Wheat stripe rust protein PstCCC2 and application thereof

Abstract

The invention belongs to the technical field of genetic engineering breeding, and discloses wheat stripe rust protein PstCCC2 and application thereof. The PstCCC2 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 PstCCC2 gene is silenced, the sensitivity of the wheat stripe rust to the triadimefon is remarkably 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 PstCCC2 and application thereof. BACKGROUND

[0002] 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 stripe rust, and triadimefon (a triazole fungicide) is a main promoted fungicide for preventing and controlling stripe rust, which has been used in a large area and single use for more than 50 years, greatly increasing the selection pressure of the emergence of resistant strains, so that the chemical prevention and control of 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 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 resistance of wheat stripe rust to triadimefon, to provide a theoretical basis for the development of management technology for delaying the development speed of stripe rust resistance and new target drugs for the prevention and control of stripe rust. SUMMARY

[0004] In order to better play the prevention and control effect of triadimefon on wheat stripe rust, the application screens a wheat stripe rust vacuolar iron ion transporter PstCCC2 by genetic engineering means, and reveals the physiological function and molecular mechanism of PstCCC2 in the process of preventing and controlling wheat stripe rust by triadimefon through the expression characteristics of the PstCCC2 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 ion transporter PstCCC2, wherein the PstCCC2 protein has an amino acid sequence as shown in SEQ ID NO: 1.

[0006] In a second aspect, the application provides a PstCCC2 gene, wherein the PstCCC2 gene comprises a nucleotide sequence encoding the PstCCC2 protein.

[0007] Further, the CDS sequence of the PstCCC2 gene is shown in SEQ ID NO: 2.

[0008] Further, silencing the PstCCC2 gene enhances the sensitivity of wheat stripe rust to triadimefon or improves the prevention and control effect of triadimefon.

[0009] In a third aspect, the present application provides a vector for silencing the PstCCC2 gene.

[0010] Further, the blank viral vector after enzyme digestion is connected with the silencing sequence PstCCC2-S1 or the silencing sequence PstCCC2-S2;

[0011] The silencing sequence PstCCC2-S1 corresponds to the 20th-252th nucleotide sequence from the 5' end of the CDS sequence of the PstCCC2 gene; and the silencing sequence PstCCC2-S2 corresponds to the 303th-506th nucleotide sequence from the 5' end of the CDS sequence of the PstCCC2 gene.

[0012] Further, the primer pair for amplifying the silencing sequence PstCCC2-S1 is SEQ ID NO: 9 and SEQ ID NO: 10; and the primer pair for amplifying the silencing sequence PstCCC2-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 PstCCC2 gene.

[0014] In a fifth aspect, the present application claims the use of the PstCCC2 protein, the PstCCC2 gene, the vector, or the expression vector in wheat breeding.

[0015] Further, in the above use, the silencing of the PstCCC2 gene and / or the reduction of the concentration of the PstCCC2 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 PstCCC2 protein by using known methods, such as the methods of directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity with the PstCCC2 gene sequence obtained by the present application, as long as they encode the PstCCC2 protein and have the same function, are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application.

[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 PstCCC2 protein (PstCCC2 gene expression cassette) refers to DNA capable of expressing PstCCC2 protein in a host cell, which can include not only a promoter to initiate PstCCC2 transcription, but also a terminator to terminate PstCCC2 transcription. 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 at least have the following beneficial effects or advantages: the present application proves that PstCCC2 gene silenced plants obtained by using transient silencing technology can effectively improve the control effect of triadimefon on Puccinia striiformis, indicating that PstCCC2 protein of Puccinia striiformis has resistance to triadimefon. The PstCCC2 protein, PstCCC2 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 2 is a diagram of the expression pattern of PstCCC2 gene after PstCCC2 gene is treated with triadimefon. Figure 1 The standard deviation is shown by error bars, and "ns" means not statistically significant; "****" means p<0.0001, extremely highly significant compared with 0h.

[0022] Figure 2 Figure 4 is a schematic diagram of the location of the PstCCC2-S1 and PstCCC2-S2 silencing sequences of PstCCC2 gene on the gene.

[0023] Figure 3Phenotype of normal wheat plants after inoculation with different recombinant virus vectors (left) and further inoculation with Puccinia striiformis and treatment with triazolone of the test groups (BSMV: gamma, BSMV: PstCCC2-S1, BSMV: PstCCC2-S2) (right).

[0024] Figure 4 Statistical results of the relative expression of PstCCC2 gene (left) and the biomass of Puccinia striiformis (right) of the test group wheat plants. "**" indicates p < 0.01, extremely significant.

[0025] Figure 5 Histological observation results of wheat leaf tissue with silenced PstCCC2 gene. Among them, a is the Puccinia striiformis mycelium expansion of wheat leaf with silenced PstCCC2 gene under fluorescence microscope at 72h after triazolone treatment; b is the mycelium area of each infection point in wheat leaf at 72h after triazolone treatment. The numerical value represents the average value ± standard deviation of three independent samples (60 infection points / sample), and the error line in the figure represents the standard deviation. "**" (p < 0.01) indicates significant difference, which is obtained by t-test analysis. DETAILED DESCRIPTION

[0026] In the following, the technical solutions of the present application will be described in conjunction with examples, but the present application is not limited to the following examples.

[0027] In the following examples, the experimental methods and detection methods are all conventional methods unless otherwise specified; the reagents and materials can be purchased on the market unless otherwise specified; and the index data are all measured by conventional methods unless otherwise specified.

[0028] Example 1

[0029] This example provides the PstCCC2 protein and its encoding gene.

[0030] Take the normal growth 14d wheat Puccinia striiformis uredospores on the wheat leaf, freeze with liquid nitrogen, and store at -80℃ for standby.

[0031] Trizol method (TianGen) is used to extract total RNA of wheat Puccinia striiformis, and reverse transcriptase XL (AMV) is used for first strand cDNA synthesis. SMART method is used to synthesize cDNA, and PCR products are detected by 1.0% agarose gel electrophoresis. The amplification primer is:

[0032] PstCCC2-F: 5'-CTCGGACGGAGATGTGATTT-3' (SEQ ID NO: 3);

[0033] PstCCC2-R: 5'-CAAACCAACCAATGACTATGATG-3' (SEQ ID NO: 4).

[0034] A 734 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 PstCCC2 gene, which encoded PstCCC2 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 PstCCC2 gene under the treatment of triazolinone.

[0037] Fresh uredospores of Puccinia striiformis YQ324 strain were inoculated on wheat seedlings of Mingxian 169 at the two-leaf stage, and the moisture and culture conditions were referred to the literature "Discovery of Lovell 10 Pathogenic New Strains 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 PstCCC2 gene and internal reference gene. The sequences of the qRT-PCR primers were as follows:

[0039] Q PstCCC2-F: 5'-TCGAATCAGCACAGATCGCA-3' (SEQ ID NO: 5);

[0040] Q PstCCC2-R: 5'-GCTGCTCCACCCACGATAAT-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 3 times, and the Ct value, average value and standard deviation of each repetition were generated by manual adjustment of the baseline by the quantitative PCR instrument. The comparative threshold method (2 –ΔΔCT ) was used to calculate the relative expression of PstCCC2 gene.

[0044] The relative expression of PstCCC2 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 PstCCC2 gene was significantly up-regulated at 12, 24, 48, 72 and 120h after triazolone treatment. Among them, "ns" means no statistical significance; "****" means p<0.0001, extremely high significant compared with 0h. Figure 1 The results show that the expression of PstCCC2 gene is significantly increased by triazolone induction.

[0045] Example 3

[0046] This example provides the verification of the anti-triazolone function of PstCCC2 gene by using HIGS (host-induced gene silencing) technology.

[0047] 1. Construction of PstCCC2 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 PstCCC2-S1

[0050] Using the 734bp PstCCC2 gene fragment amplified in Example 1 as a template, the primer pair PstCCC2-S1F, PstCCC2-S1R was used for PCR amplification, and a PCR amplification product with a size of 233bp (corresponding to the sequence of nucleotides 20-252 from the 5' end of SEQ ID NO: 2) was obtained, which was named as silencing sequence PstCCC2-S1.

[0051] The nucleotide sequences of the primer pair PstCCC2-S1F and PstCCC2-S1R are as follows (the recognition sites of restriction enzymes PacI and NotI are indicated by underlining):

[0052] PstCCC2-S1F: 5'-TAGCGAGTCCGTTAGAGCC-3' (SEQ ID NO: 9);

[0053] PstCCC2-S1R: 5'-GTAGAGACGAGACTCAGGAACTTCT-3' (SEQ ID NO: 10).

[0054] (2) Obtaining of the silencing sequence PstCCC2-S2

[0055] Using the 734 bp PstCCC2 gene fragment amplified in Example 1 as a template, the primer pair PstCCC2-S2F and PstCCC2-S2R was used for PCR amplification, and a 204 bp PCR amplification product (corresponding to the sequence of nucleotides 303-506 from the 5' end of SEQ ID NO: 2) was obtained, which was named the silencing sequence PstCCC2-S2.

[0056] The nucleotide sequences of the primer pair PstCCC2-S2F and PstCCC2-S2R are as follows (the recognition sites of restriction enzymes PacI and NotI are indicated by underlining):

[0057] PstCCC2-S2F: 5'-CATGGTATGCATTTCCCCG-3' (SEQ ID NO: 11);

[0058] PstCCC2-S2R: 5'-GAAACTCACCTTGAATACACCAA-3' (SEQ ID NO: 12).

[0059] The schematic diagram of the positions of the silencing sequences PstCCC2-S1 and PstCCC2-S2 on the PstCCC2 gene is shown in Figure 2 .

[0060] 2) Construction of the silencing vector

[0061] (1) Construction of the γ-PstCCC2-S1 silencing vector

[0062] The PstCCC2-S1 and BSMV viral vector γ obtained in step 1) were respectively digested with restriction enzymes PacI and NotI, and then the digested PstCCC2-S1 and the digested BSMV viral vector γ were ligated to obtain the recombinant vector γ-PstCCC2-S1.

[0063] The recombinant vector γ-PstCCC2-S1 can replace PstCCC2-S1 with the fragment between the PacI and NotI restriction sites of the BSMV viral vector γ, and keep other sequences of the BSMV viral vector γ unchanged, wherein the PstCCC2-S1 is opposite to the sequence direction of the PstCCC2 gene.

[0064] The recombinant vector γ-PstCCC2-S1 is subjected to PCR amplification by using the 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, which is a vector obtained by inserting the nucleotides 20-252 of SEQ ID NO: 2 from the 5' end into the PacI and NotI restriction sites of the γ chain of the BSMV viral vector γ, and keeping other sequences of the γ chain unchanged.

[0068] (2) Construction of the γ-PstCCC2-S2 silencing vector

[0069] The PstCCC2-S2 obtained in step 1) and the BSMV viral vector γ are subjected to restriction enzyme digestion by using the restriction enzymes PacI and NotI, respectively, and then the digested PstCCC2-S2 and the digested BSMV viral vector γ are connected to obtain the recombinant vector γ-PstCCC2-S2.

[0070] The recombinant vector γ-PstCCC2-S2 can replace PstCCC2-S2 with the fragment between the PacI and NotI restriction sites of the BSMV viral vector γ, and keep other sequences of the BSMV viral vector γ unchanged, wherein the PstCCC2-S2 is opposite to the sequence direction of the PstCCC2 gene.

[0071] The recombinant vector γ-PstCCC2-S2 is subjected to PCR amplification by using the primer pair γ-F and γ-R, and a positive clone is obtained by sequencing identification, which is a vector obtained by inserting the nucleotides 303-506 of SEQ ID NO: 2 from the 5' end into the PacI and NotI restriction sites of the γ chain of the BSMV viral vector γ, and keeping other sequences of the γ chain unchanged.

[0072] 3) Obtain the vector system for silencing the PstCCC2 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 and β chains and the recombinant vector γ-PstCCC2-S1 together constitute the viral vector system BSMV:PstCCC2-S1 that can silence the PstCCC2 gene. The α and β chains and the recombinant vector γ-PstCCC2-S2 together constitute the viral vector system BSMV:PstCCC2-S2 that can silence the PstCCC2 gene.

[0074] The viral vector system constructed above has the silencing fragments PstCCC2-S1 and PstCCC2-S2 inserted reversely between the restriction enzyme NotI and PacI cleavage sites of the γ chain, so as to realize the silencing of the PstCCC2 gene.

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

[0076] 1) Linearization of the vector

[0077] The BSMV viral vector α and γ chains are respectively cleaved with MluI, the recombinant vectors γ-PstCCC2-S1 and γ-PstCCC2-S2 are cleaved with BssHII, and the BSMV viral vector β chain is cleaved 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 the in vitro transcribed BSMV viral vectors α, β, γ, γ-PstCCC2-S1 and γ-PstCCC2-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 above transcription reaction system and conditions are as follows: the total reaction volume is 20.0 μL, including: linearized plasmid 6.5 μL, 5×Transcription Buffer 4.0 μL, Cap 1.5 μL (product of Promega Company, 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. Inoculation of BSMV

[0082] Wheat "water source 11" was sowed in nutrient soil, and when it grew to the two-leaf stage, 10 μL of recombinant virus vector BSMV:TaPDS, BSMV:γ, BSMV:PstCCC2-S1 and BSMV:PstCCC2-S2 solutions were respectively rubbed on the wheat leaves, and 10 min later, ddH2O was sprayed to adjust the temperature to 25°C for 24 h of moisturizing. Then, the temperature was changed to 25°C for normal condition culture, and wheat plants into which BSMV:TaPDS, BSMV:γ, BSMV:PstCCC2-S1 and BSMV:PstCCC2-S2 were transferred were obtained. In addition, mock inoculation plants were set, and 1×FES Buffer (MOCK) was applied.

[0083] In the above experiment, BSMV:TaPDS (phytoene desaturase) was used as a positive control and played an indicating role. If obvious light bleaching symptoms were observed on the wheat leaves, it indicated that the target gene had been effectively silenced. The plants into which BSMV:γ was transferred were negative controls, and the plants to which 1×FES Buffer was applied were blank control plants (MOCK). The plants into which BSMV:PstCCC2-S1 and BSMV:PstCCC2-S2 were transferred were wheat plants in which the PstCCC2 gene was silenced.

[0084] The above BSMV:TaPDS recombinant virus vector solution was 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 was 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:PstCCC2-S1 and BSMV:PstCCC2-S2 recombinant virus vector solutions were obtained by mixing equal amounts of in vitro transcribed BSMV virus vectors α, β and γ-PstCCC2-S1 and γ-PstCCC2-S2 diluted 3 times with DEPC water, and then adding 6 times the volume of 1×FES Buffer.

[0085] After inoculation with different recombinant virus vectors, the phenotypic results of the wheat leaves in each group were as shown in the left graph in FIG. 1. The wheat leaves in the MOCK group were normal, and the wheat leaves in the BSMV:TaPDS, BSMV:γ, BSMV:PstCCC2-S1 and BSMV:PstCCC2-S2 groups all showed striped chlorosis, indicating that the BSMV virus infection was successful. Figure 3

[0086] 4. qRT-PCR verification of wheat plants in which the PstCCC2 gene was silenced after inoculation with Puccinia striiformis

[0087] ​After the plants of each group obtained in step 3 above were cultured under normal conditions for 10 days, the plants of BSMV:γ, BSMV:PstCCC2-S1 and BSMV:PstCCC2-S2 groups were inoculated with urediniospores of Puccinia striiformis. 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 PstCCC2 gene in each group. Triadimefon treatment was performed 72 hours after inoculation, and samples were taken 10 days after triadimefon treatment. DNA was extracted, and the urediniospore biomass on the plants of BSMV:γ control and PstCCC2 gene-silenced plants was detected.

[0088] The leaf phenotypes of the test groups (BSMV:γ, BSMV:PstCCC2-S1 and BSMV:PstCCC2-S2) treated with triadimefon for 10 days are shown in the right graph of FIG. 3. It can be seen that the urediniospores accumulated on the leaves of the plants of BSMV:γ group are more obvious, and the urediniospores on the leaves of the plants of BSMV:PstCCC2-S1 and BSMV:PstCCC2-S2 groups are significantly less. Figure 3

[0089] The detection results of the relative expression of PstCCC2 gene after inoculation with urediniospores are shown in the left graph of FIG. 4. It can be seen that the expression levels of PstCCC2 gene in the plants of BSMV:PstCCC2-S1 and BSMV:PstCCC2-S2 groups are 42-62% of that in the plants of BSMV:γ group, indicating that the expression of PstCCC2 gene is successfully reduced, and the two selected silencing sequences PstCCC2-S1 and PstCCC2-S2 are both effective (“**” indicates p<0.01, extremely significant). Figure 4 Figure 4 The detection results of the urediniospore biomass after inoculation with urediniospores and triadimefon treatment are shown in the right graph of FIG. 4. It can be seen that the urediniospore biomass of the plants of PstCCC2-S1 and PstCCC2-S2 groups is significantly lower than that of the control leaves of BSMV:γ group 10 days after triadimefon treatment.

[0090] 5. Identification of triadimefon sensitivity of Puccinia striiformis on PstCCC2 gene-silenced plants

[0091] In addition, the plants (BSMV:PstCCC2-S1 and BSMV:PstCCC2-S2) in which PstCCC2 gene was effectively silenced and the negative control plants (BSMV:γ) obtained in step 3 were selected, and urediniospores were inoculated after the fourth leaf unfolded. Ten mL of triadimefon with a concentration of 100 μg / mL was sprayed on each wheat plant 72 hours after inoculation. The following analysis was performed:

[0092] ​​WGA (wheat germ agglutinin) staining was performed at 72 h after the application of triadimefon, and the WGA staining was used to characterize the spread of Puccinia striiformis f. sp. tritici hyphae in the wheat leaves.

[0093] At 72 h after the triadimefon treatment, the hyphal area of each infection point in the wheat leaves was counted, 60 infection points were counted for each sample, and 3 biological replicates were performed.

[0094] The WGA staining results of each group were counted as shown in a of Figure 5 The hyphal area counting results were shown in b of Figure 5 It can be seen from Figure 5 that the Puccinia striiformis f. sp. tritici infection area of the wheat plants in the BSMV:PstCCC2-S1 and BSMV:PstCCC2-S2 groups was lower than that in the control group.

[0095] The results of the example show that silencing the PstCCC2 gene improves the sensitivity of Puccinia striiformis f. sp. tritici to triadimefon.

[0096] In summary, PstCCC2 is an important triadimefon resistance-related gene in Puccinia striiformis f. sp. tritici, which can help Puccinia striiformis f. sp. tritici 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 f. sp. tritici by using genetic engineering technology, and effectively improves the sensitivity of Puccinia striiformis f. sp. tritici to triadimefon by silencing the gene or inhibiting the expression of the gene, so that the control effect of triadimefon is better.

[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. PstCCC2 protein, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. PstCCC2 gene, characterized in that It comprises a nucleotide sequence encoding the PstCCC2 protein according to claim 1.

3. The PstCCC2 gene according to claim 2, characterized in that The CDS sequence of the PstCCC2 gene is shown in SEQ ID NO:

2.

4. The PstCCC2 gene according to claim 3, characterized in that By silencing the PstCCC2 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 PstCCC2 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 PstCCC2-S1 or the silent sequence PstCCC2-S2 connected to the blank viral vector after enzyme digestion; The silent sequence PstCCC2-S1 corresponds to the nucleotide sequence from position 20 to position 252 from the 5' end of the CDS sequence of the PstCCC2 gene; The silent sequence PstCCC2-S2 corresponds to the nucleotide sequence at positions 303 to 506 from the 5' end of the CDS sequence of the PstCCC2 gene.

7. The carrier according to claim 6, characterized in that: The primer pair used to amplify the silent sequence PstCCC2-S1 is SEQ ID NO: 9 and SEQ ID NO: 10; The primer pair used to amplify the silent sequence PstCCC2-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 PstCCC2 gene.

9. Use of the PstCCC2 protein according to claim 1, or the PstCCC2 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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