CN114181956B - Wheat stripe rust resistance related metabolite and synthesis related gene and application thereof - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to wheat stripe rust resistance-related metabolites and their synthesis-related genes and applications. In the joint analysis of metabolome and transcriptome after wheat was infected by stripe rust fungus, it was found that 2,5-dihydroxyphenylacetic acid (HGA) and its synthesis-related gene TaHPD were induced and up-regulated. Exogenous spraying of HGA can improve wheat, Corn is resistant to rust and can inhibit the germination of rust fungus summer spores. Gene silencing and overexpression of TaHPD also demonstrated that changes in HGA content affect plant disease resistance. The compound HGA of the present invention can be processed into a control agent to realize green prevention and control of crop diseases.

Description

Metabolites related to wheat stripe rust resistance and their synthesis-related genes and applications

Technical field

The invention belongs to the technical field of plant genetic engineering, and in particular relates to the natural compound HGA screened out from the incompatible interaction between wheat and stripe rust and its synthesis-related genes and applications.

Background technique

Wheat is a food crop with wide planting range, high yield and long cultivation history in the world. Bread, noodles, steamed buns and other foods processed from wheat are also loved by everyone and occupy the consumer market. According to statistics, China's wheat planting area increased from 24.0716 million hectares in 2014 to 24.5104 million hectares in 2017, and the output increased from 126.2152 million tons in 2014 to 134.3406 million tons in 2017, showing an upward trend year by year. It can be seen that China's demand for wheat continues to increase. At this growth rate, it is expected that demand will increase by 60% by 2050. However, due to climate, variety and other reasons, wheat is always threatened by various diseases, resulting in reduced yields, and wheat stripe rust is one of the very important diseases.

Wheat stripe rust is a disease caused by Puccinia striiformis f.sp.tritici (Pst). Due to its rapid and frequent pathogenic mutation, it is explosive, epidemic and long-term. , recurrence and other characteristics, it is easy to cause the frequent "loss" of resistance to stripe rust in main wheat varieties, leading to the outbreak of wheat stripe rust, seriously threatening the safe production of global wheat.

At present, the prevention and control of this disease is mainly chemical control, but the long-term and excessive use of chemical agents has caused the destruction of beneficial microorganism populations in the soil, excessive heavy metals and other environmental pollution problems. Therefore, strengthening the research on the rust resistance mechanism of wheat and rationally utilizing the rust resistance of wheat are of great significance for delaying the mutation of pathogens and green control of stripe rust.

Contents of the invention

In view of this, one of the purposes of the present invention is to provide the significantly different metabolite HGA and its synthesis-related genes in the incompatible interaction between wheat and stripe rust.

The object of the present invention is achieved by the following technical means:

Through metabolome and transcriptome association analysis of the non-affinity interaction between wheat and stripe rust, the differential metabolite HGA and the gene TaHPD related to the synthesis of HGA were screened out. The gene TaHPD has three homologous copies on chromosomes 6A, 6B and 6D. The nucleotide sequences of the homologous copy genes TaHPD-6A, TaHPD-6B, and TaHPD-6D are shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3 respectively. Para-hydroxyphenylpyruvate dioxygenase (HPD) is an important enzyme in the metabolism of 2,5-dihydroxyphenylacetic acid (HGA), and TaHPD is the gene encoding p-hydroxyphenylpyruvate dioxygenase (HPD). The amino acid sequences of the protein HPD encoded by the genes TaHPD-6A, TaHPD-6B and TaHPD-6D are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ IDNO.6 respectively.

The second object of the present invention is to provide a gene silencing vector containing TaHPD, a gene related to the anabolic metabolite HGA.

The third object of the present invention is to provide a recombinant vector pCNF3-TaHPD containing the gene TaHPD related to anabolites.

The fourth object of the present invention is to provide the application of HGA in inhibiting the germination of wheat stripe rust spores and/or corn rust spores.

The fifth object of the present invention is to provide the application of HGA as a control agent for wheat stripe rust and/or corn rust, which provides a green prevention and control technology for wheat stripe rust and has important application prospects.

The sixth object of the present invention is the application of the recombinant vector pCNF3-TaHPD of the TaHPD gene in improving tobacco resistance to Sclerotinia sclerotiorum.

The seventh object of the present invention is to provide a method for preventing or inhibiting wheat stripe rust disease. HGA solution is sprayed on wheat leaves, and the concentration of HGA in the HGA solution is 1-10 mmol/L. HGA can improve the ability of wheat to resist stripe rust and can also effectively inhibit the germination of rust spores.

An eighth object of the present invention is to provide a method for preventing or inhibiting corn rust disease. HGA solution is sprayed on corn leaves, and the concentration of HAG in the HGA solution is 1-20 mmol/L. HGA can improve the resistance of corn to rust fungi and can also effectively inhibit the germination of rust fungus spores.

Compared with the existing technology, the present invention has the following beneficial technical effects: The present invention discloses for the first time that the natural compound HGA participates in the process of plant resistance to stripe rust and rust diseases, and the control effect of HGA as a control agent for wheat stripe rust and corn rust. (1) The wheat stripe rust resistance-related metabolite HGA was disclosed for the first time; (2) The synthetic HGA-related gene TaHPD and its nucleotide sequence were obtained; (3) The use of molecular biology and genetic engineering technology confirmed that HGA is involved in plant The process of resisting stripe rust and rust fungi; (4) Exogenous spraying of HGA can improve the resistance of wheat and corn to stripe rust, and can also inhibit the germination of stripe rust and rust spores. The invention confirms the effect of HGA as a control agent for wheat stripe rust and corn rust, and realizes green prevention and control of wheat stripe rust and corn rust.

Description of the drawings

Figure 1 is a differential metabolite trend analysis diagram of Example 1;

Figure 2 is a diagram showing the correlation analysis of the metabolome and transcriptome of Example 1, the real-time fluorescence quantitative PCR detection result diagram of Example 5, and the HGA content measurement diagram after inoculation;

Figure 3 is a phenotypic diagram of exogenous spraying of HGA on wheat (a) and corn (b) in Example 2;

Figure 4 is a graph showing the effect of HGA treatment in Example 3 on the germination of wheat stripe rust spores (a) and corn rust spores (b);

Figure 5 is the phenotypic observation of stripe rust fungus CYR23 after gene silencing in Example 6 (a), and the change in HGA content after gene silencing (d).

Figure 6 shows the infection of Sclerotinia sclerotiorum by overexpression of TaHPD in Example 7 (a), the diameter of the lesion after infection (b), and the change of HGA content after overexpression of TaHPD (c).

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments:

In the present invention, wheat Suwon11 (Su11), MingXian169 (MX169), corn, Nicotiana benthamiana, stripe rust physiological races CYR23, CYR34, corn rust fungus, Escherichia coli competent DH5α, Agrobacterium competent GV3101, vector pCNF3, BSMV:γ and others were provided by the Plant Fungal Disease Research Laboratory, College of Plant Protection, Southwest University.

The following is an example of fluorescent quantitative primers designed using the NCBI database, and the remaining primers were designed using Primer 5. Test primer synthesis and bacterial liquid sequencing were completed by Sangon Bioengineering (Shanghai) Co., Ltd.

The HGA used in the present invention was purchased from TCI Chemical Industry Development Co., Ltd., and other reagents are commercially available.

LB medium formula/L: 10g tryptone, 5g yeast extract, 5g NaCl, add 20g agar powder when preparing solid; PDA medium formula/L: 200g potatoes, 20g glucose, 20g agar.

Example 1 Analysis of metabolites after stripe rust fungus CYR23 infects wheat

(1)Material handling

Disinfect the Suwon11 wheat seeds with 75% alcohol, wash them 4-5 times with ultrapure water, soak them for germination, sow them in a nutrient bowl, and cultivate them in a 12°C ± 2°C environment until the wheat seedlings reach the 1-leaf stage. At that time, stripe rust fungus physiological race CYR23 was applied on the leaves and kept moisturized in the dark for 24 hours. Normally grown wheat was used as the control CK. Samples were collected at 12h, 24h, and 48h after inoculation.

(2) Detect metabolites using ultra-high performance liquid chromatography and tandem mass spectrometry

Use ultra-high performance liquid chromatography and tandem mass spectrometry to detect metabolites from the material obtained in step (1). The specific operation methods are: S1, standards and reagents. All chemical reagents are of analytical grade. The water used is ultrapure water after double deionization, and the ultrapure water instrument purification system is a Millipore product. Chemical standards were purchased from BioBioPha Company and Sigma-Aldrich Company of the United States. Standards are dissolved in dimethyl sulfoxide (DMSO) or methanol as solvent and stored at -20°C. The working samples of the standards were diluted with 70% methanol into different gradient concentrations before use for mass spectrometry analysis. S2: Metabolite extraction, take out the ultra-low temperature cryopreserved biological material sample obtained in step S1, and vacuum freeze-dry the sample. The dried sample was ground with a grinder (MM 400, Retsch) at 30 Hz for 1.5 minutes, 100 mg of powder was weighed, and extracted with 1.0 ml of 70% methanol containing 0.1 mg/l lidocaine as an internal standard at 4°C overnight. , vortex three times during this period to make the extraction more complete. After extraction, centrifuge at 10,000 g for 10 minutes, aspirate the supernatant, filter the sample with a microporous membrane (0.22 μm pore size), and store it in a sample bottle for subsequent LC-MS analysis. Quality control samples (QC) are prepared by mixing sample extracts and used to analyze the repeatability of samples under the same processing method. During the instrument analysis process, one QC sample is generally inserted into every 10 detection and analysis samples to examine the repeatability of the analysis process. S3, metabolite detection, data acquisition instrument system mainly includes ultra-performance liquid chromatography (Ultra Performance Liquid Chromatograp, UPLC) (Shim-pack UFLC SHIMADZU CBM20A, http://www.shimadzu.com.cn/) and tandem mass spectrometry ( Tandem mass spectrometry, MS/MS) (Applied Biosystems 4500QTRAP, http://www.appliedbiosystems.com.cn/). UPLC analysis conditions mainly include: chromatographic column: Waters ACQUITYUPLC HSS T3 C181.8μm, 2.1mm*100mm; mobile phase: the aqueous phase is ultrapure water (0.04% acetic acid is added), the organic phase is acetonitrile (0.04% acetic acid is added) ; Elution gradient, water: acetonitrile, 0min is 95:5V/V, 11.0min is 5:95V/V, 12.0min is 5:95V/V, 12.1min is 95:5V/V, 15.0min is 95:5V /V; the flow rate is 0.4ml/min; the column temperature is 40°C; the injection volume is 5μl. The separated samples enter ESI-QTRAP-MS for mass spectrometry analysis. In the API 4500QTRAP LC/MS/MS system, the main parameters of the linear ion trap and triple quadrupole include: the electrospray ionization source (ESI) temperature is 550°C, the mass spectrometer voltage is 5500V, and the curtain gas (CUR) is 25 psi, and the collision-activated dissociation (CD) parameter is set to high. In the triple quadrupole (QQQ), each ion pair is scanned and detected based on the optimized declustering potential (DP) and collision energy (CE). The obtained data were processed using software Analyst 1.6.1 (AB SCIEX).

The test results in this experiment found that among the significantly different metabolites after the stripe rust fungus CYR23 infected wheat, the most significantly different metabolites at 24 hours were SN-glyceryl-3-phosphocholine, orotic acid, nicotinamide, and nobiletin. , ascorbic acid, hesperidin, salicylic acid and neohesperidin were significantly down-regulated, and those that were significantly up-regulated were 2-aminoadipic acid, methoxyindoleacetic acid, N'-p-coumaroyl agmatine, N-p Coumaroylbutamide, L-histidine, L-(+)-arginine, L-yeastine and 2,5-dihydroxyphenylacetic acid were used for trend analysis of these significantly different metabolites (see attached figure 1), through metabolome and transcriptome correlation analysis, it was found (see Figure 2) that 2,5-dihydroxyphenylacetic acid (HGA) and its synthesis-related gene TaHPD in wheat water source 11 were induced and up-regulated by Triticum stripe rust CYR23, It is proved that HGA is involved in the anti-stripe rust reaction of wheat.

Example 2 Experiment on stripe rust resistance after HGA foliar spraying on wheat and corn

(1) Material cultivation

Disinfect the wheat seeds of Water Source 11 with 75% alcohol, wash them 4-5 times with ultrapure water, soak them for germination, sow them in a nutrient bowl, and cultivate them in a greenhouse at 12°C ± 2°C with 16 hours of light and 8 hours of darkness. After sterilizing the corn seeds with 75% alcohol, wash them 4-5 times with ultrapure water, soak them for germination, sow them in a nutrient bowl, and culture them in a greenhouse at 25°C ± 2°C with 16 hours of light and 8 hours of darkness.

(2)Material handling

HGA solutions with concentrations of 2.5mmol, 5mmol, and 10mmol were sprayed on the wheat leaves respectively. Each treatment was repeated three times. The control group CK was not sprayed, and then the stripe rust fungus compatible physiological race CYR34 was rubbed and inoculated. Observe the degree of disease on the wheat leaves after 12 days.

HGA solutions with concentrations of 5mmol, 10mmol, and 20mmol were sprayed on the corn leaves respectively. Each treatment was repeated three times. The control group CK was not sprayed, and then the corn rust was rubbed and inoculated, and then the disease degree of the corn leaves was observed.

This experiment found (see Figure 3) that the disease level of wheat leaves sprayed with 2.5, 5, and 10 mmol/L HGA was significantly lower than that of the control group, and the disease level of wheat leaves sprayed with 10 mmol/L HGA was the lowest. It is proved that exogenous spraying of 2.5-10mmol/L HGA on wheat can reduce the incidence of wheat stripe rust. The degree of disease on the corn leaves sprayed with 5, 10, and 20 mmol/L HGA was significantly lower than that in the control group, and the degree of disease on the corn leaves sprayed with 20 mmol/L HGA was the lowest. It is proved that exogenous spraying of HGA can reduce the occurrence of common rust in corn.

Example 3 Experiment on the influence of HGA on the germination rate of wheat stripe rust spores and corn common rust spores

Prepare HGA solutions with concentrations of 1mmol/L, 2mmol/L, 5mmol/L, and 10mmol/L respectively. Take a clean glass slide, add a drop of the above-mentioned HGA solutions of different concentrations, and add a drop of distilled water to the control group CK. Use a dissecting needle to pick out a small amount of stripe rust spores and place them in the above-mentioned solutions of different concentrations and distilled water respectively. Adjust the spore concentration under a microscope to approximately 40-60 spores/field of view. Place the slide in a petri dish lined with absorbent paper, and then culture it in an incubator at 12°C ± 2°C. Take them out for observation after 24 hours. Each treatment is repeated three times. The experimental method for corn rust spore inhibition is the same as above. The culture temperature is 20℃±2℃.

The results of this experiment show (see Figure 4) that in the spore inhibition experiment of wheat stripe rust fungus, HGA at concentrations of 1, 2, 5, and 10mmol/L can inhibit spore germination. Among them, 10mmol/L HGA can inhibit spore germination. Rust spores have the best inhibitory effect and can completely inhibit the germination of spores. At low concentrations (1-5mmol/L), the germ tubes produced by summer spores can be deformed. In the spore inhibition experiment of corn rust fungus, 1-10mmol/L HGA can deform the spores and inhibit the elongation of the germ tube. This experiment proves that 1-10mmol/L HGA can effectively inhibit the germination of wheat stripe rust and corn rust spores.

Example 4 Obtainment of gene TaHPD

(1) Extraction of total RNA and its reverse transcription

From the wheat leaves, use a plant total RNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.) to extract total RNA from wheat seedlings, and then use a reverse transcription kit (purchased from Thermo Scientific) to reverse-transcribe the RNA into cDNA. Reaction system as follows. Take 1μg total RNA and perform first-strand cDNA synthesis according to the instructions of the Thermo Scientific RevertAid First Strand cDNASynthesis Kit (Thermo). Add the following components to the nuclease-free PCR tube: total RNA 1μg 10×DNaseI Buffer 1μL DNase I 1μL nuclease -freeddH2O up to 10μL, mix well and incubate in a PCR machine at 37°C for 30 minutes, incubate at 65°C for 5 minutes to inactivate the enzyme, immediately insert it into ice to cool, and continue to add the following components to the system: Oligo (dT) 18 (0.5μg /μL) 1μL nuclease-freeddH2O 1μL, mix and incubate at 65°C for 5 minutes, cool on ice for 2 minutes, then add the following components: 5×Reaction Buffer 4μL Ribollock RNase Inhibitor (20U/μL) 1μL 10mM dNTP Mix 2μL RevertAid M-MuL V RT (200U/μL) 1μL was mixed and incubated in a PCR machine at 45°C for 1 hour, then incubated at 70°C for 5 minutes to inactivate the enzyme, and the product was stored at -20°C for later use.

(2) Amplification and recovery of the full-length sequence of TaHPD gene.

Upstream primer: 5'-ATGCCGCCCACCCCCACC-3', as shown in SEQ ID NO.7;

Downstream primer: 5'-ATCCCTGAACTGCAGCAGATTG-3', as shown in SEQ ID NO.8;

Using the cDNA of the reaction product obtained in step (1) as the template for the PCR reaction, perform PCR amplification of the TaHPD gene. The reaction system is shown in Table 1.

Table 1 Gene amplification reaction system

The PCR amplification reaction program was: 95°C for 3 min, 35 cycles of (95°C for 30 sec, 55°C for 30 sec, 72°C for 90 sec), 72°C for 10 min, and terminating the reaction at 16°C.

The PCR product was detected by agarose gel electrophoresis, and the gel with the target band was recovered and sequenced by Sangon Bioengineering (Shanghai) Co., Ltd. The gene TaHPD has three homologs on chromosomes 6A, 6B and 6D. Copy, the nucleotide sequences of the homologous copy genes TaHPD-6A, TaHPD-6B, and TaHPD-6D are shown in SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11 respectively. The amino acid sequences of the protein HPD encoded by the genes TaHPD-6A, TaHPD-6B, and TaHPD-6D are shown in SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14 respectively. HPD is an important enzyme in the metabolism of HGA.

The nucleotide sequence of TaHPD-6A is:

ATGCCGCCCACCCCCACCACCCCCGCAGCCACCGGCGCCGCCGCGGTGACGCCGGAGCACGCGCGGCCGCGCCGAATGGTCCGCTTCAACCCGCGCAGCGACCGCTTCCACACGCTCGCCTTCCACCACGTCGAGTTCTGGTGCGCGGACGCCGCCTCCGCCGCCGGCCGCTTCGCCTTCGCGCTCGGCGCCGCTCGCCGCCAGGTCCGACCTCTCCACGGGGAACTCCGTGCACGCCTCCCAGCTGCTCCGCTCG GGCAACCTCGCCTTCCTCTTCACGGCCCCCTACGCGCCAACGGCTGCGACGCCGCCACCGCCTCCCTGCCCTCCTTCCGCCGACGCCGCGCCAGTTCCTCCGCGGACCACGGCCTCGCGGTGCGCTCCATAGCGCTGCGCGTCGCGGACGCTGCCGAGGCCTTCCGCGCCAGCGTCGACGGGGGCGCGCGCCCGGCCTTCAGCCCTGTGGACCTCGGCCGCGGCTTCGGCTTCGCGGAGGTCGAGCTCTA CGGCGACGTCGTGCTCCGCTTCGTCAGCCACCCGGACGGCAGGGACGTGCCCTTCTTGCCGGGGTTCGAGGGCGTGAGCAACCCAGACGCCGTGGACTACGGCCTGACGCGGTTCGACCACGTCGTCGGCAACGTCCCGGAGCTTGCCCCCGCCGCGGCCTACGTCGCCGGGTTCACGGGGTTCCACGAGTTCGCCGAGTTCACGACGGAGGACGTGGGCACGGCCGAGAGCGGGCTCAACTCGATGGTGC TCGCCAACAACTCGGAGGGCGTGCTGCTGCCGCTCAACGAGCCGGTGCACGGCACCAAGCGCCGGAGCCAGATACAGACGTTCCTGGAACACCACGGCGGCTCGGGCGTGCAGCACATCGCGGTGGCCAGCAGCGACGTGCTCAGGACGCTCAGGGAGATGCGTGCGCGCTCCGCCATGGGCGGCTTCGACTTCCTGCCACCCCCGCTGCCGAAGTACTACGAAGGCGTGCGGCGCATCGCCGGGGATGTGCTC TCGGAGGCGCAGATCAAGGAATGCCAGGAGCTGGGGGTGCTCGTCGACAGGGACGACCAAGGGGTGTTGCTACAAATCTTCACCAAGCCAGTAGGGGACAGGCCGACGTTGTTCCTGGAGATGATCCAGAGGATCGGGTGCATGGAGAAGGACGAGAGAGGGGAAGAGTACCAGAAGGGTGGCTGCGGCGGGTTCGGCAAAGGCAACTTCTCCGAGCTGTTCAAGTCCATTGAAGATTACGAGAAGTCCCTTGAA GCCAAGCAATCTGCTGCAGTTCAGGGATCATAG

The nucleotide sequence of TaHPD-6B is:

ATGCCGCCCACCCCCACCACCCCGGCAGCTACCGGCGCCGCCGCCGCCGCCGCGGGTGACGCCGGAGCATGCACGGCCACGTAGAATGGTCCGCTTCAACCCGCGGAGCGACCGCTTCCACACGCTCGCCTTCCACCACGTCGAGTTCTGGTGCGCGGACGCCGCCTCCGCCGCCGGCCGCTTCGCCTTCGCGCTCGGCGCGCCGCTCGCCGCCAGGTCCGACCTCTCCACGGGGAACTCCGTGCACGCCTCCCAGCT GCTCCGCTCGGGCAACCTCGCCTTCCTTCACCGCGCCCTACGCGAACGGCTGCGACGCCGCCACCGCCTCCCTGCCCTCCTTCTCCGCCGACGCCGCCGCCGGTTCTCCGCGGACCACGGGCTCGCAGTGCGCTCCATAGCACTGCGCGTCGCAGACGCCGCAGAGGCCTTCCGCGCCAGCGTCGACGGAGGCGCGCGCCCGGCCTTCAGCCCCGTGGACCTCGGCCGCGGCTTCGGCTTCGCGGAGGTCGA GCTCTACGGCGACGTCGTGCTCCGCTTCGTCAGTCACCCGGATGACACGGACGTGCCCTTCTTGCCGGGGTTCGAGGGCGTGAGCAACCCGGATGCCGTGGACTACGGCCTGACGCGGTTCGACCACGTCGTCGGCAACGTCCCGGAGCTTGCCCCCGCCGCCGCATACGTCGCCGGGTTCGCGGGGTTCCACGAGTTCGCCGAGTTCACGACGGAGGACGTGGGCACGGCCGAGAGCGGGCTCAACTCGATG GTGCTCGCCAACAACTCCGAGGGCGTGCTGCTGCCGCTCAACGAGCCGGTGCACGGCACCAAGCGCCGGAGCCAGATACAGACGTTCCTGGAACACCACGGTGGCCCGGGCGTGCAGCACATCGCGGTGGCCAGCAGCGACGTGCTCAGGACGCTCAGGGAGATGCGTGCGCGCTCCGCCATGGGCGGCTTCGACTTCCTGCCACCCCCGCTGCCGAAGTACTATGAAGGCGTGCGGCGCATCGCGGGGGATGT GCTCTCGGAGGCGCAGATCAAGGAATGCCAGGAGCTGGGGGTGCTCGTCGACAGGGACGACCAAGGGGTGTTGCTCCAAATCTTCACCAAGCCAGTGGGGGACAGGCCAACGCTGTTCCTGGAGATGATCCAAAGGATCGGGTGCATGGAGAAGGACGAGAGAGGGGAAGAGTACCAGAAGGGTGGCTGCGGCGGGTTTGGCAAAGGCAACTTCTCCGAGCTGTTCAAGTCCATTGAGGATTATGAGAAATCCCTT GAAGCCAAGCAATCTGCTGCAGTTCAGGCATCATAG

The nucleotide sequence of TaHPD-6D is:

ATGCCGCCCACCCCCACCACCCCCGCAGCCACCGGCGCCGGCGCTGCCGCCGCGGTGACGCCGGAGCACGCGCGGCCGCGCCGAATGGTCCGCTTCAACCCGCGCAGCGACCGCTTCCACACGCTCTCCTTCCACCACGTCGAGTTCTGGTGCGCGGACGCCGCCTCCGCCGCCGGCCGCTTCGCCTTCGCGCTCGGCGCGCCGCTCGCCGCCAGGTCCGACCTCTCCACGGGGAACTCCGTGCACGCCTCCCAGCTGC TCCGCTCGGGCAACCTCGCCTTCCTCTTCACCGCGCCCTACGCCAACGGCTGCGACGCCGCCACCGCCTCCCTGCCCTCCTTCTCCGCCGACGCCGCGCCGCCGGTTCTCCGCGGACCACGGGCTCGCGGTGCGCTCCATAGCGCTGCGCGTCGCGGACGCCGCCGAGGCCTTCCGCGCCAGCGTCGACGGGGGCGCGCGCCCGGCCTTCAGCCCCGTGGACCTCGGCCGCGGCTTCGGCTTTGCCGGAGGTCGA GCTCTACGGCGACGTCGTGCTCCGCTTCGTCAGCCATCCGGACGGCACGGACGTGCCCTTCTTGCCGGGGTTCGAGGGCGTGAGCAACCCGGGTGCCGTGGACTACGGCCTGACACGGGTTTGACCACGTCGTCGGCAACGTCCCGGAGCTTGCTTCCGCCGCCGCCTACGTAGCCGGCTTCACGGGTTTCCATGAGTTCGCCGAGTTCACGACGGAGGACGTGGGCACGGCCGAGAGCGGGCTCAACTCGATG GTGCTCGCCAACAACTCGGAGGGCGTGCTGCTGCCGCTCAACGAGCCGGTGCACGGCACCAAGCGCCGGAGCCAGATACAGACGTTCCTGGAACCACGGCGGCCCGGGTGTGCAGCACATCGCGGTGGCCAGCAGCGACGTGCTCAGGACGCTCAGGGAGATGCGTGCGCGCTCCGCCATGGGCGGCTTCGACTTCCTGCCACCCCCGCTGCCGAAGTACTACGAAGGCGTGCGGCGCATCGCCGGGGATGT GCTCTCGGAGGCGCAGATCAAGGAATGCCAGGAGCTGGGGGTGCTCGTCGACAGGGACGACCAAGGGGTGTTGCTACAAATCTTCACAAAGCCAGTGGGGGACAGGCCAACGCTGTTCCTGGAGATGATCCAAAGGATCGGGTGCATGGAGAAGGACGAGAGAGGGGAAGAGTACCAGAAGGGTGGCTGCGGCGGGTTCGGCAAAGGCAACTTCTCCGAGCTGTTCAAGTCCATTGAAGATTACGAGAAGTCCCTT GAAGCCAAGCAATCTGCTGCAGTTCAGGGATCATAG

Example 5 Real-time fluorescence quantitative PCR detection of gene expression characteristics in wheat under stripe rust induction conditions

Disinfect the wheat seeds of Water Source 11 with 75% alcohol, wash them 4-5 times with ultrapure water, soak them for germination, sow them in a nutrient bowl, and cultivate them in an environment of 12°C ± 2°C until the wheat seedlings reach the 1-leaf stage. The stripe rust fungus physiological race CYR23 was applied on the leaves and kept moisturized in the dark for 24 hours. Normally grown wheat was used as the control CK. The seedling leaves were cut off at 12h, 24h and 48h after inoculation and placed in the refrigerator for later use. There were 3 biological replicates for each sample. Obtain the cDNA of each treatment according to the method in Example 4, and design fluorescent quantitative expression primers based on the full-length cDNA sequence:

Upstream primer 5'-CAGTAGGGGACAGGCCGA-3', as shown in SEQ ID NO.9

Downstream primer 5'-CCGCATCTTACAAACAACATCA-3', as shown in SEQ ID NO.10

Internal reference gene primers:

Upstream primer: 5'-TGGTGTCATCAAGCCTGGTATGGT-3', as shown in SEQ ID NO.11

Downstream primer: 5'-ACTCATGGTGCATCTCAACGGACT-3', as shown in SEQ ID NO.12

The real-time fluorescence quantitative PCR reaction system is shown in Table 2.

Table 2 Real-time fluorescence quantitative reaction system

The reaction program is: 95°C for 1 min, (95°C for 10 sec, 60°C for 20 sec, 72°C for 40 sec) for 40 cycles, and then the reaction is completed.

The relative expression of TaHPD gene was calculated using the relative quantification formula (2 ^-ΔΔCt ).

Relative ratio=2 ^-ΔΔCt , ΔΔCt=(C _treat ^M -C _treat ^A )-(C _control ^M -C _control ^A ).

The results of this experiment are shown in Figure 2. The relative expression of TaHPD gene showed up-regulation in wheat leaves from 0 to 48h after inoculation. At 24h after inoculation, the expression level was significantly higher than that in the control treatment. This experiment proves that the TaHPD gene is involved in the process of wheat stripe rust resistance.

Example 6 Using BSMV-mediated gene silencing experiment to verify the stripe rust resistance function of TaHPD gene

Specific fragments of TaHPD were selected and ligated with BSMV:γ to form a recombinant vector. Then linearize BSMV:α, BSMV:β, BSMV:γ and recombinant vectors, BSMV:α, BSMV:γ plasmids, BSMV:PDS and recombinant plasmids BSMV:TaHPD-V1, BSMV:TaHPD-V2 using MluI, BSMV:β Use Spe I for plasmids. Use the linearized plasmid as a template and use the Ribo MAX ^TM LargeScale RNA Production Systems-T7 in vitro transcription kit to conduct in vitro transcription of the plasmid obtained in the above reaction. Aspirate 0.5 μl of the in vitro transcription product and dilute it 10 times with nuclease-free water, and detect it by 1% agarose gel electrophoresis. Store at -80°C for later use.

Virus vaccination test:

(1) Dilute all in vitro transcription products 3 times with nuclease-free H ₂ O, take 2.5 μl of each α, β, γ or recombinant γ vector in vitro transcription products and mix them in equal amounts;

(2) Add 45 μl of FES buffer into it, mix thoroughly with a micropipette, and divide into 5 equal portions;

(3) Wear latex gloves when receiving the virus, dip your index finger into the virus liquid, select the second leaf of wheat in good growth condition and rub it back and forth 3 times from the base to the tip;

(4) Spray a small amount of nuclease-free H ₂ O on the wheat that has been grafted with the virus, place it in a greenhouse at 25±2°C to avoid light and moisturize for 24 hours, and culture it in a 16h light/8h dark cycle. In each experiment, BSMV:γ was inoculated as a control, and BSMV:γ-PDS was used as a positive control.

Stripe rust inoculation, sampling and phenotypic observation:

When the plants inoculated with BSMV:γ-PDS show bleaching and chlorosis, select wheat with good virus symptoms and inoculate the stripe rust fungus CYR23 in the marked part of the fourth leaf. The marked and inoculated leaves were cut at 0, 24, and 48 hpi, quickly frozen in liquid nitrogen, and stored at -80°C for later use. Reverse transcribed cDNA was used to detect the silencing efficiency of the target gene and the expression level of related defense genes. The cDNA of the fourth leaf of wheat inoculated with BSMV:γ virus was used as a control. After inoculation with the virus, regularly observe and take photos to record the infection symptoms of the virus; after inoculation with the stripe rust fungus, regularly observe and take photos to record the disease status of the wheat.

Phenotypic observation results: After silencing the wheat TaHPD gene, wheat became more sensitive to stripe rust fungus, and the HAG content was also significantly reduced (see Figure 5). It was proved that HGA is involved in the process of wheat stripe rust resistance.

Example 7 Construction of recombinant vector pCNF3-TaHPD

Design primers based on the complete open reading frame of the TaHPD gene:

Upstream primer:

5'-TGC TCTAGA ATGCCGCCCACCCCCACC-3', as shown in SEQ ID NO.13

Downstream primers:

5'-CGG GGTACC GTGGTGGTGGTGGTGGTGTGATCCCTGAACTGCAGCAGATTG-3', as shown in SEQ IDNO.14

The PCR amplification product in Example 4 was connected to pCNF3. Then transform the ligation product into Escherichia coli DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), add the ligation product to 50ul of the freshly thawed competent cells, mix well, incubate on ice for 30 minutes, heat shock in 42°C water bath for 45 seconds, and immediately Place on ice for 2 minutes. Add 500ul of LB liquid culture medium without antibiotics, 200rpm, and incubate at 37°C for 1 hour. Centrifuge for 1 minute, discard part of the supernatant with a pipette, flick to suspend the bacterial cells, retain 200ul of bacterial liquid, spread evenly on the LB agar medium containing Kan, invert the plate, and culture at 37°C overnight. Pick multiple single colonies with normal status and culture them in LB liquid medium containing Kan at 37°C and 200 rpm. Shake the bacteria for 12h-16h until the bacterial liquid becomes turbid. Sequence three independent clones in both directions and save the bacteria for later use.

Shake the correctly sequenced bacterial solution and extract the plasmid. After the plasmid extraction is completed, conduct concentration measurement and agarose gel electrophoresis detection, and select plasmid candidates with high concentration and bright bands for later use. Use the plasmid as a template and perform PCR with the designed cloning primers. The PCR program is as follows: 95°C for 3 minutes, (95°C for 30 seconds, 55°C for 30 seconds, 72°C for 90 seconds) for 35 cycles, 72°C for 10 minutes, and 16°C to terminate the reaction.

After agarose gel electrophoresis, the PCR product was recovered, and after concentration determination, it was stored at -20°C for later use. Shake the bacterial culture of the pCNF3 expression vector, extract the plasmid, and select the one with a high concentration for the enzyme digestion test. The enzyme digestion system is shown in Table 3.

Table 3 Enzyme digestion system

After digestion at 37°C for 4 hours, the digested vector fragment was recovered from the gel and the concentration was determined. The target fragment and the linearized vector fragment were recombined to obtain a tobacco transient expression vector. By constructing a tobacco transient expression vector, GV3101 Agrobacterium was used to inject into Nicotiana benthamiana for transient expression.

Transform the constructed tobacco transient expression vector into Agrobacterium competent cells, transfer Agrobacterium to liquid culture medium and culture it to the logarithmic growth phase, collect the cell pellet by centrifugation, add buffer, and resuspend until the OD 600 value is about 0.6 -0.8, infected wheat by Agrobacterium infiltration method. Freshly activated S. sclerotiorum 1980 was inoculated 48 hours after constructing the tobacco transient expression vector.

The results of this experiment (see Figure 6): 24 hours later, the diseased area in the area injected with pCNF3-TaHPD was significantly smaller than the area injected with pCNF3-eGFP. The HGA content in pCNF3-TaHPD overexpression plants was significantly higher than that in pCNF3-eGFP. This experiment shows that overexpression of TaHPD can improve tobacco resistance to Sclerotinia sclerotiorum.

The above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently substituted. Without departing from the purpose and scope of the technical solutions of the present invention, they should all be covered by the claims of the present invention. The technology, shape, and structural parts not described in detail in the present invention are all known technologies.

sequence list

<110> Southwest University

<120> Metabolites related to wheat stripe rust resistance and their synthesis-related genes and applications

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1302

<212> DNA

<213> Wheat (Triticum aestivum L.)

<400> 1

atgccgccca cccccaccac ccccgcagcc accggcgccg ccgcggtgac gccggagcac 60

gcgcggccgc gccgaatggt ccgcttcaac ccgcgcagcg accgcttcca cacgctcgcc 120

ttccaccacg tcgagttctg gtgcgcggac gccgcctccg ccgccggccg cttcgccttc 180

gcgctcggcg cgccgctcgc cgccaggtcc gacctctcca cggggaactc cgtgcacgcc 240

tcccagctgc tccgctcggg caacctcgcc ttcctcttca cggccccccta cgccaacggc 300

tgcgacgccg ccaccgcctc cctgccctcc ttctccgccg acgccgcgcg ccagttctcc 360

gcggaccacg gcctcgcggt gcgctccata gcgctgcgcg tcgcggacgc tgccgaggcc 420

ttccgcgcca gcgtcgacgg gggcgcgcgc ccggccttca gccctgtgga cctcggccgc 480

ggcttcggct tcgcggaggt cgagctctac ggcgacgtcg tgctccgctt cgtcagccac 540

ccggacggca gggacgtgcc cttcttgccg gggttcgagg gcgtgagcaa cccagacgcc 600

gtggactacg gcctgacgcg gttcgaccac gtcgtcggca acgtcccgga gcttgccccc 660

gccgcggcct acgtcgccgg gttcacgggg ttccacgagt tcgccgagtt cacgacggag 720

gacgtgggca cggccgagag cgggctcaac tcgatggtgc tcgccaacaa ctcggagggc 780

gtgctgctgc cgctcaacga gccggtgcac ggcaccaagc gccggagcca gatacagacg 840

ttcctggaac accacggcgg ctcgggcgtg cagcacatcg cggtggccag cagcgacgtg 900

ctcaggacgc tcaggggagat gcgtgcgcgc tccgccatgg gcggcttcga cttcctgcca 960

cccccgctgc cgaagtacta cgaaggcgtg cggcgcatcg ccggggatgt gctctcggag 1020

gcgcagatca aggaatgcca ggagctgggg gtgctcgtcg acagggacga ccaaggggtg 1080

ttgctacaaa tcttcaccaa gccagtaggg gacaggccga cgttgttcct ggagatgatc 1140

cagaggatcg ggtgcatgga gaaggacgag agaggggaag agtaccagaa gggtggctgc 1200

ggcgggttcg gcaaaggcaa cttctccgag ctgttcaagt ccattgaaga ttacgagaag 1260

tcccttgaag ccaagcaatc tgctgcagtt cagggatcat ag 1302

<210> 2

<211> 1311

<212> DNA

<213> Wheat (Triticum aestivum L.)

<400> 2

atgccgccca cccccaccac cccggcagct accggcgccg ccgccgccgc cgcggtgacg 60

ccggagcatg cacggccacg tagaatggtc cgcttcaacc cgcggagcga ccgcttccac 120

acgctcgcct tccaccacgt cgagttctgg tgcgcggacg ccgcctccgc cgccggccgc 180

ttcgccttcg cgctcggcgc gccgctcgcc gccaggtccg acctctccac ggggaactcc 240

gtgcacgcct cccagctgct ccgctcgggc aacctcgcct tcctcttcac cgcgccctac 300

gcgaacggct gcgacgccgc caccgcctcc ctgccctcct tctccgccga cgccgcgcgc 360

cggttctccg cggaccacgg gctcgcagtg cgctccatag cactgcgcgt cgcagacgcc 420

gcagaggcct tccgcgccag cgtcgacgga ggcgcgcgcc cggccttcag ccccgtggac 480

ctcggccgcg gcttcggctt cgcggaggtc gagctctacg gcgacgtcgt gctccgcttc 540

gtcagtcacc cggatgacac ggacgtgccc ttcttgccgg ggttcgaggg cgtgagcaac 600

ccggatgccg tggactacgg cctgacgcgg ttcgaccacg tcgtcggcaa cgtcccggag 660

cttgcccccg ccgccgcata cgtcgccggg ttcgcggggt tccacgagtt cgccgagttc 720

acgacggagg acgtgggcac ggccgagagc gggctcaact cgatggtgct cgccaacaac 780

tccgagggcg tgctgctgcc gctcaacgag ccggtgcacg gcaccaagcg ccggagccag 840

atacagacgt tcctggaaca ccacggtggc ccgggcgtgc agcacatcgc ggtggccagc 900

agcgacgtgc tcaggacgct cagggagatg cgtgcgcgct ccgccatggg cggcttcgac 960

ttcctgccac ccccgctgcc gaagtactat gaaggcgtgc ggcgcatcgc gggggatgtg 1020

ctctcggagg cgcagatcaa ggaatgccag gagctggggg tgctcgtcga cagggacgac 1080

caaggggtgt tgctccaaat cttcaccaag ccagtggggg acaggccaac gctgttcctg 1140

gagatgatcc aaaggatcgg gtgcatggag aaggacgaga gaggggaaga gtaccagaag 1200

ggtggctgcg gcgggtttgg caaaggcaac ttctccgagc tgttcaagtc cattgaggat 1260

tatgagaaat cccttgaagc caagcaatct gctgcagttc aggcatcata g 1311

<210> 3

<211> 1311

<212> DNA

<213> Wheat (Triticum aestivum L.)

<400> 3

atgccgccca cccccaccac ccccgcagcc accggcgccg gcgctgccgc cgcggtgacg 60

ccggagcacg cgcggccgcg ccgaatggtc cgcttcaacc cgcgcagcga ccgcttccac 120

acgctctcct tccaccacgt cgagttctgg tgcgcggacg ccgcctccgc cgccggccgc 180

ttcgccttcg cgctcggcgc gccgctcgcc gccaggtccg acctctccac ggggaactcc 240

gtgcacgcct cccagctgct ccgctcgggc aacctcgcct tcctcttcac cgcgccctac 300

gccaacggct gcgacgccgc caccgcctcc ctgccctcct tctccgccga cgccgcgcgc 360

cggttctccg cggaccacgg gctcgcggtg cgctccatag cgctgcgcgt cgcggacgcc 420

gccgaggcct tccgcgccag cgtcgacggg ggcgcgcgcc cggccttcag ccccgtggac 480

ctcggccgcg gcttcggctt tgcggaggtc gagctctacg gcgacgtcgt gctccgcttc 540

gtcagccatc cggacggcac ggacgtgccc ttcttgccgg ggttcgaggg cgtgagcaac 600

ccgggtgccg tggactacgg cctgacacgg tttgaccacg tcgtcggcaa cgtcccggag 660

cttgcttccg ccgccgccta cgtagccggc ttcacgggtt tccatgagtt cgccgagttc 720

acgacggagg acgtgggcac ggccgagagc gggctcaact cgatggtgct cgccaacaac 780

tcggagggcg tgctgctgcc gctcaacgag ccggtgcacg gcaccaagcg ccggagccag 840

atacagacgt tcctggaaca ccacggcggc ccgggtgtgc agcacatcgc ggtggccagc 900

agcgacgtgc tcaggacgct cagggagatg cgtgcgcgct ccgccatggg cggcttcgac 960

ttcctgccac ccccgctgcc gaagtactac gaaggcgtgc ggcgcatcgc cggggatgtg 1020

ctctcggagg cgcagatcaa ggaatgccag gagctggggg tgctcgtcga cagggacgac 1080

caaggggtgt tgctacaaat cttcacaaag ccagtggggg acaggccaac gctgttcctg 1140

gagatgatcc aaaggatcgg gtgcatggag aaggacgaga gaggggaaga gtaccagaag 1200

ggtggctgcg gcgggttcgg caaaggcaac ttctccgagc tgttcaagtc cattgaagat 1260

tacgagaagt cccttgaagc caagcaatct gctgcagttc agggatcata g 1311

<210> 4

<211> 433

<212> PRT

<213> Wheat (Triticum aestivum L.)

<400> 4

Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Ala Ala Val

1 5 10 15

Thr Pro Glu His Ala Arg Pro Arg Arg Met Val Arg Phe Asn Pro Arg

20 25 30

Ser Asp Arg Phe His Thr Leu Ala Phe His His Val Glu Phe Trp Cys

35 40 45

Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala Leu Gly Ala

50 55 60

Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser Val His Ala

65 70 75 80

Ser Gln Leu Leu Arg Ser Gly Asn Leu Ala Phe Leu Phe Thr Ala Pro

85 90 95

Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser Leu Pro Ser Phe Ser

100 105 110

Ala Asp Ala Ala Arg Gln Phe Ser Ala Asp His Gly Leu Ala Val Arg

115 120 125

Ser Ile Ala Leu Arg Val Ala Asp Ala Ala Glu Ala Phe Arg Ala Ser

130 135 140

Val Asp Gly Gly Ala Arg Pro Ala Phe Ser Pro Val Asp Leu Gly Arg

145 150 155 160

Gly Phe Gly Phe Ala Glu Val Glu Leu Tyr Gly Asp Val Val Leu Arg

165 170 175

Phe Val Ser His Pro Asp Gly Arg Asp Val Pro Phe Leu Pro Gly Phe

180 185 190

Glu Gly Val Ser Asn Pro Asp Ala Val Asp Tyr Gly Leu Thr Arg Phe

195 200 205

Asp His Val Val Gly Asn Val Pro Glu Leu Ala Pro Ala Ala Ala Tyr

210 215 220

Val Ala Gly Phe Thr Gly Phe His Glu Phe Ala Glu Phe Thr Thr Glu

225 230 235 240

Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Met Val Leu Ala Asn

245 250 255

Asn Ser Glu Gly Val Leu Leu Pro Leu Asn Glu Pro Val His Gly Thr

260 265 270

Lys Arg Arg Ser Gln Ile Gln Thr Phe Leu Glu His His Gly Gly Ser

275 280 285

Gly Val Gln His Ile Ala Val Ala Ser Ser Asp Val Leu Arg Thr Leu

290 295 300

Arg Glu Met Arg Ala Arg Ser Ala Met Gly Gly Phe Asp Phe Leu Pro

305 310 315 320

Pro Pro Leu Pro Lys Tyr Tyr Glu Gly Val Arg Arg Ile Ala Gly Asp

325 330 335

Val Leu Ser Glu Ala Gln Ile Lys Glu Cys Gln Glu Leu Gly Val Leu

340 345 350

Val Asp Arg Asp Asp Gln Gly Val Leu Leu Gln Ile Phe Thr Lys Pro

355 360 365

Val Gly Asp Arg Pro Thr Leu Phe Leu Glu Met Ile Gln Arg Ile Gly

370 375 380

Cys Met Glu Lys Asp Glu Arg Gly Glu Glu Tyr Gln Lys Gly Gly Cys

385 390 395 400

Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu

405 410 415

Asp Tyr Glu Lys Ser Leu Glu Ala Lys Gln Ser Ala Ala Val Gln Gly

420 425 430

Ser

<210> 5

<211> 436

<212> PRT

<213> Wheat (Triticum aestivum L.)

<400> 5

Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Ala Ala Ala

1 5 10 15

Ala Ala Val Thr Pro Glu His Ala Arg Pro Arg Arg Met Val Arg Phe

20 25 30

Asn Pro Arg Ser Asp Arg Phe His Thr Leu Ala Phe His His Val Glu

35 40 45

Phe Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala

50 55 60

Leu Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser

65 70 75 80

Val His Ala Ser Gln Leu Leu Arg Ser Gly Asn Leu Ala Phe Leu Phe

85 90 95

Thr Ala Pro Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser Leu Pro

100 105 110

Ser Phe Ser Ala Asp Ala Ala Arg Arg Phe Ser Ala Asp His Gly Leu

115 120 125

Ala Val Arg Ser Ile Ala Leu Arg Val Ala Asp Ala Ala Glu Ala Phe

130 135 140

Arg Ala Ser Val Asp Gly Gly Ala Arg Pro Ala Phe Ser Pro Val Asp

145 150 155 160

Leu Gly Arg Gly Phe Gly Phe Ala Glu Val Glu Leu Tyr Gly Asp Val

165 170 175

Val Leu Arg Phe Val Ser His Pro Asp Asp Thr Asp Val Pro Phe Leu

180 185 190

Pro Gly Phe Glu Gly Val Ser Asn Pro Asp Ala Val Asp Tyr Gly Leu

195 200 205

Thr Arg Phe Asp His Val Val Gly Asn Val Pro Glu Leu Ala Pro Ala

210 215 220

Ala Ala Tyr Val Ala Gly Phe Ala Gly Phe His Glu Phe Ala Glu Phe

225 230 235 240

Thr Thr Glu Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Met Val

245 250 255

Leu Ala Asn Asn Ser Glu Gly Val Leu Leu Pro Leu Asn Glu Pro Val

260 265 270

His Gly Thr Lys Arg Arg Ser Gln Ile Gln Thr Phe Leu Glu His His

275 280 285

Gly Gly Pro Gly Val Gln His Ile Ala Val Ala Ser Ser Asp Val Leu

290 295 300

Arg Thr Leu Arg Glu Met Arg Ala Arg Ser Ala Met Gly Gly Phe Asp

305 310 315 320

Phe Leu Pro Pro Pro Leu Pro Lys Tyr Tyr Glu Gly Val Arg Arg Ile

325 330 335

Ala Gly Asp Val Leu Ser Glu Ala Gln Ile Lys Glu Cys Gln Glu Leu

340 345 350

Gly Val Leu Val Asp Arg Asp Asp Gln Gly Val Leu Leu Gln Ile Phe

355 360 365

Thr Lys Pro Val Gly Asp Arg Pro Thr Leu Phe Leu Glu Met Ile Gln

370 375 380

Arg Ile Gly Cys Met Glu Lys Asp Glu Arg Gly Glu Glu Tyr Gln Lys

385 390 395 400

Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys

405 410 415

Ser Ile Glu Asp Tyr Glu Lys Ser Leu Glu Ala Lys Gln Ser Ala Ala

420 425 430

Val Gln Ala Ser

435

<210> 6

<211> 436

<212> PRT

<213> Wheat (Triticum aestivum L.)

<400> 6

Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Gly Ala Ala

1 5 10 15

Ala Ala Val Thr Pro Glu His Ala Arg Pro Arg Arg Met Val Arg Phe

20 25 30

Asn Pro Arg Ser Asp Arg Phe His Thr Leu Ser Phe His His Val Glu

35 40 45

Phe Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala

50 55 60

Leu Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser

65 70 75 80

Val His Ala Ser Gln Leu Leu Arg Ser Gly Asn Leu Ala Phe Leu Phe

85 90 95

Thr Ala Pro Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser Leu Pro

100 105 110

Ser Phe Ser Ala Asp Ala Ala Arg Arg Phe Ser Ala Asp His Gly Leu

115 120 125

Ala Val Arg Ser Ile Ala Leu Arg Val Ala Asp Ala Ala Glu Ala Phe

130 135 140

Arg Ala Ser Val Asp Gly Gly Ala Arg Pro Ala Phe Ser Pro Val Asp

145 150 155 160

Leu Gly Arg Gly Phe Gly Phe Ala Glu Val Glu Leu Tyr Gly Asp Val

165 170 175

Val Leu Arg Phe Val Ser His Pro Asp Gly Thr Asp Val Pro Phe Leu

180 185 190

Pro Gly Phe Glu Gly Val Ser Asn Pro Gly Ala Val Asp Tyr Gly Leu

195 200 205

Thr Arg Phe Asp His Val Val Gly Asn Val Pro Glu Leu Ala Ser Ala

210 215 220

Ala Ala Tyr Val Ala Gly Phe Thr Gly Phe His Glu Phe Ala Glu Phe

225 230 235 240

Thr Thr Glu Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Met Val

245 250 255

Leu Ala Asn Asn Ser Glu Gly Val Leu Leu Pro Leu Asn Glu Pro Val

260 265 270

His Gly Thr Lys Arg Arg Ser Gln Ile Gln Thr Phe Leu Glu His His

275 280 285

Gly Gly Pro Gly Val Gln His Ile Ala Val Ala Ser Ser Asp Val Leu

290 295 300

Arg Thr Leu Arg Glu Met Arg Ala Arg Ser Ala Met Gly Gly Phe Asp

305 310 315 320

Phe Leu Pro Pro Pro Leu Pro Lys Tyr Tyr Glu Gly Val Arg Arg Ile

325 330 335

Ala Gly Asp Val Leu Ser Glu Ala Gln Ile Lys Glu Cys Gln Glu Leu

340 345 350

Gly Val Leu Val Asp Arg Asp Asp Gln Gly Val Leu Leu Gln Ile Phe

355 360 365

Thr Lys Pro Val Gly Asp Arg Pro Thr Leu Phe Leu Glu Met Ile Gln

370 375 380

Arg Ile Gly Cys Met Glu Lys Asp Glu Arg Gly Glu Glu Tyr Gln Lys

385 390 395 400

Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys

405 410 415

Ser Ile Glu Asp Tyr Glu Lys Ser Leu Glu Ala Lys Gln Ser Ala Ala

420 425 430

Val Gln Gly Ser

435

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 7

atgccgcccacccccacc 18

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 8

atccctgaac tgcagcagat tg 22

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 9

cagtagggga caggccga 18

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 10

ccgcatctta caaacaacat ca 22

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 11

tggtgtcatc aagcctggta tggt 24

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 12

actcatggtg catctcaacg gact 24

<210> 13

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 13

tgctctagaa tgccgcccac ccccacc 27

<210> 14

<211> 51

<212> DNA

<213> Artificial Sequence

<400> 14

cggggtaccg tggtggtggt ggtggtgtga tccctgaact gcagcagatt g 51

Claims (4)

1. The application of the 2, 5-dihydroxyphenylacetic acid HGA in inhibiting the germination of wheat or corn rust spores is characterized in that the related genes for synthesizing the HGA areTaHPD，The geneTaHPDThere are three homologous copies on the 6A,6B and 6D chromosomes, the homologous copies of the geneTaHPD-6A、TaHPD-6B、TaHPD-The nucleotide sequences of the 6D are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
2. The application of 2.2,5-dihydroxyphenylacetic acid HGA as wheat or corn stripe rust preventing and treating agent is characterized in that the related gene for synthesizing the HGA isTaHPD，The geneTaHPDThere are three homologous copies on the 6A,6B and 6D chromosomes, the homologous copies of the geneTaHPD-6A、TaHPD-6B、TaHPD-The nucleotide sequences of the 6D are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
3. 3. A method for preventing and treating wheat stripe rust disease is characterized in that 2, 5-dihydroxyphenylacetic acid HGA solution is sprayed on wheat leaves, and the concentration of the HGA solution is 1-10mmol/L.
4. 4. The method for preventing and treating corn stripe rust disease is characterized in that 2, 5-dihydroxyphenylacetic acid HGA solution is sprayed on corn leaves, and the concentration of the HGA solution is 1-20mmol/L.