Wheat stripe rust protein PstCC3 and application thereof

Abstract

The invention belongs to the technical field of genetic engineering breeding, and discloses wheat stripe rust protein PstCC3 and application thereof. The PstCC3 protein has an amino acid sequence as shown in SEQ ID NO: 1, and is coded by a nucleotide sequence as shown in SEQ ID NO: 2. Through verification, after the PstCC3 gene is silenced, the sensitivity of the wheat stripe rust to the triadimefon is obviously enhanced, the control effect of the triadimefon is improved, and stripe rust spore piles generated on wheat leaves are obviously reduced. The invention provides a theoretical basis for research and development of a treatment technology taking delaying of the development speed of the drug resistance of the puccinia striiformis as a core and a new target drug for preventing and treating the puccinia striiformis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering breeding, and particularly relates to a wheat stripe rust protein PstCCC3 and application thereof. BACKGROUND

[0002] Wheat stripe rust caused by Puccinia striiformis f.sp.tritici is listed as a class of crop diseases, which seriously threatens the safety of wheat production and causes huge yield and economic losses. Applying chemical fungicides is an important measure for preventing and controlling wheat stripe rust, and triadimefon (a triazole fungicide) is a main promoted fungicide for preventing and controlling wheat stripe rust, which has been used in a large area and singly for more than 50 years, greatly increasing the selection pressure of the emergence of resistant strains, so that the chemical prevention and control of wheat stripe rust faces severe risks and challenges.

[0003] At present, about 6.79% of the wheat stripe rust strains in the main wheat production areas have developed resistance to triadimefon, and the resistant strains are mainly concentrated in the winter breeding area and the northwest summering area, but the resistance mechanism is not clear. The emergence of the resistant strains reduces the prevention and control effect of triadimefon by about 12.35-23.87%, increases the prevention and control cost, and causes environmental pollution problems. Therefore, it is urgent to clarify the molecular mechanism of the wheat stripe rust resistance to triadimefon, so as to provide a theoretical basis for the development of management technology for delaying the development speed of the wheat stripe rust resistance and new target drugs for the prevention and control of wheat stripe rust. SUMMARY

[0004] In order to better exert the prevention and control effect of triadimefon on wheat stripe rust, the application screens a wheat stripe rust vacuolar iron transporter PstCCC3 by a genetic engineering method, and reveals the physiological function and molecular mechanism of PstCCC3 in the process of preventing and controlling wheat stripe rust by triadimefon through the expression characteristic research of the PstCCC3 protein coding gene. In order to achieve this technical purpose, the application provides the following technical scheme.

[0005] In a first aspect, the application provides a wheat stripe rust vacuolar iron transporter PstCCC3, wherein the PstCCC3 protein has an amino acid sequence as shown in SEQ ID NO: 1.

[0006] In a second aspect, the application provides a PstCCC3 gene, wherein the PstCCC3 gene comprises a nucleotide sequence coding the PstCCC3 protein.

[0007] Further, the CDS sequence of the PstCCC3 gene is shown in SEQ ID NO: 2.

[0008] Further, the sensitivity of the wheat stripe rust to triadimefon is enhanced or the prevention and control effect of triadimefon is improved by silencing the PstCCC3 gene.

[0009] In a third aspect, the present application provides a vector for silencing the PstCCC3 gene.

[0010] Further, the blank viral vector after enzyme digestion is connected with the silencing sequence PstCCC3-S1 or the silencing sequence PstCCC3-S2;

[0011] The silencing sequence PstCCC3-S1 corresponds to the nucleotide sequence from 90th to 293th nucleotide from the 5' end of the CDS sequence of the PstCCC3 gene; and the silencing sequence PstCCC3-S2 corresponds to the nucleotide sequence from 691th to 891th nucleotide from the 5' end of the CDS sequence of the PstCCC3 gene.

[0012] Further, the primer pair for amplifying the silencing sequence PstCCC3-S1 is SEQ ID NO: 9 and SEQ ID NO: 10; and the primer pair for amplifying the silencing sequence PstCCC3-S2 is SEQ ID NO: 11 and SEQ ID NO: 12.

[0013] In a fourth aspect, the present application provides an expression vector for silencing the PstCCC3 gene.

[0014] In a fifth aspect, the present application claims the use of the PstCCC3 protein, the PstCCC3 gene, the vector or the expression vector in wheat breeding.

[0015] Further, in the above use, the silencing of the PstCCC3 gene and / or the reduction of the concentration of the PstCCC3 protein enhances the sensitivity of the Puccinia striiformis to triadimefon or improves the control effect of triadimefon.

[0016] In addition, those skilled in the art can easily mutate the nucleotide sequence encoding the PstCCC3 protein by using known methods such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity with the PstCCC3 gene sequence obtained by the present application are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application, as long as they encode the PstCCC3 protein and have the same function.

[0017] The term "identity" as used herein refers to sequence similarity with a native nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with SEQ ID NO: 2. Identity can be assessed by eye or by computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%) which can be used to assess identity between related sequences.

[0018] The 75% or more identity described above can be 80%, 85%, 90% or 95% or more identity.

[0019] The expression cassette containing the nucleic acid molecule encoding PstCCC3 protein (PstCCC3 gene expression cassette) refers to DNA capable of expressing PstCCC3 protein in a host cell, which can include not only a promoter to initiate transcription of PstCCC3, but also a terminator to terminate transcription of PstCCC3. Further, the expression cassette can also include an enhancer sequence. The promoters that can be used in the present application include but are not limited to: constitutive promoters; tissue, organ and development specific promoters and inducible promoters. Suitable transcription terminators include but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), Cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcSE9 terminator and nopaline and opine synthase terminator.

[0020] Compared with the prior art, the technical solutions provided by the present application have at least the following beneficial effects or advantages: the present application proves that PstCCC3 gene silenced plants obtained by using transient silencing technology can effectively improve the control effect of triadimefon on Puccinia striiformis, indicating that PstCCC3 protein of Puccinia striiformis has resistance to triadimefon. The PstCCC3 protein, PstCCC3 gene and application thereof provided by the present application provide a basis for scientific and efficient use of triadimefon, and will play an important role in wheat breeding and research and development of new control agents for Puccinia striiformis. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Figure 4 is a diagram of the expression pattern of PstCCC3 gene after PstCCC3 gene in Puccinia striiformis is treated with triadimefon. Figure 1 The standard deviation is shown by error bars, "ns" means not statistically significant, "*" means p<0.05, significant compared with 0h; "****" means p<0.0001, extremely highly significant compared with 0h.

[0022] Figure 2 Figure 6 is a schematic diagram of the location of the silencing sequences PstCCC3-S1 and PstCCC3-S2 of PstCCC3 gene on the gene.

[0023] Figure 3 The phenotypes of normal wheat plants after inoculation with different recombinant viral vectors (left) and the phenotypes of the experimental groups (BSMV:γ, BSMV:PstCCC3-S1, BSMV:PstCCC3-S2) further inoculated with stripe rust and treated with triadimefon (right).

[0024] Figure 4 The statistical results of the relative expression of the PstCCC3 gene in wheat plants (left) and the biomass of stripe rust (right) in the experimental group are shown. “**” indicates p < 0.01, which is extremely significant.

[0025] Figure 5 These are the histological observation results of wheat leaves with PstCCC3 gene silenced. Figure 5 Figure (a) shows the expansion of stripe rust hyphae in wheat leaves with PstCCC3 gene silenced after 72 hours of triadimefon treatment, as viewed under a fluorescence microscope. Figure (b) shows the mycelial area per infection site on wheat leaves after 72 hours of triadimefon treatment. Values ​​represent the mean ± standard deviation of three independent samples (60 infection sites / sample); error bars indicate standard deviation. **(p<0.01) indicates significant difference, analyzed using a t-test. DETAILED DESCRIPTION

[0026] The technical solutions of the present invention are described below with reference to the following embodiments, but the present invention is not limited to the following embodiments.

[0027] The experimental methods and detection methods in the following embodiments are all conventional methods unless otherwise specified; the agents and materials are all commercially available unless otherwise specified; the indicator data are all based on conventional measurement methods unless otherwise specified.

[0028] Example 1

[0029] This example provides the acquisition of PstCCC3 protein and its encoding gene.

[0030] Summer spores of wheat stripe rust that had grown normally for 14 days on wheat leaves were taken, quickly frozen with liquid nitrogen, and stored at -80°C for later use.

[0031] Total RNA from wheat stripe rust was extracted using the Trizol method (TianGen), and first-strand cDNA was synthesized using reverse transcriptase XL (AMV). cDNA was synthesized using the SMART method, and PCR products were detected by 1.0% agarose gel electrophoresis. The amplification primers were:

[0032] PstCCC3-F: 5'-AGTCTCCCCTAAAAATCCATAAC-3' (SEQ ID NO: 3);

[0033] PstCCC3-R: 5'-GTGGATGGGAATGAGTATGAGG-3' (SEQ ID NO: 4).

[0034] A 1194 bp PCR product was obtained. Sequencing showed that the PCR product had a nucleotide sequence as shown in SEQ ID NO: 2, which was named as PstCCC3 gene, which encoded PstCCC3 protein, and the amino acid sequence of which was shown in SEQ ID NO: 1.

[0035] Example 2

[0036] This example provides qRT-PCR detection of the expression of Puccinia striiformis PstCCC3 gene under the treatment of triazolinone.

[0037] Fresh uredospores of Puccinia striiformis YQ324 strain were inoculated on "Mingxian 169" wheat seedlings at the two-leaf stage, and the moisture and culture conditions were referred to the literature "Discovery of Lovell 10 pathogenic new strain at room temperature [J]. Kang Zhen-sheng, Li Zhen-qi. Journal of Northwest A&F University (Natural Science Edition), 1984 (04): 18-28." After 3 days of inoculation, triazolinone with a concentration of 100 μg / mL was sprayed at a dosage of 10 mL per pot (15 plants per pot), and acetone was used as a control. Each treatment was set up in triplicate. After treatment, samples were taken at 0, 6, 12, 24, 48, 72, and 120 hours and immediately frozen in liquid nitrogen, and then stored in a -80°C refrigerator for later use. Total RNA was extracted from wheat leaves using the Trizol method (TianGen), and first-strand cDNA was synthesized using reverse transcriptase XL (AMV). cDNA was synthesized using the SMART method.

[0038] The elongation factor gene PstEF-1a was used as an internal reference gene, and specific qRT-PCR primers were designed according to the sequences of PstCCC3 gene and internal reference gene. The qRT-PCR primer sequences were as follows:

[0039] Q PstCCC3-F: 5'-ACCGCCGGTCTCTCATCG-3' (SEQ ID NO: 5);

[0040] Q PstCCC3-R: 5'-TCACTCGGTCTCGGGTCG-3' (SEQ ID NO: 6).

[0041] PstEF-1a-F: 5'-TTCGCCGTCCGTGATATGAGACAA-3' (SEQ ID NO: 7);

[0042] PstEF-1a-R: 5'-ATGCGTATCATGGTGGTGGAGTGA-3' (SEQ ID NO: 8).

[0043] qRT-PCR primers were tested for their specificity and amplification efficiency (≥ 90%) before use. Using AceQ Universal SYBR qPCR MasterMix (Vazyme, Nanjing, China) and Bio-Rad CFX Manager quantitative PCR instrument (Bio-rad, Hercules, California), according to the instructions, the cDNA of each sampling point was used as a template for qRT-PCR amplification. Each reaction was repeated at least three times, and the Ct value, average value and standard deviation of each repeat were generated by manually adjusting the baseline by the quantitative PCR instrument. The comparative threshold method (2 –ΔΔCT ) was used to calculate the relative expression of PstCCC3 gene.

[0044] The relative expression of PstCCC3 gene at different time points after triazolone treatment is shown in Table 1. Figure 1 Compared with the control group (treated with acetone), the expression of PstCCC3 gene was significantly up-regulated at 12, 24, 48 and 72h after triazolone treatment. Among them, "ns" means no statistical significance, "*" means p < 0.05, significant; "****" means p < 0.0001, extremely high significant. Figure 1 The results show that the expression of PstCCC3 gene is significantly increased by triazolone induction.

[0045] Example 3

[0046] This example provides the use of HIGS (host-induced gene silencing) technology to verify the anti-triazolone function of PstCCC3 gene of Puccinia striiformis.

[0047] 1. Construction of PstCCC3 gene silencing vector by barley stripe mosaic virus-induced gene silencing (BSMV-HIGS)

[0048] 1) Obtaining of silencing sequence

[0049] (1) Obtaining of silencing sequence PstCCC3-S1

[0050] Using the 1194bp PstCCC3 gene fragment amplified in Example 1 as a template, the primer pair PstCCC3-S1F, PstCCC3-S1R was used for PCR amplification, and a 204bp PCR amplification product (corresponding to the sequence of nucleotides 90-293 from the 5' end of SEQ ID NO: 2) was obtained, which was named as silencing sequence PstCCC3-S1.

[0051] The nucleotide sequences of the primer pair PstCCC3-S1F and PstCCC3-S1R are as follows (the recognition sites of restriction enzymes PacI and NotI are indicated by underlining):

[0052] PstCCC3-S1F: 5'-ATTGAGACCATCAGCCTCATTG-3' (SEQ ID NO: 9);

[0053] PstCCC3-S1R: 5'-TGAGGGGTGATGTTATTTTTG-3' (SEQ ID NO: 10).

[0054] (2) Obtaining of the silencing sequence PstCCC3-S2

[0055] Using the 1194 bp PstCCC3 gene fragment amplified in Example 1 as a template, the primer pair PstCCC3-S2F and PstCCC3-S2R was used for PCR amplification, and a 201 bp PCR amplification product (corresponding to the sequence of SEQ ID NO: 2 from the 5' end 691~891 nucleotides) was obtained, which was named as the silencing sequence PstCCC3-S2.

[0056] The nucleotide sequences of the primer pair PstCCC3-S2F and PstCCC3-S2R are as follows (the recognition sites of restriction enzymes PacI and NotI are indicated by underlining):

[0057] PstCCC3-S2F: 5'-GGTTGCAGCCGCACTACTT-3' (SEQ ID NO: 11);

[0058] PstCCC3-S2R: 5'-CCTTCGCCGAATTTCAAG-3' (SEQ ID NO: 12).

[0059] The schematic diagram of the positions of the silencing sequences PstCCC3-S1 and PstCCC3-S2 on the PstCCC3 gene is shown in Figure 2 .

[0060] 2) Construction of the silencing vector

[0061] (1) Construction of the γ-PstCCC3-S1 silencing vector

[0062] The PstCCC3-S1 obtained in step 1) and the BSMV viral vector γ are respectively digested by restriction enzymes PacI and NotI, and then the digested PstCCC3-S1 is connected with the digested BSMV viral vector γ to obtain a recombinant vector γ-PstCCC3-S1.

[0063] The recombinant vector γ-PstCCC3-S1 can replace the fragment between the PacI and NotI digestion sites of the BSMV viral vector γ with the PstCCC3-S1, and keep other sequences of the BSMV viral vector γ unchanged, wherein the PstCCC3-S1 is opposite to the sequence direction of the PstCCC3 gene.

[0064] The recombinant vector γ-PstCCC3-S1 is subjected to PCR amplification by using a primer pair γ-F and γ-R, and the sequences of the γ-F and γ-R primers are as follows:

[0065] γ-F: 5'-GTGAGGTTAACGCAATACG-3' (SEQ ID NO: 13);

[0066] γ-R: 5'-TCAGGCATCGTTTTCA-3' (SEQ ID NO: 14).

[0067] A positive clone is obtained by sequencing identification, and the positive clone is a vector obtained by inserting the nucleotides 90-292 of SEQ ID NO: 2 from the 5' end into the PacI and NotI digestion sites of the γ chain of the BSMV viral vector γ, and keeping other sequences of the γ chain unchanged.

[0068] (2) Construction of γ-PstCCC3-S2 silencing vector

[0069] The PstCCC3-S2 obtained in step 1) and the BSMV viral vector γ are respectively digested by restriction enzymes PacI and NotI, and then the digested PstCCC3-S2 is connected with the digested BSMV viral vector γ to obtain a recombinant vector γ-PstCCC3-S2.

[0070] The recombinant vector γ-PstCCC3-S2 can replace the fragment between the PacI and NotI digestion sites of the BSMV viral vector γ with the PstCCC3-S2, and keep other sequences of the BSMV viral vector γ unchanged, wherein the PstCCC3-S2 is opposite to the sequence direction of the PstCCC3 gene.

[0071] The positive clone is obtained by PCR amplification of the recombinant vector γ-PstCCC3-S2 with primer pair γ-F and γ-R, and is identified by sequencing. The positive clone is obtained by inserting SEQ ID NO: 2 from the 691st to 891st nucleotide at the 5' end into the PacI and NotI enzyme cutting sites of the BSMV viral vector γ chain, and keeping other sequences of the γ chain unchanged.

[0072] 3) Obtaining the vector system for silencing PstCCC3 gene

[0073] The BSMV vector system is composed of three genomic RNA chains (α, β, γ), wherein the γ chain is the core component for realizing the function of HIGS (host-induced gene silencing). In the present application, the α, β chains and the recombinant vector γ-PstCCC3-S1 together constitute the viral vector system BSMV:PstCCC3-S1 for silencing PstCCC3 gene. The α, β chains and the recombinant vector γ-PstCCC3-S2 together constitute the viral vector system BSMV:PstCCC3-S2 for silencing PstCCC3 gene.

[0074] The viral vector system constructed above reversely inserts the silencing fragments PstCCC3-S1 and PstCCC3-S2 between the enzyme cutting sites of the restriction endonuclease NotI and PacI of the γ chain, so as to realize the silencing of PstCCC3 gene.

[0075] 2. Linearization of BSMV vector fragments in vitro transcription

[0076] 1) Linearization of the vector

[0077] The BSMV viral vector α and γ chains are respectively digested with MluI, the recombinant vectors γ-PstCCC3-S1 and γ-PstCCC3-S2 are digested with BssHII, and the BSMV viral vector β chain is digested with SpeI, so as to obtain linearized plasmids.

[0078] 2) In vitro transcription

[0079] The linearized plasmids obtained in step 1) are used as templates for in vitro transcription, so as to obtain in vitro transcribed BSMV viral vectors α, β, γ, γ-PstCCC3-S1 and γ-PstCCC3-S2. The in vitro transcription reaction is performed according to the instructions of RiboMAX TM Large Scale RNA Production System-T7 (product of Promega Company, item number: P1300).

[0080] The transcription reaction system and conditions are as follows: the total volume of the reaction is 20.0 μL, including: linearized plasmid 6.5 μL, 5×Transcription Buffer 4.0 μL, Cap 1.5 μL (Promega product, item number: P1718), rNTP PreMix 6.0 μL, Enzyme Mix 2.0 μL; 37°C reaction for 4 h, and the transcription product is stored at -80°C for standby.

[0081] 3. BSMV inoculation

[0082] The "water source 11" wheat is sowed in nutrient soil, and is grown to the two-leaf stage. 10 μL of recombinant virus vector BSMV:TaPDS, BSMV:γ, BSMV:PstCCC3-S1 and BSMV:PstCCC3-S2 solutions are taken respectively, and are rubbed on the wheat leaves. After 10 min, ddH2O is sprayed, the temperature is adjusted to 25°C, and the moisture is maintained for 24 h. Then, the temperature is adjusted to 25°C, and the normal condition culture is carried out. The wheat plants into which BSMV:TaPDS, BSMV:γ, BSMV:PstCCC3-S1 and BSMV:PstCCC3-S2 are transferred are obtained respectively. In addition, the simulation inoculation plants are set, and 1×FES Buffer (MOCK) is applied.

[0083] In the above experiment, BSMV:TaPDS (phytoene desaturase) is used as a positive control, and has an indicating effect. If obvious light bleaching symptoms of the wheat leaves are observed, it indicates that the target gene has been effectively silenced. The plants into which BSMV:γ is transferred are used as a negative control, and the plants to which 1×FES Buffer is applied are blank control plants (MOCK). The plants into which BSMV:PstCCC3-S1 and BSMV:PstCCC3-S2 are transferred are wheat plants in which the PstCCC3 gene is silenced.

[0084] The above BSMV:TaPDS recombinant virus vector solution is obtained by mixing equal amounts of in vitro transcribed BSMV virus vectors α, β and γ-PDS diluted 3 times with DEPC water, and then adding 6 times the volume of 1×FES Buffer. The above BSMV:γ recombinant virus vector solution is obtained by mixing equal amounts of in vitro transcribed BSMV virus vectors α, β and γ diluted 3 times with DEPC water, and then adding 6 times the volume of 1×FES Buffer. The above BSMV:PstCCC3-S1 and BSMV:PstCCC3-S2 recombinant virus vector solutions are obtained by mixing equal amounts of in vitro transcribed BSMV virus vectors α, β and γ-PstCCC3-S1, γ-PstCCC3-S2 diluted 3 times with DEPC water, and then adding 6 times the volume of 1×FES Buffer.

[0085] The phenotype of the wheat leaves in each group after inoculation with different recombinant virus vectors is shown in the left graph of FIG. 1. The leaves of the wheat in the MOCK group were normal, and the leaves of the wheat in the BSMV:TaPDS, BSMV:γ, BSMV:PstCCC3-S1, and BSMV:PstCCC3-S2 groups all showed striped chlorosis, indicating that the BSMV virus was successfully infected. Figure 3

[0086] 4. qRT-PCR verification of the PstCCC3 gene-silenced wheat after inoculation with Puccinia striiformis

[0087] After the wheat plants in each group obtained in Step 3 above were cultured under normal conditions for 10 days, the plants in the BSMV:γ, BSMV:PstCCC3-S1, and BSMV:PstCCC3-S2 groups were inoculated with Puccinia striiformis urediniospores. Samples were taken at 0, 48, 72, and 120 hours after inoculation, and RNA was extracted and reverse transcribed to synthesize cDNA. The synthesized cDNA was used as a template, and qRT-PCR was performed according to the method of Example 1 to detect the relative expression of the PstCCC3 gene in each group. Triadimefon treatment was performed 72 hours after inoculation with Puccinia striiformis, and samples were taken 10 days after triadimefon treatment, and DNA was extracted to detect the biomass of Puccinia striiformis spores on the BSMV:γ control and the PstCCC3 gene-silenced plants.

[0088] The phenotype of the leaves of the test groups (BSMV:γ, BSMV:PstCCC3-S1, and BSMV:PstCCC3-S2) after 10 days of triadimefon treatment is shown in the right graph of FIG. 1. It can be seen that the accumulation of Puccinia striiformis spores on the leaves of the BSMV:γ group was more obvious, and compared therewith, the Puccinia striiformis spores on the leaves of the BSMV:PstCCC3-S1 and BSMV:PstCCC3-S2 groups were significantly less. Figure 3

[0089] The detection results of the relative expression of the PstCCC3 gene after inoculation with Puccinia striiformis are shown in the left graph of FIG. 2. It can be seen that the expression level of the PstCCC3 gene in the BSMV:PstCCC3-S1 and BSMV:PstCCC3-S2 groups was 38-64% of that in the BSMV:γ group, indicating that the expression of the PstCCC3 gene was successfully reduced, and the two selected silencing sequences PstCCC3-S1 and PstCCC3-S2 were both effective (“**” indicates p<0.01, extremely significant). Figure 4 The detection results of the biomass of Puccinia striiformis spores after inoculation with Puccinia striiformis and triadimefon treatment are shown in the right graph of FIG. 2. It can be seen that, after 10 days of triadimefon treatment, the spore biomass of the PstCCC3-S1 and PstCCC3-S2 silenced strains was significantly lower than that of the control leaves in the BSMV:γ group. Figure 4

[0090] ​​​5. Triadimefon sensitivity identification of Puccinia striiformis f. sp. tritici in PstCCC3 gene silenced plants

[0091] In addition, the plants (BSMV:PstCCC3-S1 and BSMV:PstCCC3-S2) obtained in step 3 in which the PstCCC3 gene is effectively silenced, and the negative control plants (BSMV:γ) are inoculated with Puccinia striiformis urediniospores after the fourth leaf unfolds, and 10 mL of triadimefon with a concentration of 100 μg / mL is sprayed on each pot of wheat plants 72 hours after inoculation. The following analysis is performed:

[0092] WGA (wheat germ agglutinin) staining is performed 72 hours after spraying triadimefon, and WGA staining is used to characterize the spread of Puccinia striiformis mycelium in wheat leaves.

[0093] At 72 hours after triadimefon treatment, the mycelial area of each infection point in the wheat leaf is counted, 60 infection points are counted for each sample, and three biological replicates are performed.

[0094] The WGA staining results of each group are shown in a of Figure 5 , and the mycelial area counting results are shown in b of Figure 5 . As can be seen from Figure 5 , the Puccinia striiformis infection area of the BSMV:PstCCC3-S1 and BSMV:PstCCC3-S2 wheat plants is lower than that of the control group.

[0095] The results of this example show that silencing the PstCCC3 gene improves the sensitivity of Puccinia striiformis to triadimefon.

[0096] In summary, PstCCC3 is an important triadimefon resistance-related gene in Puccinia striiformis, which can help Puccinia striiformis resist the toxicity of triadimefon when normally expressed. The present application comprehensively analyzes the physiological function of the gene in the process of using triadimefon to control Puccinia striiformis, and effectively improves the sensitivity of Puccinia striiformis to triadimefon by silencing the gene or inhibiting the expression of the gene, so as to better play the control effect of triadimefon.

[0097] The above examples can better illustrate the technical solutions of the present application, but only describe the preferred embodiments of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various changes and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the scope of protection of the present application.

Claims

1. PstCCC3 protein, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. PstCCC3 gene, characterized in that It comprises a nucleotide sequence encoding the PstCCC3 protein according to claim 1.

3. The PstCCC3 gene according to claim 2, characterized in that The CDS sequence of the PstCCC3 gene is shown in SEQ ID NO:

2.

4. The PstCCC3 gene according to claim 3, characterized in that By silencing the PstCCC3 gene, the sensitivity of wheat stripe rust to triadimefon is enhanced, or the control effect of triadimefon is improved.

5. A vector for silencing the PstCCC3 gene according to any one of claims 2 to 4.

6. The carrier according to claim 5, characterized in that It is composed of the silent sequence PstCCC3-S1 or the silent sequence PstCCC3-S2 connected to the blank viral vector after enzyme digestion; The silent sequence PstCCC3-S1 corresponds to the nucleotide sequence from position 90 to position 293 from the 5' end of the CDS sequence of the PstCCC3 gene; The silent sequence PstCCC3-S2 corresponds to the 691st to 891st nucleotide sequence of the CDS sequence of the PstCCC3 gene from the 5' end.

7. The carrier according to claim 6, characterized in that: The primer pair used to amplify the silent sequence PstCCC3-S1 is SEQ ID NO: 9 and SEQ ID NO: 10; The primer pair used to amplify the silent sequence PstCCC3-S2 is SEQ ID NO: 11 and SEQ ID NO:

12.

8. An expression vector, characterized in that The expression vector is used to silence the PstCCC3 gene.

9. Use of the PstCCC3 protein according to claim 1, or the PstCCC3 gene according to any one of claims 2 to 4, or the vector according to claim 5, or the expression vector according to claim 8 in wheat breeding.

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