Application of wheat TaPP2C32 protein or coding gene thereof in regulating wheat stem rust resistance

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of wheat TaPP2C32 protein or a coding gene thereof to regulation and control of wheat stem rust resistance. The invention provides an application of a wheat TaPP2C32 protein or a coding gene thereof in regulation and control of wheat stem rust resistance. The amino acid sequence of the wheat TaPP2C32 protein is as shown in SEQ ID NO: 2. It is found that TaPP2C32 protein coding can regulate and control expression of a PR gene and an ROS scavenging gene, compared with control, wheat TaPP2C32 protein coding gene plants can be silenced, wheat stalk rust resistance can be weakened, wheat plants can be over-expressed, the wheat stalk rust resistance can be enhanced, and the TaPP2C32 protein coding gene participates in regulation and control of the wheat stalk rust resistance.

Description

Technical Field

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

[0002] Wheat Stem Rust is a disease of wheat caused by the wheat-specific form of Puccinia graminis. It mainly harms the stems and leaf sheaths of wheat, and can also harm the leaves and ears. The summer spores are oblong, high-raised, brown-yellow, and scattered irregularly; when the summer spores mature, the epidermis cracks in large areas and turns outward like lips, emitting rust-brown summer spore powder. In the later stage, black winter spores are produced, and the epidermis breaks to emit black rust-like winter spores. The use and planting of disease-resistant varieties is the most economical and effective strategy for preventing and controlling stem rust, but the population structure and virulence of wheat stem rust fungi frequently vary, which can easily lead to the loss of resistance of varieties.

[0003] During the interaction between wheat and stem rust, the plant 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 signal transduction. However, their role in the interaction between wheat and stem rust is still unclear. Therefore, exploring genes related to wheat stem rust resistance is of great value in enriching the resistance mechanism of wheat to stem rust and in wheat production and breeding. Summary of the invention

[0004] The purpose of the present invention is to provide an 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 a foundation for genetic breeding of stem rust-resistant wheat.

[0005] The present invention provides 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.

[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 a wheat TaPP2C32 protein encoding gene, the nucleotide sequence of which is shown in SEQ ID NO:25.

[0010] The present invention also provides a BSMV gene silencing vector of wheat TaPP2C32 protein encoding gene, 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.

[0011] The present invention also provides a recombinant Agrobacterium for silencing a gene encoding a wheat TaPP2C32 protein. The recombinant Agrobacterium comprises a basic Agrobacterium and the BSMV gene silencing vector described in the above technical solution introduced into the basic Agrobacterium.

[0012] The present invention also provides a method for regulating wheat stem rust resistance, comprising the following steps:

[0013] Regulate the expression of the gene encoding the wheat TaPP2C32 protein in wheat;

[0014] The amino acid sequence of the wheat TaPP2C32 protein is shown in SEQ ID NO:2.

[0015] Preferably, the resistance of wheat to stem rust is improved by increasing the expression level of the wheat TaPP2C32 protein encoding gene in the wheat;

[0016] By silencing the wheat TaPP2C32 protein encoding gene in the wheat, the wheat's resistance to stem rust is reduced.

[0017] 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.

[0018] Beneficial effects:

[0019] The present invention provides an 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. The present invention finds by silencing the TaPP2C32 protein encoding gene in wheat that the PR genes (TaPR1, TaPR2 and TaPR5) specifically induced by pathogens or exogenous hormones in wheat are significantly downregulated, ROS scavenging genes (TaCAT and TaSOD) are significantly upregulated, and after inoculation with stem rust, the spore piles increase and the colony area of ​​stem rust increases; the TaPP2C32 protein encoding gene is overexpressed in wheat and it is found that the PR genes (TaPR1, TaPR2 and TaPR5) specifically induced by pathogens or exogenous hormones in wheat are significantly upregulated, ROS scavenging genes (TaCAT and TaSOD) are significantly downregulated, and after inoculation with stem rust, the hyphae growth is inhibited and the colony area of ​​stem rust is significantly reduced. Compared with the control varieties, silencing the wheat TaPP2C32 protein encoding gene can weaken the resistance to wheat stem rust, and overexpressing the wheat TaPP2C32 protein encoding gene can enhance the resistance to wheat stem rust. The wheat TaPP2C32 protein or its encoding gene has a regulatory effect on the resistance to wheat stem rust. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 The electrophoresis diagram of TaPP2C32 connected to the pCa-γbLIC vector (BSMVγ) (A) and the phenotype of tobacco injected with different BSMV vectors (B);

[0022] Figure 2 The resistance of wheat to stem rust after TaPP2C32 was silenced; A is the phenotype of the wheat silenced strain 10 days after inoculation with BSMV virus; B is the relative expression of TaPP2C32 detected by qPCR after 5 days of inoculation with BSMV virus; C is the phenotype of the wheat silenced strain 14 days after inoculation with wheat stem rust; D is the relative expression of TaPP2C32 during the infection of wheat silenced strain; n = 3, mean ± standard error, **P < 0.01, *** 0.0001 <P≤0.001,****P<0.0001;

[0023] 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, *** 0.0001 <P≤0.001,****P<0.0001;

[0024] Figure 4 The resistance of wheat to stem rust after overexpression of TaPP2C32; A is the electrophoresis of CDS sequence of TaPP2C32; B is the expression level of TaPP2C32 in TaPP2C32 overexpression strain detected by qPCR; C is the phenotype of TaPP2C32 overexpression strain 14 days after inoculation with wheat stem rust; D is the relative expression level of TaPP2C32 in TaPP2C32 overexpression strain during stem rust infection; n=3, mean±SE, **P<0.01, ***0.0001 <P≤0.001,****P<0.0001;

[0025] Figure 5 The hyphae growth of wheat TaPP2C32 overexpressing strain after inoculation with wheat stem rust; A is the result of laser confocal microscopy observation; B is the hyphae length of stem rust; C is the colony area of ​​stem rust; n=3, mean ± standard error, ****P<0.0001;

[0026] Figure 6 The expression of related genes in the wheat TaPP2C32 overexpression 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, *** 0.0001 <P≤0.001,****P<0.0001。 DETAILED DESCRIPTION

[0027] The present invention provides 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.

[0028] 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.

[0029] 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.

[0030] 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.).

[0031] As an embodiment, the nucleotide sequence of the wheat TaPP2C32 protein encoding gene of the present invention is shown as SEQ ID NO:1.

[0032] The sequence information of SEQ ID NO: 1-2 of the present invention is as follows:

[0033]

[0034] SEQ ID NO:2:MSCSVAIPSSPVFSPSRRPLSCKAASASPESVSVSSPAPSTAGSPLRPFGLLRAQIREEASPSPKTSSAAPSVAAGSVLKRRRPAPLMVPVDGAAAAAAAAAAVAAVESDPSNEVEEEGDEFAAYCRRGRGRRRVEMEDRHVAKVALGGDPEVALFAVFDGHGGKNAAEFAAQNMPKFMAEVVRKVDGGD SDEIEGAVKKCYLKTDEEFLKREESGGACCVTALLQKGGLTVSNTGDCRAVLSRAGTAEALTSDHRASREDERIENLGGFVVNNRGTWRVQGSLAVSRGIGDAHLKQWVVADPDTRLLVDPQCEFLVLASDGLWDKVDNQEAIDIARPLCIGNDKTSRIAACRRLVETAGSRGSTDDISVLIIQLQKFSGSS.

[0035] The present invention also provides a silent fragment of a wheat TaPP2C32 protein encoding gene, the nucleotide sequence of which is shown in SEQ ID NO:25.

[0036] The present invention also provides a BSMV gene silencing vector of wheat TaPP2C32 protein encoding gene, 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.

[0037] The present invention also provides a recombinant Agrobacterium for silencing a gene encoding a wheat TaPP2C32 protein. The recombinant Agrobacterium comprises a basic Agrobacterium and the BSMV gene silencing vector described in the above technical solution introduced into the basic Agrobacterium.

[0038] As an embodiment, the basic Agrobacterium of the present invention includes GV3101. As an 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.

[0039] The present invention also provides a method for regulating wheat stem rust resistance, comprising the following steps:

[0040] Regulate the expression of the gene encoding the wheat TaPP2C32 protein in wheat;

[0041] The amino acid sequence of the wheat TaPP2C32 protein is shown in SEQ ID NO:2.

[0042] As an embodiment, the present invention improves the wheat resistance to stem rust by increasing the expression of the wheat TaPP2C32 protein encoding gene in the wheat; or reduces the wheat resistance to stem rust by silencing the wheat TaPP2C32 protein encoding gene in the wheat. As an embodiment, the method of silencing the wheat TaPP2C32 protein encoding gene in the wheat of the present invention comprises: infecting wheat with the recombinant Agrobacterium described in the above technical solution. As an embodiment, the present invention infects wheat leaves with the recombinant Agrobacterium described in the above technical solution. The present invention has no strict requirements on the specific steps of the infection, and conventional operations in the art can be used.

[0043] 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 in conjunction with the drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] The biological materials used in the following examples are as follows.

[0045] (1) “Sr6” and “Sr10” wheat: single gene lines for resistance to stem rust, published in “Li Tianya, Cao Yuanyin, Li Weihua, et al. Analysis of stem rust resistance genes in 52 important wheat varieties from four provinces in northern my country [J]. Henan Agricultural Science, 2011, 40(10): 84-87.”

[0046] (2) 21C3: physiological race of wheat stem rust, published in “Li Tianya, Cao Yuanyin, Li Weihua, et al. Analysis of stem rust resistance genes in 52 important wheat varieties from four provinces in northern my country [J]. Henan Agricultural Science, 2011, 40(10): 84-87.”

[0047] (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 the barley stripe mosaic virus (BSMV), and at the same time, a LIC connection site is introduced downstream of the γb gene in the plasmid for cloning foreign gene fragments. In the embodiment of the present invention, the pCa-γbLIC plasmid is referred to as the BSMVγ vector;

[0048] (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.”

[0049] The culture medium used in the following examples is as follows.

[0050] (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;

[0051] (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, make up to 200 mL, and sterilize at 120°C for 20 min.

[0052] The PCR primers used in the following examples are shown in Table 1.

[0053] Table 1 PCR primers used in the examples

[0054]

[0055]

[0056] The main instruments used in the following examples are shown in Table 2.

[0057] Table 2 Main instruments used in the examples

[0058]

[0059] If not otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure. If not 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 meanings of all technical and scientific terms used in the present invention are the same as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0060] Example 1

[0061] 1. Extraction of Total RNA from Wheat

[0062] The total RNA of wheat was extracted by Trizol method, and the specific steps are as follows: put the wheat leaves into a mortar, grind them into powder in liquid nitrogen, put them into a pre-cooled 1.5mL centrifuge tube and place them on ice, quickly add 1mL RNAiso Plus (Takara), mix them upside down until the sample is completely dissolved in RNAiso Plus; add 200μL chloroform, shake for 15s, centrifuge at 12000rpm for 5min, take the supernatant (about 530μL) and transfer it to a new 1.5mL centrifuge tube; add an equal volume of isopropanol, mix them upside down by hand, let them stand at 4℃ for 20min, centrifuge at 12000rpm for 20min, discard the supernatant and keep the white precipitate; add 350μL 75% ethanol, blow, centrifuge at 12000rpm for 5min, and aspirate the ethanol; open the lid and place it in a clean bench and blow for 5min; add 20μL sterile water and mix them to obtain RNA solution, which is stored in a -20℃ refrigerator.

[0063] 2. Synthesis of wheat cDNA

[0064] The total RNA of wheat obtained in step 1 was reverse transcribed using the RNA reverse transcription kit (Cat. No. R423-01) of Novozymes according to the kit instructions to synthesize the first strand of cDNA as a template for gene cloning. The obtained wheat cDNA was stored in a -20°C refrigerator.

[0065] Example 2

[0066] Construction of TaPP2C32 silencing vector

[0067] 1. Cloning of target gene

[0068] (1) Using the wheat cDNA obtained in Example 1 as a template, the primers V-TaPP2C32-F and V-TaPP2C32-R (Tm is 58° C.) in Table 1 were used to amplify the TaPP2C32 silencing target fragment V-TaPP2C32 (250 bp); wherein the PCR reaction system and procedure are as follows:

[0069] PCR reaction system: 10×KOD buffer 5μL, 2mM dNTPs 5μL, 25mM MgSO 4 2μL, 10pmol / μLV-TaPP2C32-F 1.5μL, 10pmol / μLV-TaPP2C32-R 1.5μL, cDNA≤100ng(RNAequiv.) / 50μL 2μL, KOD Plus (1.0U / μL) 1μL, DMSO (Solerbo, Cat. No. D8370) 1μL and ddH 2 O was added to 50 μL; KOD Plus was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.

[0070] 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.

[0071] (2) Detect the PCR product by 1.0% agarose gel electrophoresis (200 V, 15 min). Use a DNA purification recovery kit (Takara, Cat. No. 9761) to recover the target band according to the kit instructions to obtain the TaPP2C32 silencing target fragment V-TaPP2C32, which was stored in a -20°C refrigerator.

[0072] 2. Use ApaⅠ endonuclease (Takara) to digest the BSMVγ vector. The digestion system and conditions are as follows: 10× Buffer 3μL, ApaⅠ 1μL, BSMVγ vector 5μL and ddH 2 O 21μL; the prepared enzyme digestion reaction system was placed in a 37℃ metal bath for 1h;

[0073] After the reaction, the digestion product was detected by 1.0% agarose gel electrophoresis. A DNA purification recovery kit (Takara, item number 9761) was used to recover the purified BSMVγ linear vector according to the kit instructions and stored in a -20°C refrigerator.

[0074] 3. Use Vazyme's T4 DNA Polymerase to connect the purified BSMVγ linear vector with the TaPP2C32 silencing target fragment V-TaPP2C32 as follows.

[0075] (1) Treatment of purified BSMVγ linear vector

[0076] The mixture was prepared by mixing 2 μL of 10×T4 DNApol Buffer, 1 μL of 0.1% BSA, 1 μL of dTTP (100 nM), 0.5 μL of T4 DNA Polymerase, 200 ng of purified BSMVγ linear vector and ddH 2 O to make up to 20 μL to prepare the reaction system; add the above system into a PCR tube, 25°C, 90 min; 72°C, 20 min; the obtained reaction solution is cooled on ice for 5 to 10 min.

[0077] (2) Processing of TaPP2C32 silencing target fragment

[0078] The 10×T4 DNApol Buffer (2 μL), 0.1% BSA (1 μL), dATP (100 nM) (1 μL), T4 DNA Polymerase (0.5 μL), purified TaPP2C3 silencing target fragment (100 ng) and ddH 2 O to a ratio of 20 μL to prepare the reaction system; add the above system to a PCR tube, 25°C, 90 min; 72°C, 20 min. The obtained reaction solution is cooled on ice for 5 to 10 min.

[0079] (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 min, then place at room temperature for 10 min to obtain a ligation product. The ligation product was transformed into E. coli DH5α competent cells by heat shock method and screened with kanamycin. Pick 6 to 8 monoclonal clones, use 2×Taq MasterMix (Dye Plus) of Nanjing Novogene Biotechnology Co., Ltd., use primers BSMV-11 and BSMV-32 in Table 1, and perform bacterial liquid PCR verification according to the following reaction system and reaction procedure.

[0080] PCR reaction system: 2×TaqMasterMix (Dye Plus) 10μL, 10μM BSMV-11 1μL, 10μM BSMV-32 1μL, bacterial solution 1μL and ddH 2 O to make up to 20 μL;

[0081] PCR reaction program: 98℃30s; 98℃30s, 58℃30s, 72℃10s, 35 cycles; 72℃5min. After the reaction, the amplified product was sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing verification.

[0082] (4) The positive colonies with correct sequencing were extracted with a rapid plasmid extraction kit (Takara, catalog number: 9760) and detected by 1.0% agarose gel electrophoresis (200 V, 15 min) to obtain the TaPP2C32 silencing vector BSMVγ:TaPP2C32 ( Figure 1 (A).

[0083] Comparative Example 1

[0084] Construction of NbPDS silencing vector

[0085] NbPDS is the tobacco phytoene dehydrogenase PDS gene, and its GenBank accession number is ABE99707.1. Using the leaf cDNA of Nicotiana benthamiana and the primers V-NbPDS-F and V-NbPDS-R in Table 1, the NbPDS silencing target fragment V-NbPDS was amplified in the manner 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'.

[0086] Example 3

[0087] Construction of TaPP2C32 overexpression vector

[0088] 1. Cloning of target gene

[0089] (1) Using the wheat cDNA obtained in Example 1 as a template, the primers OE-TaPP2C32-F and OE-TaPP2C32-R (Tm is 58° C.) in Table 1 were used to amplify the full-length TaPP2C32 gene coding sequence (CDS) OE-TaPP2C32 (1158 bp); wherein the PCR reaction system and procedure are as follows:

[0090] PCR reaction system: 10×KOD buffer 5μL, 2mM dNTPs 5μL, 25mM MgSO 4 2μL, 10pmol / μL OE-TaPP2C32-F 1.5μL, 10pmol / μL OE-TaPP2C32-R 1.5μL, cDNA≤100ng(RNAequiv.) / 50μL2μL, KOD Plus (1.0U / μL) 1μL, DMSO (Solerbo, Cat. No. D8370) 1μL and ddH 2 O was added to 50 μL; KOD Plus was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.

[0091] PCR reaction program: 94°C for 2 min; 94°C for 15 s, 58°C for 30 s, 68°C for 60 s, 35 cycles; stored at 4°C.

[0092] (2) PCR products were detected by 1.0% agarose gel electrophoresis (200 V, 15 min). The target band was recovered using a DNA purification recovery kit (Takara, Cat. No. 9761) according to the kit instructions to obtain the full-length OE-TaPP2C32 CDS of the TaPP2C32 gene (overexpressed gene fragment), which was stored in a -20°C refrigerator.

[0093] 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. The enzyme digestion system and conditions are as follows:

[0094] 10× Buffer 3μL, Kpn Ⅰ 0.5μL, Spe I 0.5μL, pLGY-02 vector / OE-TaPP2C32 fragment 5μL and ddH 2 O 21μL; the prepared enzyme digestion reaction system was placed in a 37℃ metal bath for 1h;

[0095] After the reaction, the digestion products were detected by 1.0% agarose gel electrophoresis. A DNA purification and recovery kit (Takara, item number 9761) was used for recovery according to the kit instructions to obtain the pLGY-02 linear vector and OE-TaPP2C32 fragment purified by double digestion, respectively, and stored in a -20°C refrigerator.

[0096] 3. Use Vazyme's T4 DNALigase to connect the pLGY-02 linear vector and the OE-TaPP2C32 fragment after double digestion and purification to obtain the ligation product. The connection system and conditions are as follows: 1.5μL of pLGY-02 linear vector after double digestion and purification, 6.5μL of OE-TaPP2C32 fragment after double digestion and purification, 1μL of T4 DNALigase Buffer and 1μL of T4 DNALigase; 22℃, 3h; 70℃, 10min.

[0097] 4. The ligation product was transformed into E. coli DH5α competent cells by heat shock method and screened with kanamycin. 6 to 8 monoclonal clones were selected and verified by bacterial liquid PCR using 2×Taq MasterMix (DyePlus) of Nanjing Novozyme Biotechnology Co., Ltd. according to the following reaction system and reaction procedure. PCR reaction system: 2×TaqMasterMix (Dye Plus) 10μL, 10μM BSMV-111μL, 10μM BSMMV-321μL, bacterial liquid 1μL and ddH 2 O was added to 20 μL; PCR reaction program: 98℃30s; 98℃30s, 58℃30s, 72℃10s, 35 cycles; 72℃5min. After the reaction, the amplified product was sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing verification.

[0098] 5. The positive colonies with correct sequencing 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 (A).

[0099] Example 4

[0100] Transformation of Agrobacterium competent cells

[0101] The plasmids BSMVγ, BSMVγ: TaPP2C32, BSMVγ: NbPDS, pCaBS-α, pCaBS-β, pLGY-02 and pLGY-02: TaPP2C32 were used to transform Agrobacterium GV3101 competent cells. The Agrobacterium transformation method is as follows: take out the GV3101 competent cells from the -80℃ refrigerator, melt on ice, add 5μL of plasmid, gently pipette to mix, and stand on ice for 20min; quick freeze with liquid nitrogen for 1min, 37℃ for 5min, and stand on ice for 5min; add 400μL LB liquid culture medium to the clean bench, shake at 30℃ and 200rpm for 2.5h; centrifuge at 5000rpm for 1min, discard most of the supernatant, resuspend the bacteria with the remaining small amount of liquid and spread on LB solid culture medium containing kanamycin, seal and invert and culture at 30℃ for 48h.

[0102] According to the introduced plasmids, 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.

[0103] Example 5

[0104] TaPP2C32 gene silencing

[0105] (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 in a shaking incubator 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 Agrobacterium suspension prepared in the dark, and then the cells were suspended, and the OD of the bacterial liquid was adjusted. 600nm The formula of Agrobacterium suspension is: 500 μL 10 mM MES, 50 μL 150 mM As, 5 mL 10 mM MgCl 2 , add sterile water to make up to 50 mL, mix well and set aside.

[0106] (2) Adjust the OD 600nmThe bacterial solutions of BSMVγ recombinant Agrobacterium (GV3101 / BSMVγ, GV3101 / BSMVγ:TaPP2C32, GV3101 / BSMVγ:NbPDS), GV3101 / pCaBS-α recombinant Agrobacterium and GV3101 / pCaBS-β recombinant Agrobacterium were mixed in a volume ratio of 1:1:1 to obtain three mixed bacterial solutions. The mixed bacterial solutions were placed at room temperature for 2 to 3 hours for activation, and then injected into leaves of Nicotiana benthamiana, respectively, and the specific groups were as follows:

[0107] Empty vector control tobacco (BSMVγ): injected with a mixed bacterial solution of GV3101 / BSMVγ, GV3101 / pCaBS-α, and GV3101 / pCaBS-β at a volume ratio of 1:1:1;

[0108] 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.

[0109] 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.

[0110] (3) After the tobacco leaves have dried, place the injected tobacco back into the light incubator and incubate until the virus appears in the tobacco before inoculating wheat. The phenotype of tobacco leaves 10 days after virus inoculation is as follows: Figure 1 As shown in B. Figure 1 As can be seen in B, compared with the 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.

[0111] The specific steps of wheat inoculation are as follows: add 5 mL of PBS buffer and approximately 0.5 g of tobacco leaves 10 days after virus inoculation into a sterilized mortar, grind into juice, mix thoroughly by pipetting, and place in a 2 mL centrifuge tube and store on ice. Sprinkle a little quartz sand on the surface of the wheat "Sr10" leaves, then put on latex gloves and dip into the juice of the tobacco leaves to inoculate the wheat leaves with BSMV virus. Set up empty vector control wheat (wheat inoculated with BSMVγ), wheat silent strain (inoculated with BSMVγ: TaPP2C32) and positive control wheat (wheat inoculated with BSMVγ: NbPDS). After inoculation, the wheat was cultured in a 24°C incubator. After 10 days, the symptoms of BSMV infection were clearly observed ( Figure 2 (A).

[0112] (4) Extract 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, real-time fluorescence quantitative PCR was performed using the specific primers q-TaPP2C32-F / q-TaPP2C32-R (Tm value is 58°C) of the TaPP2C32 gene in Table 1 to detect the silencing effect of TaPP2C32. At the same time, real-time fluorescence quantitative PCR was performed using the primers q-TaGAPDH-F and q-TaGAPDH-R (Tm value is 58°C) of the internal reference gene TaGAPDH (GenBank accession number LOC123160238) in Table 1. Using 2 -ΔΔCt The relative expression of TaPP2C32 gene was calculated by the analysis method. The results are shown in Figure 2 As shown in B; wherein, the system and procedure of real-time fluorescence quantitative PCR are as follows:

[0113] Fluorescence quantitative PCR reaction system: ChamQ Universal SYBR qPCR MasterMix (Novozyme) 10 μL, forward primer (F) 2 μL, reverse primer (R) 2 μL and cDNA 6 μL;

[0114] 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.

[0115] according to Figure 2 As can be seen in 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.

[0116] Example 6

[0117] TaPP2C32 gene overexpression

[0118] The 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 in a shaking incubator at 30°C for 12-16 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 Agrobacterium suspension prepared in the dark, and then the OD of the bacterial liquid was adjusted. 600nm The formula of Agrobacterium suspension is: 500 μL 10 mM MES, 50 μL 150 mM As, 5 mL 10 mM MgCl 2 , add sterile water to make up to 50 mL, mix well and set aside.

[0119] Place the bacterial solution at room temperature and in the dark 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 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 real-time fluorescence quantitative PCR was used to detect the effect of TaPP2C32 overexpression. The fluorescence quantitative PCR detection method is the same as the detection method in Example 5. The results are as shown in Figure 4 As shown in B.

[0120] 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.

[0121] Example 7

[0122] Resistance test of TaPP2C32 silenced strain and TaPP2C32 overexpression strain to stem rust

[0123] (1) Wheat plants 10 days after inoculation with BSMVγ and BSMVγ:TaPP2C32, and 3 days after injection with GV3101 / pLGY-02 and GV3101 / pLGY-02:TaPP2C32 were selected and inoculated with wheat stem rust 21C3. The inoculation method was as follows: Tween and water were mixed evenly in a volume ratio of 20:1000 and put into a spray bottle, 1 g of summer spores of wheat stem rust 21C3 were added, and the mixture was shaken and inoculated on the leaves of the above wheat plants, kept moisturized at 16-18°C for 16-20 hours, and then moved into a greenhouse for cultivation.

[0124] (2) After the wheat plants were inoculated with wheat stem rust, total RNA was extracted from 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 in the same manner as in Example 5. The results are as follows: Figure 2 Medium D and Figure 4 As shown in D. Figure 2 As shown in D, within 96 hours of inoculation, the relative expression of TaPP2C32 in the wheat BSMVγ:TaPP2C32 silenced strain was significantly reduced during stem rust infection compared with the BSMVγ empty vector control wheat. Figure 4 As can be seen from D, within 96 hours of inoculation, 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.

[0125] (3) Total RNA from the leaves of the wheat plants at 24h, 48h and 120h after inoculation with wheat stem rust was extracted 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 at 24h, 48h and 120h after inoculation with wheat stem rust. The fluorescence quantitative PCR detection method is the same as the detection method in Example 5. The PCR primers for TaPR1 are q-TaPR1-F / q-TaPR1-R (Tm value is 59°C), the PCR primers for TaPR2 are q-TaPR2-F / q-TaPR2-R (Tm value is 64°C), the PCR primers for TaPR5 are q-TaPR5-F / q-TaPR5-R (Tm value is 62°C), the PCR primers for TaCAT are q-TaCAT-F / q-TaCAT-R (Tm value is 58°C), and the PCR primers for TaSOD are q-TaSOD-F / q-TaSOD-R (Tm value is 58°C). The nucleotide sequences are shown in Table 1.

[0126] 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 3In A-C), reactive oxygen species (ROS) scavenging genes (TaSOD and TaCAT) were significantly upregulated ( 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-C), the expression levels of ROS scavenging genes (TaCAT and TaSOD) were significantly downregulated ( Figure 6 The above PR genes (Pathogenesis-related genes) are a type of genes specifically induced by pathogens or exogenous hormones and are closely related to systemic acquired resistance.

[0127] (4) Laser confocal microscopy was used to observe the mycelial infection of the wheat TaPP2C32 overexpression strain at 12h, 24h, 48h and 96h after inoculation with wheat stem rust. Compared with the empty vector control (pLGY-02), the mycelial infection in the wheat TaPP2C32 overexpression strain (pLGY-02:TaPP2C32) was significantly inhibited ( Figure 5 (A~B).

[0128] (5) The colony area of ​​wheat stem rust of TaPP2C32-silenced strains and TaPP2C32-overexpressing strains was counted 96 hours after inoculation with wheat stem rust. The phenotypes of wheat TaPP2C32-silenced strains and TaPP2C32-overexpressing strains were observed 14 days after inoculation with wheat stem rust to identify the infection type of wheat stem rust. The grading standard of wheat stem rust infection type 0 to 4 is as follows:

[0129] 0: No allergic spots occur;

[0130] 0;: no summer spores but yellow-white allergic spots can be seen;

[0131] 1: There are tiny summer spores but they are surrounded by obvious yellow-white allergic dead spots;

[0132] 2: Small to medium-sized summer spores but often inhabit green islands surrounded by spindle-shaped allergic dieback;

[0133] 3: Medium-sized summer spores, rarely fused; there should be no allergic dieback around the spores, but there may be chlorosis;

[0134] 4: Summer spores are large, often fused, without allergic dieback, but may also produce chlorosis;

[0135] In the infection type levels 1, 2, 3, and 4, if the lesions of the same infection type are larger, add "+", and 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).

[0136] 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 on the TaPP2C32 overexpression 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.

[0137] 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.

[0138] 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 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. Application 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 a wheat TaPP2C32 protein encoding gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

25.

6. A BSMV gene silencing vector for wheat TaPP2C32 protein encoding gene, characterized in that: 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 according to claim 5.

7. A recombinant Agrobacterium for silencing the gene encoding wheat TaPP2C32 protein, characterized in that: The recombinant Agrobacterium comprises a basic Agrobacterium and the BSMV gene silencing vector of 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 gene encoding the wheat TaPP2C32 protein 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 By increasing the expression level of the wheat TaPP2C32 protein encoding gene in the wheat, the wheat stem rust resistance is improved; 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 described in claim 7 to infect wheat.

Images