Application of TaNAC29 Gene in Regulating Resistance to Wheat Stem Rust

Abstract

The present invention discloses the application of the TaNAC29 gene in regulating wheat stem rust resistance, belonging to the technical field of gene functions. The nucleotide sequence of the TaNAC29 gene in the present invention is shown as SEQ ID NO:1. The present invention discovers that by silencing the TaNAC29 gene of wheat, the wheat stem rust resistance can be weakened, and by overexpressing the TaNAC29 gene of wheat, the wheat stem rust resistance can be enhanced. The present invention also correspondingly provides a method for enhancing wheat stem rust resistance, providing a new target gene for molecular improvement breeding of wheat against stem rust, and having important application value in wheat production and breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of gene functions, and particularly relates to the application of TaNAC29 gene in regulating wheat stem rust resistance. Background Art

[0002] Wheat is an important food crop. Wheat stem rust is a major disaster-causing disease in wheat production. Utilizing and planting disease-resistant varieties is the most economical and effective strategy for controlling stem rust. However, the population structure and virulence variation of wheat stem rust fungi are frequent, which easily leads to the loss of variety resistance. During the interaction between wheat and stem rust fungi, plants will produce a series of defense mechanisms to resist the invasion of stem rust fungi. At present, it has been reported that TaNAC transcription factors play an important role in the invasion process of pathogenic bacteria, but the research on the interaction between wheat and stem rust fungi is not deep enough. Therefore, enriching the resistance mechanism of wheat to stem rust has important application value in the research of wheat stem rust resistance in wheat production and breeding. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide the application of TaNAC29 gene in regulating wheat stem rust resistance, and it is found that overexpression of TaNAC29 gene enhances wheat stem rust resistance, and silencing of TaNAC29 gene weakens wheat stem rust resistance.

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides the application of TaNAC29 gene in regulating wheat stem rust resistance, and the nucleotide sequence of the TaNAC29 gene is as shown in SEQ ID NO:1.

[0006] Preferably, the amino acid sequence of the protein encoded by the TaNAC29 gene is as shown in SEQ ID NO:2.

[0007] Preferably, overexpression of the TaNAC29 gene enhances wheat stem rust resistance.

[0008] Preferably, silencing of the TaNAC29 gene weakens wheat stem rust resistance.

[0009] The present invention also provides a method for enhancing wheat stem rust resistance, and the method is to overexpress the TaNAC29 gene in wheat.

[0010] Preferably, the overexpression method includes: transferring an expression vector overexpressing the TaNAC29 gene into wheat.

[0011] Preferably, the method for transferring the expression vector is Agrobacterium infection.

[0012] The present invention also provides the application of the method in wheat breeding.

[0013] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0014] The present invention discovers for the first time that the TaNAC29 gene in wheat is related to wheat stem rust resistance. By silencing the TaNAC29 gene through virus induction, the wheat stem rust resistance can be weakened, and the wheat plants are more susceptible to the disease. By overexpressing the TaNAC29 gene through the Agrobacterium-mediated transient overexpression technology, the wheat stem rust resistance can be enhanced, and the wheat plants are more disease-resistant. That is, the TaNAC29 gene can positively participate in regulating the resistance of wheat to stem rust, providing a new target gene for molecular improvement breeding of wheat against stem rust, and having important application value in wheat production and breeding. Description of the Drawings

[0015] Figure 1 : Gel electrophoresis diagrams of TaNAC29 and the silencing fragments BSMV-TaNAC29-1as and BSMV-TaNAC29-2as;

[0016] Figure 2 : Symptoms after injecting BSMV into Nicotiana benthamiana;

[0017] Figure 3 : Detection of the silencing effect of TaNAC29 by RT-qPCR;

[0018] Figure 4 : Detection of the transient expression effect of TaNAC29 by RT-qPCR;

[0019] Figure 5 : Changes in the expression levels of TaPR1, TaPR2, and TaPR after TaNAC29 silencing;

[0020] Figure 6 : Changes in the expression levels of TaPR1, TaPR2, and TaPR5 after transient expression of TaNAC29;

[0021] Figure 7 : Mycelial growth after TaNAC29 silencing;

[0022] Figure 8 : Mycelial growth after transient expression of TaNAC29;

[0023] Figure 9 : Inoculation phenotype after TaNAC29 silencing;

[0024] Figure 10 : Inoculation phenotype after transient expression of TaNAC29;

[0025] Figure 11: Colony area after 144 h of TaNAC29 silencing and transient expression. Detailed implementation manner

[0026]

[0027] The amino acid sequence of the protein encoded by the TaNAC29 gene of the present invention is: MAMAQGQGQGAATSLPPGFRFHPTDEELILHYLRNRAAAAPCPVSIIADVDIYKFDPWDLPSQAVYGDCEWYFFSPRDRKYPNGIRPNRAAGSGYWKATGTDKPIHDPATGQGVGVKKALVFYKGRPPKGTKTAWIMHEYRLAADPLTTAVNTYKPIKFRNVSMRLDDWVLCRIYKKTGLASPMVPPLSDYDHMADHDDLSGGGSTFDDAACSFYAHSSSSSSASRTMITQQPPHAGGLPTIPSFSELFDDYSLAQILDAEAEHGATHHLAVHPSLNMLLPVGDNAHGVQPSYYAPSSSSPDASGGSAGKRKAASPEESSAKRLNGSCFDAPPQSANSWQGAASVLGGLGHQMLPQF, as shown in SEQ ID NO:2.

[0028] In the present invention, the wheat varieties include Little Club (LC) and Mianzi 52.

[0029] By virus-induced silencing of the TaNAC29 gene in the present invention, the resistance of wheat to stem rust can be weakened, and wheat plants are more susceptible to the disease; by transient overexpression of the TaNAC29 gene through Agrobacterium-mediated transient overexpression technology, the resistance of wheat to stem rust can be enhanced, and wheat plants are more disease-resistant. The present invention discovers that the TaNAC29 gene can positively participate in regulating the resistance of wheat to stem rust.

[0030] The present invention also provides a method for enhancing the resistance of wheat to stem rust, and the method is to overexpress the TaNAC29 gene in wheat. The overexpression method of the present invention includes: transferring an expression vector overexpressing the TaNAC29 gene into wheat, and the transfer method of the expression vector is Agrobacterium infection.

[0031] As an alternative embodiment, the present invention uses the cDNA obtained by reverse transcription of wheat RNA as a template, and performs PCR amplification with the amplification primer pair of the TaNAC29 gene to obtain an amplification product; the amplification product is ligated to an expression vector to obtain an overexpression vector; the overexpression vector is transferred into wheat to obtain wheat overexpressing the TaNAC29 gene, and the wheat overexpressing the TaNAC29 gene has high resistance to wheat stem rust. The amplification primer pair of the present invention is shown in SEQ ID NO:9 to SEQ ID NO:10, and the amplification system includes: 5 μL of 10×KOD buffer, 5 μL of dNTPs, MgSO 4 2 μL, 1.5 μL of Forward Primer, 1.5 μL of Reverse Primer, 2 μL of cDNA, 1 μL of DMSO, 1 μL of KOD Plus, and ddH 2 O is supplemented to 50 μL. The procedure of the PCR includes pre-denaturation at 94 °C for 2 min; denaturation at 94 °C for 15 s, annealing at 58 °C for 30 s, extension at 68 °C at 1 kb / min, for 32 cycles; storage at 4 °C. The expression vector of the present invention is the pLGY-02 vector. The transient overexpression vector of the present invention is transferred into wheat by Agrobacterium infiltration.

[0032] The present invention also provides the application of the method in wheat breeding. By overexpressing the TaNAC29 gene in wheat, wheat with high resistance to wheat stem rust can be obtained, and new germplasms with high resistance to wheat stem rust can be screened.

[0033] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] In the embodiments of the present invention, the wheat varieties are Little Club (LC) and Mianzi 52; the tobacco is Nicotiana benthamiana; the wheat stem rust fungus is 34MKGQM; the competent cells of Escherichia coli DH5α and Agrobacterium tumefaciens GV3101 are purchased from Sangon Biotech Co., Ltd.

[0035] In the embodiments of the present invention, the used Barley stripe mosaic virus (BSMV) VIGS vector system is the pCa-γbLIC plasmid (BSMVγ vector), the pCaBS-α plasmid and the pCaBS-β plasmid, which are described in the article "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". The information of the used pLGY-02 transient overexpression vector is described in the article "Liang Fang, Liu Yifei, Cui Zhongchi, etc. Functional identification of wheat pathogenesis-related protein gene TaPR1 by Agrobacterium-mediated method [J]. Journal of Agricultural University of Hebei, 2019, 42(2): 12-17."

[0036] In the embodiments of the present invention, the rapid plasmid miniprep kit (Takara, product number: 9760), 2×Taq MasterMix (Dye Plus), DNA purification and recovery kit (Takara, product number: 9761), and Phanta Super-Fidelity DNA Polymerase P501 were purchased from Nanjing Novozymes Biotech Co., Ltd. The high-fidelity enzyme KOD-Plus was purchased from Toyobo (Shanghai) Biotech Co., Ltd. Antibiotics such as Yeast Extract, Tryptone, agar powder, 50×TAE buffer, Kanamycin, Rifampin, and Spectinomycin hydrochloride were purchased from Sangon Biotech (Shanghai) Co., Ltd. The fast-cut enzyme was purchased from Takara Co., Ltd.

[0037] In the embodiments of the present invention, the solvents used are:

[0038] (1) 1×TAE Buffer buffer: 20 mL of 50×TAE Buffer is made up to 1000 mL with water.

[0039] (2) LB medium: Weigh 2 g of sodium chloride, 1 g of yeast extract powder, and 2 g of tryptone, make up to 200 mL, and sterilize at 120 °C for 20 min (4 g of agar powder needs to be added for solid medium).

[0040] (3) Kanamycin stock solution (50 mg / ml): Weigh 2.5 g of kanamycin and place it in a 50 mL centrifuge tube. First, add 40 mL of sterilized distilled water, mix well until completely dissolved, then make up the volume to 50 mL. Filter and sterilize with a 0.22 μm filter membrane, and store in small aliquots at -20 °C.

[0041] (4) Rifampicin stock solution (20 mg / mL): Weigh 0.2 g of rifampicin into a 50 mL centrifuge tube, add 10 mL of DMSO (dimethyl sulfoxide), filter and sterilize, then aliquot and store at -20 °C.

[0042] (5) Antibiotic-LB medium: Add 200 μL of the corresponding antibiotic to 200 mL of liquid LB medium (4 g of agar powder needs to be added for solid medium).

[0043] (6) Tobacco injection solution (Buffer): Take 500 μL of 10 mM MES, 50 μL of 150 mM As, 5 mL of 10 mM MgCl 2 , add sterile water to make up the volume to 50 mL, and mix well for later use.

[0044] In the examples of the present invention, the primer sequences used are shown in Table 1.

[0045] Table 1 Primer sequences

[0046]

[0047]

[0048] In the examples of the present invention, ** represents P < 0.01, *** represents P < 0.001, and **** represents P ≤ 0.0001.

[0049] Example 1

[0050] 1. Extraction of wheat / tobacco RNA and synthesis of cDNA

[0051] Extract wheat / tobacco RNA using the Trizol method. The specific steps are as follows:

[0052] (1) Take 1 g of wheat leaf / tobacco leaf of No. 52 rabbit characters and put it into a mortar for grinding (gently press when there is a lot of liquid nitrogen to prevent splashing, and grind quickly when the liquid nitrogen is about to run out), then put the ground sample into a pre-cooled 1.5 mL centrifuge tube and place it in liquid nitrogen;

[0053] (2) Take out the sample from liquid nitrogen and place it on ice. Before it turns green, add 1 mL of RNAiso Plus and invert it up and down to mix well until the sample is completely dissolved in RNAiso Plus;

[0054] (3) Add 200 μL of chloroform, shake for 15 s, centrifuge at 12000 rpm for 5 min, and transfer the supernatant (about 530 μL) to a new 1.5 mL centrifuge tube.

[0055] (4) Add an equal volume of isopropanol, mix well by inverting the tube up and down by hand, let it stand at 4 °C for 20 min, centrifuge at 12000 rpm for 20 min, discard the supernatant, and leave the white precipitate.

[0056] (5) Add 350 μL of 75% ethanol, pipette up and down, centrifuge at 12000 rpm for 5 min, and aspirate the alcohol.

[0057] (6) Set the blower of the laminar flow hood to level 3 and blow for 5 min.

[0058] (7) Add 20 μL of sterile water and mix well, then store it in a -20 °C refrigerator.

[0059] Use the RNA reverse transcription kit (product number: R423-01) of Novoprotein Scientific Inc. to reverse transcribe the total RNA of wheat / tobacco obtained according to the kit instructions to synthesize cDNA strands, which are used as templates for gene cloning. The obtained wheat / tobacco cDNA is placed in a -20 °C refrigerator for storage.

[0060] 2. Cloning of the target gene

[0061] Using the reverse transcribed wheat cDNA as a template, the primer pairs are used to perform PCR amplification on the target gene fragments respectively. The silencing fragment BSMV-TaNAC29-1as is amplified using BSMV-TaNAC29-1as-F and BSMV-TaNAC29-1as-R, the silencing fragment BSMV-TaNAC29-2as is amplified using BSMV-TaNAC29-2as-F and BSMV-TaNAC29-2as-R, and the TaNAC29 gene is amplified using TaNAC29-F and TaNAC29-R. Using the reverse transcribed tobacco cDNA as a template, the NbPDS gene is amplified using BSMV-NbPDS-F and BSMV-NbPDS-R. The primer sequences are entrusted to Sangon Biotech (Shanghai) Co., Ltd. for primer synthesis. The PCR reaction system is shown in Table 2.

[0062] Table 2 PCR reaction system for the target gene

[0063] Reagent Dosage 10×KOD buffer 5μL dNTPs 5μL <![CDATA[MgSO 4 > 2μL Forward Primer 1.5μL Reverse Primer 1.5μL cDNA 2μL KOD Plus 1μL DMSO 1μL <![CDATA[ddH 2 O]]> Make up to 50μL

[0064] The PCR reaction program was as follows: 94°C for 2 min; 94°C for 15 s, Tm for 30 s, 68°C for 60 s / 1 kb, for 35 cycles; store at 4°C (the reaction program was modified accordingly based on the primer annealing temperature and fragment size). The PCR products were detected by 1.0% agarose gel electrophoresis (200 V, 15 min), and the results were as Figure 1 shown. The target bands were recovered using a DNA purification and recovery kit according to the kit instructions, and the BSMV-TaNAC29-1as, BSMV-TaNAC29-2as silencing target gene fragments, the full-length CDS of the TaNAC29 gene, and the NbPDS gene fragment were obtained and stored in a -20°C refrigerator.

[0065] 3. Digestion of vectors

[0066] (1) Digestion of the BSMVγ vector

[0067] The BSMVγ vector was digested with ApaⅠ, and the digestion system and time were as follows:

[0068] Table 3 ApaⅠ digestion system

[0069] Reagent Dosage <![CDATA[ddH 2 O]]> 21μL Buffer 3μL ApaⅠ 1μL BSMVγ 5μL

[0070] The prepared digestion reaction system was placed in a 37°C metal bath for 1 h. After the reaction ended, the digestion products were detected by 1.0% agarose gel electrophoresis. They were recovered using a DNA purification and recovery kit according to the kit instructions, and the linear BSMVγ vector was obtained and stored in a -20°C refrigerator.

[0071] (2) Digestion of the pLGY-02 vector

[0072] The pLGY-02 vector was digested with SacⅠ and Kpn I, and the digestion system and time were as follows:

[0073] Table 4 SacⅠ and Kpn I digestion system

[0074] Reagent Dosage <![CDATA[ddH 2 O]]> 21μL Buffer 3μL SacⅠ 0.5μL KpnI 0.5μL pLGY-02 5μL

[0075] The prepared digestion reaction system was placed in a 37°C metal bath for 1 h. After the reaction ended, the digestion products were detected by 1.0% agarose gel electrophoresis. They were recovered using a DNA purification and recovery kit according to the kit instructions, and the linear pLGY-02 vector was obtained and stored in a -20°C refrigerator.

[0076] 4. Ligation of the target gene and the vector

[0077] (1) Ligation of the BSMVγ vector and the gene

[0078] Prepare the ligation systems for the BSMVγ linear vector, the BSMV-TaNAC29-1as silencing target gene fragment, the BSMV-TaNAC29-2as silencing target gene fragment, and the NbPDS gene fragment respectively. The ligation systems are configured as follows:

[0079] Table 5 Ligation system of BSMVγ linear vector

[0080] Reagent Dosage 10×T4 DNA pol Buffer 2μL 0.1%BSA 1μL dTTP(100nM) 1μL T4 DNA Polymerase 0.5μL BSMVγ linear vector 200ng <![CDATA[ddH 2 O]]> Make up to 20μL

[0081] Add the above systems into a PCR tube, incubate at 25 °C for 90 min; then at 72 °C for 20 min.

[0082] Table 6 Ligation system of gene fragments

[0083] Reagent Dosage 10×T4 DNA pol Buffer 2μL 0.1%BSA 1μL dATP(100nM) 1μL T4 DNA Polymerase 0.5μL BSMV-TaNAC29-1as or BSMV-TaNAC29-2as or NbPDS 100ng <![CDATA[ddH 2 O]]> Make up to 20μL

[0084] Add the above systems into a PCR tube, incubate at 25 °C for 90 min; then at 72 °C for 20 min.

[0085] Use T4 DNA Polymerase from Vazyme to ligate the purified BSMVγ linear vector with the BSMV-TaNAC29-1as, BSMV-TaNAC29-2as, and NbPDS target gene fragments respectively: Pipette 10 μL of the treated BSMVγ linear vector ligation system and 20 μL of the treated gene fragment ligation system (BSMV-TaNAC29-1as or BSMV-TaNAC29-2as or NbPDS), react at 66 °C for 2 min, and then place at room temperature for 10 min to obtain the ligation product. Perform Escherichia coli transformation after ligation.

[0086] (2) Ligation of pLGY-02 vector and TaNAC29

[0087] After double-digesting the pLGY-02 vector and the TaNAC29 amplification product with SacⅠ and Kpn I, perform purification and recovery (the double-digestion method of the TaNAC29 amplification product is the same as that of the pLGY-02 vector). Use T4 DNA Ligase from Vazyme to ligate the purified pLGY-02 linear vector with the TaNAC29 target gene fragment. The ligation system is as follows:

[0088] Table 7 Ligation reaction system of pLGY-02 and target gene

[0089] Reagent Dosage pLGY-02 after double digestion and purification 1.5μL TaNAC29 after double digestion and purification 6.5μL T4 DNA Ligase Buffer 1μL T4 DNA Ligase 1μL

[0090] Incubate at 22 °C for 3 h; then at 70 °C for 10 min. Perform Escherichia coli transformation after ligation.

[0091] (3) Transformation method of Escherichia coli DH5α competent cells

[0092] Take out the DH5α competent cells from the -80 °C refrigerator, thaw them on ice, add 10 μL of the ligation product, gently pipette and mix well, and let it stand on ice for 20 min; heat shock at 42 °C for 90 s, and let it stand on ice for 1 min. Add 350 μL of LB liquid medium in the laminar flow hood, shake it on a shaker at 37 °C and 200 rpm for 1 h; centrifuge at 4000 rmp for 1 min, aspirate and discard 350 μL of the supernatant, resuspend the remaining liquid and spread it on the corresponding antibiotic solid medium, and incubate it upside down at 37 °C for 14 h. Pick 8 monoclonal colonies and culture them in test tubes containing LB liquid medium with kanamycin (50 mg / L) at 37 °C on a shaker for 14 h. After the bacterial solution becomes turbid, aspirate 1 μL of the bacterial solution for bacterial liquid PCR verification. The PCR reaction system is shown in Table 8. The positive bacterial liquid PCR primers for silencing TaNAC29 and NbPDS are BSMV-11 and BSMV-32; the positive PCR primers for overexpression are TaNAC29-F and TaNAC29-R, and then extract the plasmids.

[0093] Table 8 Bacterial liquid PCR reaction system

[0094] Reagent Dosage 2×Taq Master Mix(Dye Plus) 10μL 10μM Forward Primer 1μL 10μM Reverse Primer 1μL Bacterial solution 1μL <![CDATA[ddH 2 O]]> Make up to 20μL

[0095] The PCR reaction program is as follows: 98 °C for 30 s; 98 °C for 30 s, 58 °C for 30 s, 72 °C for 10 s, 35 cycles; 72 °C for 5 min. After the reaction is completed, send the amplification product to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing verification. Extract the plasmids from the positive colonies with correct sequencing to obtain the TaNAC29 silencing vector plasmids BSMVγ:TaNAC29-1as and BSMVγ:TaNAC29-2as, the NbPDS silencing vector plasmid BSMVγ:NbPDS, and the TaNAC29 overexpression vector plasmid pLGY-02:TaNAC29.

[0096] 5. Transformation of Agrobacterium competent cells

[0097] Transform the Agrobacterium GV3101 (pJICSA-Rep) competent cells with the recombinant plasmids BSMVγ:TaNAC29-1as, BSMVγ:TaNAC29-2as, pLGY-02:TaNAC29, BSMVγ:NbPDS, and the empty vector plasmids BSMVγ, pCaBS-α, pCaBS-β, pLGY-02 respectively.

[0098] The Agrobacterium transformation method is as follows:

[0099] GV3101 competent cells were taken out from an -80 °C refrigerator, melted on ice, 5 μL of plasmid was added, gently pipetted and mixed well, and left standing on ice for 20 min; quick-frozen in liquid nitrogen for 1 min, incubated at 37 °C for 5 min, and left standing on ice for 5 min; 400 μL of LB liquid medium was added in a laminar flow hood, shaken at 200 rpm on a shaker at 30 °C for 2.5 h; centrifuged at 5000 rpm for 1 min, 400 μL of the supernatant was aspirated and discarded, and the remaining liquid was resuspended and spread on solid LB medium containing kanamycin, sealed and incubated upside down at 30 °C for 48 h. The obtained recombinant agrobacteria were named GV3101 / BSMVγ, GV3101 / BSMVγ:TaNAC29-1as, GV3101 / BSMVγ:TaNAC29-2as, GV3101 / BSMVγ:NbPDS, GV3101 / pCaBS-α, GV3101 / pCaBS-β, GV3101 / pLGY-02 and GV3101 / pLGY-02:TaNAC29 respectively.

[0100] 6. Agrobacterium infiltration of tobacco

[0101] In a laminar flow hood, Agrobacterium tumefaciens GV3101 / BSMVγ, GV3101 / BSMVγ:TaNAC29-1as, GV3101 / BSMVγ:TaNAC29-2as, GV3101 / BSMVγ:NbPDS, GV3101 / pCaBS-α, GV3101 / pCaBS-β were respectively inoculated into 2 mL of LB liquid medium containing 2 μL of kanamycin at a concentration of 50 mg / L and 3 μL of rifampicin at a concentration of 20 mg / L. Then, Agrobacterium colonies were picked with a 10 μL pipette tip and the tip was vertically inserted into a 50 mL glass tube, sealed and placed in a shaker at 30 °C for 16 h. The Agrobacterium liquid was taken out, centrifuged at 4000 rpm at room temperature for 10 min, the supernatant was discarded, and then the OD of the bacterial liquid was adjusted with an Agrobacterium suspension 600nm to 0.5 (the formula of the Agrobacterium suspension is: 500 μL of 10 mM MES, 50 μL of 150 mM As, 5 mL of 10 mM MgCl 2 , made up to 50 mL with sterile water).

[0102] The following Agrobacterium combinations:

[0103] Negative control: GV3101 / BSMVγ, GV3101 / pCaBS-α and GV3101 / pCaBS-β were mixed at a volume ratio of 1:1:1;

[0104] Positive control: GV3101 / BSMVγ:NbPDS, GV3101 / pCaBS-α and GV3101 / pCaBS-β were mixed at a volume ratio of 1:1:1;

[0105] Experimental group 1: GV3101 / BSMVγ:TaNAC29-1as, GV3101 / pCaBS-α and GV3101 / pCaBS-β were mixed evenly at a volume ratio of 1:1:1;

[0106] Experimental group 2: GV3101 / BSMVγ:TaNAC29-2as, GV3101 / pCaBS-α and GV3101 / pCaBS-β were mixed evenly at a volume ratio of 1:1:1

[0107] Let it stand at room temperature for more than 3 h, inject tobacco at the 6-8 leaf stage, air-dry the excess injection solution on the tobacco leaves at room temperature, then put the tobacco back into the light incubator for cultivation, and inoculate wheat after the tobacco shows virus phenotypes. The cultivation results are shown in Figure 2 , Figure 2 Among them, BSMVγ was used as the negative control, BSMVγ-TaNAC29-1as was used as experimental group 1, BSMVγ-TaNAC29-2as was used as experimental group 2, and BSMVγ-NbPDS was used as the positive control. When the tobacco shows symptoms of mottled mosaic, it indicates that the virus has been successfully recombined in the tobacco. The results show that BSMVγ can induce gene silencing.

[0108] 7. Method for rubbing and inoculating wheat with virus

[0109] Add 5 mL of PBS buffer to a sterilized mortar, add 0.5 g of tobacco leaves inoculated with virus for 10 d (take the tobacco leaves obtained from the negative control and experimental groups in step 6 respectively), grind them into juice, fully pipette and mix evenly, then transfer them into a 2 mL centrifuge tube and store them on ice. Sprinkle a little quartz sand on the surface of the wheat leaves of Mianzi 52 at the one-leaf-one-heart stage, and dip the juice with latex gloves for inoculation. After inoculation, put the wheat into an incubator at 24 °C for cultivation. After 14 d, the symptoms of BSMV infection were clearly observed. Take 0.1 g of wheat leaves from each group to extract RNA, then reverse transcribe to synthesize the first-strand cDNA. Using the specific primers q-TaNAC29-F / q-TaNAC29-R (Tm value is 58 °C) of the TaNAC29 gene as the template, perform real-time fluorescence quantitative PCR to detect the silencing effect of TaNAC29. At the same time, use the primers q-TaGAPDH-F and q-TaGAPDH-R (Tm value is 58 °C) of the internal reference gene TaGAPDH (GenBank accession number: LOC123160238) to perform real-time fluorescence quantitative PCR. Reaction procedure: Pre-denaturation: 95 °C, 30 s; PCR reaction: 95 °C, 10 s; 58 °C, 30 s; 40 cycles; Dissolution curve: 95 °C, 15 s; 60 °C, 60 s; 95 °C, 15 s. Use 2 -ΔΔCt The analysis method was used to calculate the relative expression level of the TaNAC29 gene. The nucleotide sequences of the primers are shown in Table 1, and the real-time quantitative fluorescence PCR system is as follows.

[0110] Table 9 Fluorescent quantitative PCR reaction system

[0111] Reagent Dosage ChamQ Universal SYBR qPCR Master Mix (Novizan) 5μL Forward Primer (F) 1μL Reverse Primer (R) 1μL cDNA 3μL

[0112] The results are as Figure 3 shown. The significantly reduced relative expression level of the TaNAC29 gene indicates that it has been successfully silenced, and wheat plants with silenced TaNAC29 have been obtained.

[0113] 8. Agrobacterium injection into wheat

[0114] Using a white pipette tip, pick Agrobacterium tumefaciens GV3101 / pLGY-02 and GV3101 / pLGY-02:TaNAC29 in a laminar flow hood and transfer them into a 2 mL LB liquid medium containing 2 μL of kanamycin at a concentration of 50 mg / L and 3 μL of rifampicin at a concentration of 20 mg / L. Seal the medium and place it in a shaker at 30 °C for 16 h. Take out the Agrobacterium liquid, centrifuge it at 4000 rpm at room temperature for 10 min, discard the supernatant, and then adjust the OD of the bacterial liquid with an Agrobacterium suspension 600nm to 1.0. Prepare the Agrobacterium suspension Buffer according to the method in step 6 and inject it into the leaves of LC wheat at the one-leaf-one-heart stage. After the wheat leaves dry, put them back into the light incubator for cultivation. At 24 h, 48 h, 72 h, and 144 h after injection, take 0.1 g of wheat leaves in the injection area to extract RNA, reverse transcribe it to synthesize the first-strand cDNA, and detect the expression effect by RT-qPCR. The detection method is the same as that for detecting the silencing effect of TaNAC29 in step 7. The results are as Figure 4 shown. It was found through fluorescence quantitative PCR detection that 3 days after injection, the expression level of TaNAC29 increased significantly compared with the control (P < 0.0001), indicating that TaNAC29 has been successfully transiently expressed, and wheat plants with transient expression of TaNAC29 have been obtained.

[0115] 9. Inoculation of wheat with Puccinia graminis f. sp. tritici

[0116] Select wheat plants inoculated with BSMVγ, BSMVγ:TaNAC29-1as, and BSMVγ:TaNAC29-2as respectively, as well as wheat plants injected with GV3101 / pLGY-02 and GV3101 / pLGY-02:TaNAC29 respectively, and inoculate them with Puccinia graminis f. sp. tritici 34MKGQM. Method for inoculating Puccinia graminis f. sp. tritici: Prepare it in a spray bottle according to the ratio of Tween: water being 20:1000, add 1 g of urediniospores of Puccinia graminis f. sp. tritici 34MKGQM, and shake well. Spray it evenly on the above-mentioned wheat leaves, keep them moist in a dark environment for 15 h, place them in an incubator at 20 ± 1 °C and a light intensity of 5 - 6.0 klx for cultivation, and start investigating the hyphal infection status and infection type at 14 d.

[0117] (1) The grading standards of 0-4 types for the infection type of wheat stem rust are as follows:

[0118] 0: No hypersensitive necrotic spots occur;

[0119] 0; : No uredinia, but yellowish-white hypersensitive necrotic spots can be seen;

[0120] 1: Tiny uredinia are produced, but there is obvious yellowish-white hypersensitive necrosis around;

[0121] 2: Small to medium-sized uredinia, but often located in green islands, and the green islands are surrounded by fusiform hypersensitive necrosis;

[0122] 3: Medium-sized uredinia, rarely fused; there should be no hypersensitive necrosis around the uredinia, but chlorosis may occur;

[0123] 4: Large uredinia, often fused, no hypersensitive necrosis, but chlorosis may also occur;

[0124] If the lesions of the same infection type are on the larger side, add "+", and if they are on the smaller side, add "-". Among them, 0, 0;, 1-, 1, 1+, 2-, 2, and 2+ belong to the low infection type (disease-resistant), and 3-, 3, 3+, 4-, 4, and 4+ belong to the high infection type (disease-susceptible).

[0125] (2) Steps for preparing fluorescent staining samples:

[0126] Take wheat leaves at 12 h, 72 h, and 144 h after inoculation, and soak them in a decolorizing solution with a volume ratio of acetic acid to glacial acetic acid of 1:1 for 2 d (the decolorizing solution can be placed in a constant temperature water bath at 40 °C to accelerate decolorization);

[0127] Rinse the decolorized wheat leaves with distilled water 3-5 times;

[0128] Soak the rinsed wheat leaves in a chloral hydrate solution for more than 15 min, rinse the leaves soaked in chloral hydrate with distilled water 3-5 times, and if stored for a long time, they need to be stored in 50% glycerol;

[0129] Take out the leaves stored in glycerol, rinse them with distilled water 3-5 times, and then soak them in 0.5 mol / L KOH solution for 5-8 min;

[0130] Take out the leaves, rinse them with distilled water 3-5 times, stain them with WGA-Alexa 488 fluorescent dye solution (20 μg / mL) in the dark for 40 min, and then rinse them thoroughly with distilled water.

[0131] Using RT-qPCR (the PCR system and reaction procedure were the same as the detection method for the silencing effect of TaNAC29 in step 7, with wheat plants injected with the BSMVγ empty vector as the control), the expression levels of TaPR1, TaPR2, and TaPR5 at 24 h, 48 h, 72 h, and 144 h after inoculation were detected. The results were as Figures 5 - 6 shown. Figure 5 It was shown that, compared with the control, the expression of the three PR genes (TaPR1, TaPR2, and TaPR5) in TaNAC29-silenced plants was significantly downregulated. Figure 6 It was shown that the expression levels of the three PR genes (TaPR1, TaPR2, and TaPR5) in plants with transient expression of TaNAC29 were significantly upregulated.

[0132] The hyphal infection conditions at 12 h, 72 h, and 144 h after inoculation in silenced and transient expression plants were observed using a laser confocal microscope. The results of the hyphal growth conditions after TaNAC29 silencing (GT: germ tube; SV: substomatal vesicle; IH: primary infection hypha; SH: secondary infection hypha) were as Figure 7 shown, Figure 7 in which, for the BSMV:γ group and the BSMV:γ-TaNAC29 group, the scale bar was 40 μm at 12 hpi, 60 μm at 72 hpi, and 100 μm at 144 hpi. It could be seen that the number of wheat hyphae was significantly higher in the TaNAC29-silenced group than in the control group. The results of the hyphal growth conditions after transient expression of TaNAC29 (GT: germ tube; SV: substomatal vesicle) were as Figure 8 shown, Figure 8 in which, for the pLGY-02 group and the pLGY-TaNAC29 group, the scale bar was 40 μm at 12 hpi, 50 μm at 72 hpi, and 100 μm at 144 hpi. It could be seen that the number of wheat hyphae was significantly lower in the TaNAC29 transient expression group than in the control group.

[0133] The wheat inoculation phenotypes after TaNAC29 silencing were as Figure 9 shown, and the wheat inoculation phenotypes after transient expression of TaNAC29 were as Figure 10 shown. From the phenotypes, it could be seen that the infection type of BSMVγ silencing was 0, the infection type after TaNAC29 silencing was 2, and the spore heap after silencing was larger than that of the control; the infection type decreased from 4 to 2 after transient expression of TaNAC29.

[0134] The colony areas of TaNAC29-silenced wheat and TaNAC29 transient expression wheat after inoculation were statistically analyzed. The results were as Figure 11 shown, Figure 11 indicating that, compared with the control, the colony area in TaNAC29-silenced plants increased significantly, while the colony area in transient expression plants decreased significantly.

[0135] The above results indicate that silencing TaNAC29 reduces the resistance of wheat to Puccinia graminis f. sp. tritici, and transient expression of TaNAC29 enhances the resistance of wheat to Puccinia graminis f. sp. tritici.

[0136] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. TaNAC29 The use of a gene in regulating wheat stem rust resistance is characterized in that: Said TaNAC29 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; TaNAC29 Gene overexpression enhances wheat stem rust resistance.

2. The use according to claim 1, characterized in that: Said TaNAC29 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

3. A method for enhancing wheat stem rust resistance, characterized in that: The method is to overexpress wheat TaNAC29 gene; TaNAC29 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

4. The method according to claim 3, characterized in that The overexpression method comprises: TaNAC29 The gene expression vector was transferred into wheat.

5. The method according to claim 4, characterized in that The expression vector is introduced by Agrobacterium infection.

6. Application of the method according to any one of claims 3 to 5 in wheat breeding, characterized in that: The application is to overexpress wheat TaNAC29 Genes that enhance wheat stem rust resistance.

Images