγ:TaPP2C32, GV3101 / BSMVγ:NbPDS, GV3101 / pCaBS-α, GV3101 / pCaBS-β, GV3101 / pLGY-02 and GV3101 / pLGY-02:TaPP2C32 according to the introduced plasmids.

[0106] Example 5

[0107] TaPP2C32 gene silencing

[0108] (1) The recombinant Agrobacterium GV3101 / BSMVγ, GV3101 / BSMVγ:TaPP2C32, GV3101 / BSMVγ:NbPDS, GV3101 / pCaBS-α, and GV3101 / pCaBS-β obtained in Example 4 were inoculated into LB liquid culture medium containing kanamycin (50 mg / L) and cultured on a shaking platform at 30°C for 12 to 16 hours. The Agrobacterium liquid was taken out and centrifuged at 4000 rpm for 10 minutes at room temperature. The supernatant was discarded, and the cells were washed three times with the prepared Agrobacterium suspension in the dark, and then the cells were suspended. The OD of the bacterial liquid was then adjusted. 600nm The formula of Agrobacterium suspension is as follows: take 500 μL 10mM MES, 50 μL 150mM AAs, 5mL 10mM MgCl2, add sterile water to 50mL, mix well and set aside.

[0109] (2) Adjust the OD 600nmThree mixed bacterial solutions were prepared by mixing BSMVγ recombinant Agrobacterium (GV3101 / BSMVγ, GV3101 / BSMVγ:TaPP2C32, GV3101 / BSMVγ:NbPDS), GV3101 / pCaBS-α recombinant Agrobacterium, and GV3101 / pCaBS-β recombinant Agrobacterium at a volume ratio of 1:1:1. The mixed bacterial solutions were left at room temperature for 2–3 hours to activate, and then injected into leaves of Nicotiana benthamiana plants, grouped as follows:

[0110] Empty vector control tobacco (BSMVγ): injection of a mixed bacterial solution of GV3101 / BSMVγ, GV3101 / pCaBS-α, and GV3101 / pCaBS-β at a volume ratio of 1:1:1;

[0111] Positive control (BSMVγ:NbPDS): Inject a mixed bacterial solution of GV3101 / BSMVγ:NbPDS, GV3101 / pCaBS-α and GV3101 / pCaBS-β in a volume ratio of 1:1:1.

[0112] Tobacco silenced strain (BSMVγ:TaPP2C32): Inject a mixed bacterial solution of GV3101 / BSMVγ:TaPP2C32, GV3101 / pCaBS-α and GV3101 / pCaBS-β in a volume ratio of 1:1:1.

[0113] (3) After the tobacco leaves have dried, place the injected tobacco leaves back into the light incubator and culture them until the virus appears in the tobacco leaves before inoculating wheat. The phenotype of the tobacco leaves 10 days after virus inoculation is as follows: Figure 1 As shown in B. Figure 1 As can be seen in Figure B, compared with healthy Nicotiana benthamiana, the positive control (BSMVγ:NbPDS) has obvious albinism, and the empty vector control tobacco (BSMVγ) and the tobacco silenced strain (BSMVγ:TaPP2C32) both have obvious mottled chlorosis, indicating that BSMV has successfully invaded tobacco.

[0114] The specific steps of wheat inoculation are as follows: add 5mL PBS buffer and about 0.5g of tobacco leaves 10 days after virus inoculation into a sterilized mortar, grind into juice, mix thoroughly by pipetting, and place in a 2mL centrifuge tube and store on ice. Sprinkle a little quartz sand on the surface of wheat "Sr10" leaves, then put on latex gloves and dip into the juice of tobacco leaves to inoculate the wheat leaves with BSMV virus. Set up empty vector control wheat (wheat inoculated with BSMVγ), wheat silenced strain (inoculated with BSMVγ:TaPP2C32) and positive control wheat (wheat inoculated with BSMVγ:NbPDS). After inoculation, the wheat was placed in a 24℃ incubator for culture. After 10 days, the symptoms of BSMV infection were clearly observed ( Figure 2 Middle A).

[0115] (4) Extract the total RNA from the leaves of the empty vector control wheat and the wheat silenced strain after virus inoculation and reverse transcribe it into cDNA. Using cDNA as a template, the specific primers q-TaPP2C32-F / q-TaPP2C32-R (Tm value is 58°C) of the TaPP2C32 gene in Table 1 were used for real-time fluorescence quantitative PCR to detect the silencing effect of TaPP2C32. At the same time, the primers q-TaGAPDH-F and q-TaGAPDH-R (Tm value is 58°C) of the internal reference gene TaGAPDH (GenBank accession number is LOC123160238) in Table 1 were used for real-time fluorescence quantitative PCR. -ΔΔCt The relative expression of TaPP2C32 gene was calculated by the analysis method. The results are shown in Figure 2. Figure 2 As shown in Figure B, the system and procedure of real-time fluorescence quantitative PCR are as follows:

[0116] Fluorescence quantitative PCR reaction system: ChamQ Universal SYBR qPCR Master Mix (Novozymes) 10 μL, forward primer (F) 2 μL, reverse primer (R) 2 μL and cDNA 6 μL;

[0117] Fluorescence quantitative PCR reaction program: 95°C for 30 s; 95°C for 10 s, 58°C for 10 s, 60°C for 40 s, 35 cycles; 95°C for 1 min; 55°C for 30 s; 95°C for 30 s.

[0118] according to Figure 2 As can be seen in Figure B, compared with the BSMVγ empty vector control wheat, the relative expression level of TaPP2C32 in the wheat BSMVγ:TaPP2C32 silenced strain was significantly reduced, indicating that the TaPP2C32 gene was successfully silenced.

[0119] Example 6

[0120] TaPP2C32 gene overexpression

[0121] Recombinant Agrobacterium GV3101 / pLGY-02: TaPP2C32 and GV3101 / pLGY-02 were inoculated into LB liquid medium containing kanamycin (50 mg / L) in a clean bench and cultured on a shaking platform at 30°C for 12-16 hours. The Agrobacterium liquid was removed and centrifuged at 4000 rpm for 10 minutes at room temperature. The supernatant was discarded and the cells were washed three times with the prepared Agrobacterium suspension in the dark and then suspended. The OD of the bacterial liquid was then adjusted. 600nm The formula of Agrobacterium suspension is as follows: take 500 μL 10 mM MES, 50 μL 150 mM AAs, 5 mL 10 mM MgCl2, add sterile water to 50 mL, mix well and set aside.

[0122] Place the bacterial solution at room temperature and dark conditions for 2 to 3 hours for activation, and then inject it into the leaves of wheat "Sr6" respectively. The wheat injected with GV3101 / pLGY-02:TaPP2C32 is a TaPP2C32 overexpression strain. The wheat injected with GV3101 / pLGY-02 is an empty vector control plant. After the wheat leaves become dry, put them back into the light incubator for culture. 72 hours after the injection, the total RNA of the leaves of the wheat TaPP2C32 overexpression strain and the empty vector control plant was extracted and reverse transcribed into cDNA, and the TaPP2C32 overexpression effect was detected by real-time fluorescence quantitative PCR. The fluorescence quantitative PCR detection method is the same as the detection method in Example 5. The results are as follows. Figure 4 As shown in B.

[0123] The results are as follows Figure 4 As shown in B, 72 h after injection, the relative expression level of TaPP2C32 in the TaPP2C32 overexpression strain was significantly increased compared with that in the empty vector control plant, indicating that the TaPP2C32 gene was successfully overexpressed.

[0124] Example 7

[0125] Resistance testing of TaPP2C32 silenced and TaPP2C32 overexpressing strains to stem rust

[0126] (1) Wheat plants 10 days after inoculation with BSMVγ or BSMVγ:TaPP2C32, and 3 days after injection with GV3101 / pLGY-02 or GV3101 / pLGY-02:TaPP2C32, were selected and inoculated with wheat stem rust 21C3. The inoculation method was as follows: Tween and water were mixed at a volume ratio of 20:1000 and placed in a spray bottle. 1 g of uredurea of ​​wheat stem rust 21C3 was added and shaken well. The mixture was inoculated onto the leaves of the wheat plants. The plants were kept moist at 16-18°C for 16-20 hours and then moved to a greenhouse for incubation.

[0127] (2) After the wheat plants were inoculated with wheat stem rust, total RNA was extracted from the wheat leaves at different inoculation times and reverse transcribed into cDNA. The relative expression levels of the TaPP2C32 gene at different inoculation times were detected according to the method of Example 5. The results are as follows: Figure 2 D and Figure 4 As shown in D. Figure 2 As shown in Figure D, within 96 hours of inoculation, the relative expression level of TaPP2C32 in the wheat BSMVγ:TaPP2C32 silenced strain was significantly reduced during the infection of stem rust compared with the BSMVγ empty vector control wheat. Figure 4 As shown in D, within 96 hours of inoculation, the relative expression level of TaPP2C32 in the TaPP2C32 overexpression strain was significantly increased compared with the empty vector control plant, indicating that the TaPP2C32 gene was successfully overexpressed.

[0128] (3) Total RNA was extracted from the leaves of the wheat plants 24 h, 48 h, and 120 h after inoculation with wheat stem rust and reverse transcribed into cDNA. Real-time fluorescence quantitative PCR was used to detect the expression levels of related genes such as TaPR1 (GenBank accession number LOC543437), TaPR2 (GenBank accession number LOC123080647), TaPR5 (GenBank accession number LOC543342), TaCAT (GenBank accession number LOC123168517), and TaSOD (GenBank accession number LOC101290631) in the wheat plants 24 h, 48 h, and 120 h after inoculation with wheat stem rust. The fluorescence quantitative PCR detection method was the same as that of Example 5. The PCR primers for TaPR1 are q-TaPR1-F / q-TaPR1-R (Tm value: 59°C), the PCR primers for TaPR2 are q-TaPR2-F / q-TaPR2-R (Tm value: 64°C), the PCR primers for TaPR5 are q-TaPR5-F / q-TaPR5-R (Tm value: 62°C), the PCR primers for TaCAT are q-TaCAT-F / q-TaCAT-R (Tm value: 58°C), and the PCR primers for TaSOD are q-TaSOD-F / q-TaSOD-R (Tm value: 58°C). The nucleotide sequences are shown in Table 1.

[0129] The results showed that the expression of three PR genes (TaPR1, TaPR2 and TaPR5) in the wheat TaPP2C32 silenced strain (BSMVγ:TaPP2C32) was significantly downregulated compared with the empty vector control (BSMVγ). Figure 3A to C), reactive oxygen species (ROS) scavenging genes (TaSOD and TaCAT) were significantly up-regulated ( Figure 3 D and E). Compared with the empty vector control (pLGY-02), the expression levels of three PR genes (TaPR1, TaPR2 and TaPR5) in the wheat TaPP2C32 overexpression strain (pLGY-02:TaPP2C32) were significantly upregulated ( Figure 6 A to C), the expression levels of ROS scavenging genes (TaCAT and TaSOD) were significantly downregulated ( Figure 6 The above-mentioned PR genes (Pathogenesis-related genes) are a type of genes that are specifically induced by pathogens or exogenous hormones and are closely related to systemic acquired resistance.

[0130] (4) Laser confocal microscopy was used to observe the mycelial infection of wheat TaPP2C32 overexpression strains at 12h, 24h, 48h and 96h after inoculation with wheat stem rust. Compared with the empty vector control (pLGY-02), mycelial infection in the wheat TaPP2C32 overexpression strain (pLGY-02:TaPP2C32) was significantly inhibited ( Figure 5 (A~B).

[0131] (5) The wheat stem rust colony area of ​​the TaPP2C32-silenced and TaPP2C32-overexpressing strains was counted 96 hours after inoculation with wheat stem rust. The phenotypes of the wheat TaPP2C32-silenced and TaPP2C32-overexpressing strains were observed 14 days after inoculation with wheat stem rust to identify the wheat stem rust infection type. The wheat stem rust infection type grading standard 0 to 4 is as follows:

[0132] 0: No allergic freckles occur;

[0133] 0;: no summer spores but yellow-white allergic spots can be seen;

[0134] 1: There are tiny summer spores but they are surrounded by obvious yellow-white allergic dead spots;

[0135] 2: Small to medium-sized summer spores but often inhabit green islands surrounded by spindle-shaped allergic dieback;

[0136] 3: Medium-sized uredia, rarely fused; there should be no allergic dieback around the uredia, but there may be chlorosis;

[0137] 4: The summer spores are large and often fused. There is no allergic dieback but chlorosis may occur.

[0138] In the infection type levels 1, 2, 3, and 4, if the lesions of the same infection type are larger, add "+"; if they are smaller, add "-". Among them, 0, 0;, 1-, 1, 1+, 2-, 2, and 2+ belong to low infection types (disease-resistant), and 3-, 3, 3+, 4-, 4, and 4+ belong to high infection types (susceptible).

[0139] The results showed that the infection type of the BSMVγ empty vector control plant was 2, and the infection type of the TaPP2C32 silenced strain BSMVγ:TaPP2C32 was 3. The spore piles on the TaPP2C32 silenced strain were much larger than those on the BSMVγ empty vector control plant ( Figure 2 The infection type of the pLGY-02 empty vector control plant was 4, and the infection type of the TaPP2C32 overexpression strain pLGY-02:TaPP2C32 was 1 ( Figure 4 C), the colony area of ​​stem rust fungi on the TaPP2C32 overexpressing strain was significantly smaller than that on the pLGY-02 empty vector control plant ( Figure 5 This indicates that the TaPP2C32 gene has a positive regulatory effect on wheat stem rust resistance.

[0140] Based on the above content, it can be seen that wheat TaPP2C32 protein or its encoding gene is involved in the positive regulation of wheat resistance to stem rust.

[0141] Although the above embodiment provides a detailed description of the present invention, 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 scope of protection of the present invention.

Claims

1. Application of wheat TaPP2C32 protein or its encoding gene in regulating wheat stem rust resistance; the amino acid sequence of the wheat TaPP2C32 protein is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that The regulation includes positively regulating the expression level of the wheat TaPP2C32 protein or its encoding gene to improve the wheat's resistance to stem rust; or negatively regulating the expression level of the wheat TaPP2C32 protein or its encoding gene to reduce the wheat's resistance to stem rust.

3. Use of wheat TaPP2C32 protein or its encoding gene in genetic breeding of stem rust-resistant wheat and / or preparation of stem rust-resistant transgenic wheat; the amino acid sequence of the wheat TaPP2C32 protein is shown in SEQ ID NO:

2.

4. The use according to any one of claims 1 to 3, characterized in that The nucleotide sequence of the wheat TaPP2C32 protein encoding gene is shown in SEQ ID NO:

1.

5. A silent fragment of the wheat TaPP2C32 protein encoding gene, characterized in that: The silencing fragment was amplified using wheat cDNA as a template using primers V-TaPP2C32-F and V-TaPP2C32-R; The nucleotide sequence of the primer V-TaPP2C32-F is shown in SEQ ID NO: 3; The nucleotide sequence of the primer V-TaPP2C32-R is shown in SEQ ID NO:

4.

6. A BSMV gene silencing vector encoding the wheat TaPP2C32 protein gene, characterized in that: The BSMV gene silencing vector includes a pCa-γbLIC vector, a pCaBS-α vector and a pCaBS-β vector; the pCa-γbLIC vector contains the silencing fragment according to claim 5.

7. A recombinant Agrobacterium for silencing the wheat TaPP2C32 protein encoding gene, characterized in that: The recombinant Agrobacterium comprises a basic Agrobacterium and the BSMV gene silencing vector according to claim 6 introduced into the basic Agrobacterium.

8. A method for regulating wheat stem rust resistance, characterized in that: The steps include: Regulate the expression of the wheat TaPP2C32 protein encoding gene in wheat; The amino acid sequence of the wheat TaPP2C32 protein is shown in SEQ ID NO:

2.

9. The method according to claim 8, characterized in that Improving the wheat's resistance to stem rust by increasing the expression level of the wheat TaPP2C32 protein encoding gene in the wheat; By silencing the wheat TaPP2C32 protein encoding gene in the wheat, the wheat's resistance to stem rust is reduced.

10. The method according to claim 9, characterized in that The method for silencing the wheat TaPP2C32 protein encoding gene in the wheat comprises: using the recombinant Agrobacterium according to claim 7 to infect wheat.

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