PCR (Polymerase Chain Reaction) primer and method for specifically detecting peronospora sojae and application of PCR primer and method

Abstract

The invention belongs to the technical field of biology, and relates to a PCR primer and method for specifically detecting peronospora sojae and application of the PCR primer and the method, and the specific PCR primer is WS180F and WS180R; the nucleotide sequence of the WS180F is as shown in SEQ ID NO. 1; the nucleotide sequence of the WS180R is as shown in SEQ ID NO. 2. According to the present invention, the sequence comparison is performed according to the ITS sequence of Perospora manshurica and the ITS sequences of nine different bacterial genus such as Chrysozyma flavifolia, Colacogloea rhododendri, Glaciozyma litoria and the like in GenBank, such that the specific primers WS180F and WS180R are designed, and the specific primers WS180F and WS180R can be used for the detection of the bacterial genus of the Perospora manshurica and the specific primers WS180R can be used for the detection of the bacterial genus of the Perospora manshurica and the bacterial genus of the Perospora manshurica. The PCR primer has the advantages of strong specificity, high sensitivity and capability of quickly and accurately detecting soybean leaves carrying peronospora sojae, and has great significance in prevention and treatment of soybean downy mildew.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a PCR primer, a method and an application thereof for specifically detecting soybean downy mildew. Background Art

[0002] The pathogen of soybean downy mildew is Peronospora manchurianensis ( Peronospora manschurica (Naum.) Syd.), belongs to Oomycetes, Peronosporales. The sporangium stalks of the pathogen extend from the stomata, solitary or clustered, colorless, gray to light purple, and cross-branched. The size is 240 microns to 424 microns long and 6 microns to 10 microns wide, with 3 to 4 dichotomous branches at the top. The main branches are symmetrical, curved or slightly curved, without septa, and the twigs are at right angles or acute angles. The tip of the last twig is sharper, and each twig has a sporangium. The sporangium is colorless, transparent or light gray-purple, single-celled, elliptical, 14 microns to 26 microns long, 14 microns to 20 microns wide, and without papillae. The oospores are yellow-brown, nearly spherical, thick-walled, and contain one ovum with a diameter of 29 to 50 microns. The oospore is irregular and light yellow.

[0003] Soybean downy mildew, also known as Downy mildew, is caused by the northeastern downy mildew fungus [ Peronospora manshurica A fungal disease caused by Soybean (Naum.) Syd. Ex Gaum) mainly harms soybean seedlings, leaves, pods and grains, causing leaves to fall off or wilt prematurely, seeds to become damaged and mildewed, and generally diseased fields can reduce yields by 6% to 15%, with a seed damage rate of about 10%, and severely diseased fields reducing yields by 30% to 50%; the germination rate of diseased seeds drops by more than 10%, the oil content decreases by 0.6% to 1.7%, and the oil yield decreases by 2.7% to 7.6%, posing a great threat to soybean yield and quality. The disease can occur throughout the entire growth period of soybeans, especially in areas with lower temperatures and higher humidity, and is more common and serious. It has now become a potential epidemic disease.

[0004] The classification and identification of soybean downy mildew are mainly based on morphological characteristics and pathogenicity determination, which are time-consuming, low-sensitivity and highly empirical. It often takes several days to isolate and identify pathogens from diseased plants, which makes it difficult to monitor the occurrence of diseases in a timely manner and to effectively control the spread of pathogens and the prevalence of diseases. Therefore, it is extremely important to simply and quickly detect the presence and degree of downy mildew infection in soybean samples for evaluating the severity of the disease and the resistance to soybean downy mildew. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a PCR primer, method and application thereof for specifically detecting soybean downy mildew.

[0006] The present invention is achieved through the following technical solutions: A PCR primer for specifically detecting soybean downy mildew, the specific PCR primer comprises WS180F and WS180R; the nucleotide sequence of the WS180F is shown in SEQ ID NO.1; the nucleotide sequence of the WS180R is shown in SEQ ID NO.2.

[0007] The PCR primers are used in the specific detection of soybean downy mildew.

[0008] Preferably, the specific detection object is soybean leaves.

[0009] A method for specifically detecting soybean downy mildew uses the PCR primers for detection.

[0010] Preferably, the specific steps include: (1) Extract DNA from soybean leaf samples.

[0011] (2) Using the sample DNA extracted in step (1) as a template, PCR amplification is performed on the extracted sample DNA using the PCR primers WS180F and WS180R.

[0012] (3) The results were determined based on the amplified band. When the size of the amplified band was 536 bp, it was determined that soybean downy mildew was present; otherwise, soybean downy mildew was not present.

[0013] Preferably, the concentration of the sample DNA is 10-100 ng / μL.

[0014] Preferably, the reaction system for PCR amplification is 25 μL: 2×Premix Taq™ Mix 12.5 μL, WS180F and WS180R primers 0.5 μL each, DNA 1 μL, and sterile ultrapure water 10.5 μL.

[0015] Preferably, the reaction conditions for the PCR amplification are: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 30 cycles; and extension at 72°C for 10 min.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a PCR primer for specifically detecting soybean downy mildew, wherein the specific PCR primer comprises WS180F and WS180R; the nucleotide sequence of the WS180F is shown in SEQ ID NO.1; the nucleotide sequence of the WS180R is shown in SEQ ID NO.2. Peronospora manshurica The ITS sequence and Chrysozyma flava, Colacogloea rhododendri and Glaciozyma litoralis By comparing the ITS sequences of 9 different fungi, we designed the WS180F and WS180R primers, which have strong specificity and high sensitivity. They can quickly detect whether soybean leaf samples are infected by soybean downy mildew, which is of great significance for the prevention and control of soybean downy mildew. At the same time, according to the pathogenicity law, by detecting the relative amount of soybean downy mildew genome components on leaves, the severity of downy mildew can be judged, which can be used for resistance evaluation and provide a reference for the screening of disease-resistant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 Electrophoresis diagram of soybean downy mildew DNA amplification at different concentrations of the present invention. Wherein, M is DNA Marker II, 1 is negative control, ddH 2 O, 2 are the host normal soybean leaf DNA, and 3~8 are the lanes of soybean downy mildew leaf DNA amplification at 0.1ng / μL, 1ng / μL, 10ng / μL, 25ng / μL, 50ng / μL, and 100ng / μL, respectively.

[0019] Figure 2 The electrophoresis diagram of the amplification of soybean downy mildew DNA and 5 outgroup pathogen DNA. Wherein, M is DNA Marker II, 1 is negative control, ddH 2 O, 2~4 are the DNA of soybean downy mildew leaves, and 5~9 are the lanes for amplification of DNA of 5 outgroup pathogens.

[0020] Figure 3 The electrophoresis diagram of soybean downy mildew DNA amplification from different regions of the present invention. Wherein, M is DNA Marker II, 1 is negative control, ddH 2 Lanes 0, 2~24 are DNA amplifications of 23 different soybean downy mildew leaves.

[0021] Figure 4 The electrophoresis diagram of DNA amplification of soybean downy mildew with different resistance levels in the present invention. Wherein, M is DNA Marker II, 1 is negative control, ddH 2O, 2, and 9 are level 0; 8 and 20 are level 1; 3, 11, 14, and 21 are level 3; 5, 10, 15, and 22 are level 5; 4, 7, 16, 19, and 23 are level 7; 6, 12, 13, 17, 18, and 24 are level 9 lanes for DNA amplification of soybean downy mildew leaves.

[0022] Figure 5 The specific primers designed by the present invention are compared with the specific primers Pm1 and Pm2 for amplification electrophoresis. 2 0, 2-5 are lanes for amplifying DNA from soybean downy mildew leaves using the specific primers Pm1 and Pm2, and lanes 6-9 are lanes for amplifying DNA from soybean downy mildew leaves using the specific primers designed by the present invention. DETAILED DESCRIPTION

[0023] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0025] The inventive concept of the present invention is as follows: Soybean downy mildew is a fungal disease caused by Peronospora mandshurica. It can occur throughout the entire growth period of soybeans, leading to reduced soybean yields and quality. It is particularly common and serious in low-temperature and high-humidity areas, and has become a potential epidemic disease. Traditional soybean downy mildew classification and identification are mainly based on morphological characteristics and pathogenicity determination, which are time-consuming, low-sensitivity, and highly empirical, making it difficult to achieve timely monitoring of the disease and effectively control the spread of pathogens and the prevalence of the disease. Therefore, there is an urgent need for a simple and rapid method to detect the presence of downy mildew infection and the degree of infection in soybean samples, which can be used to evaluate the severity of the disease and the resistance of soybeans to downy mildew.

[0026] Based on this, the present invention provides a PCR primer for specifically detecting soybean downy mildew, wherein the specific PCR primers include WS180F and WS180R; the nucleotide sequence of the WS180F is shown in SEQ ID NO.1; the nucleotide sequence of the WS180R is shown in SEQ ID NO.2. Peronospora manshurica ) with the ITS sequence Chrysozyma flava, Colacogloea rhododendri and Glaciozyma litoralis The ITS sequences of 9 different bacterial genera were aligned using clustalx1.8, and then the specific primers WS180F / WS180R were designed using Oligo 6.0 software.

[0027] PCR amplification of soybean leaf sample DNA was performed using the designed specific primers WS180F and WS180R. The primers can bind to specific regions of soybean downy mildew DNA and amplify under appropriate reaction systems and conditions. The PCR amplification products were detected by agarose gel electrophoresis. If a positive amplification band appeared, it indicated that soybean downy mildew was present in the sample, otherwise soybean downy mildew was not present. In addition, since the content of pathogens in the leaves of soybeans with different resistance levels was different after being infected with soybean downy mildew, the brightness of the PCR amplification bands was also different. Therefore, the resistance to soybean downy mildew can be preliminarily graded and evaluated based on the brightness of the bands.

[0028] Example 1. Design of specific primers and detection of primer sensitivity and specificity 1. Materials and Methods 1.1 Test materials 1.1.1 Primer sensitivity test materials: soybean leaves 14 days after artificial inoculation with soybean downy mildew, as shown in Table 2.

[0029] 1.1.2 Primer specificity detection materials: 3 soybean downy mildew leaves, 1 soybean gray spot pathogen leaf, 1 soybean powdery mildew pathogen leaf, 1 soybean mosaic virus leaf, 1 soybean rust pathogen leaf and 1 soybean root rot pathogen, as shown in Table 3.

[0030] 1.2 Design of specific primers The gene version number is GenBank: DQ202400.1 Peronospora manshurica ITS sequences and GenBank Chrysozyma flava , Colacogloea rhododendri and Glaciozyma litoralis The ITS sequences of 9 different bacterial genera were aligned using clustalx1.8, as shown in Table 1. Chrysozyma flava The gene version number is GenBank: NR_174798.1, Colacogloea rhododendri The gene version number is GenBank: NR_174804.1, Glaciozyma litoralis The gene version number is GenBank: NR_155110.1. Specific primers were designed using Oligo6.0 software. The primer sequences are as follows: The nucleotide sequence of WS180F is shown in SEQ ID NO. 1, which is: 5'-TATCAAGGCAAGTGCTCGGA-3'.

[0031] The nucleotide sequence of WS180R is shown in SEQ ID NO. 2, which is: 5'-GAATGTCCAATAAGCGTCTG-3'.

[0032] Table 1 Latin names of soybean downy mildew and its different genera

[0033] 1.3 DNA extraction The DNA of the test materials was extracted by the modified CTAB method, and the purity and concentration of the obtained DNA were detected by NanoDrop 2000C ultra-micro spectrophotometer. 260 / OD 280 The best ratio is 1.9.

[0034] 1.4 PCR amplification using specific primers WS180F and WS180R The reaction system for PCR amplification was 25 μL: 2×Premix Taq™ Mix 12.5 μL, WS180F and WS180R primers 10 μM each 0.5 μL, 50 ng / μL DNA 1 μL, and ultrapure water 10.5 μL.

[0035] The amplification reaction conditions were as follows: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s; annealing at 58°C for 30 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0036] Take 5 μL of PCR reaction solution and 6 μL of DNA Marker II, the 700 bp band is about 100 ng, and the remaining bands are about 50 ng. Use 1% agarose gel electrophoresis to detect the PCR amplification product. Use a gel imager to observe whether there are target bands, and use gel imaging analysis software to image and analyze the relative amount of the bands.

[0037] 1.5 Primer sensitivity and primer specificity detection DNA was extracted from soybean leaves 14 days after artificial inoculation with soybean downy mildew. 2 0 The DNA template was diluted to 100 ng / μL, 50 ng / μL, 25 ng / μL, 10 ng / μL, 1 ng / μL, and 0.1 ng / μL. The sensitivity of specific primers WS180F and WS180R was detected by PCR amplification and agarose gel electrophoresis, and the optimal DNA concentration for detection was determined.

[0038] The specificity of WS180F and WS180R primers was tested using leaves infected with soybean gray leaf spot, soybean powdery mildew, soybean mosaic virus, soybean rust, soybean root rot, host soybean leaves not infected with the pathogen, and normal soybean leaves.

[0039] 2. Results and Analysis 2.1 Detecting primer sensitivity and determining the optimal DNA concentration Specific primers WS180F and WS180R were used to amplify the DNA of leaves infected with soybean downy mildew at different concentrations. The target bands could be amplified at DNA concentrations of 100ng / μL, 50ng / μL, 25ng / μL, and 10ng / μL. DNA at concentrations of 1ng / μL, 0.1ng / μL, normal soybean leaf DNA from the host, and ddH 2 O could not amplify the target band, as shown in Table 2 and Figure 1 The detection system designed by the present invention has high sensitivity and is not interfered by host DNA. The DNA concentration is between 10ng / μL and 100ng / μL, and soybean downy mildew can be detected.

[0040] Using leaf DNA infected with soybean downy mildew as a template, PCR amplification was performed with specific primers WS180F and WS180R, and the positive amplified band was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results were compared and a positive band of 536 bp was detected in soybean downy mildew.

[0041] Table 2 Amplification and amplified bands of soybean downy mildew DNA at different concentrations

[0042] Note: “+” indicates that the target band was amplified; “-” indicates that the target band was not amplified.

[0043] 2.2 Primer specificity detection The designed specific primers were used to perform PCR amplification on the tested soybean downy mildew DNA and the DNA of five outgroup pathogens. The 25μL PCR amplification system included 1μL of 50ng / μL DNA. The soybean downy mildew DNA was able to amplify bands in the PCR amplification, while the five outgroup pathogen DNAs were unable to amplify bands in the PCR amplification, as shown in Table 3 and Figure 2 As shown, the primers WS180F and WS180R designed in this study have good specificity, stability and reliability, and can well detect soybean downy mildew.

[0044] Table 3 Amplified bands of DNA from soybean downy mildew and five outgroup pathogens

[0045] Note: “+” indicates that the target band was amplified; “-” indicates that the target band was not amplified; “ / ” indicates that there is no such item.

[0046] Comparative Example 1 The specific primers in the paper “Liu Yan, et al. Design and application of molecular probes for rDNA ITS region of soybean downy mildew pathogen, Acta Agriculturae Boreali-Sinica, 2012, 27(2): 230-233” similar to the present invention are as follows: The nucleotide sequence of Pm1 is shown in SEQ ID NO. 3, which is: 5'-TCAGTGAGTCATCGATCATT-3'.

[0047] The nucleotide sequence of Pm2 is shown in SEQ ID NO.4, which is: 5'-CGTAGTCATGTGTGACTCA-3'.

[0048] The results are as follows Figure 5 As shown, the target bands 2 to 5 lanes could not be amplified using Pm1 and Pm2. Figure 5 In the table, M is DNA Marker II, 1 is negative control, ddH 2 0, 2-5 are lanes for amplifying DNA from soybean downy mildew leaves using the specific primers Pm1 and Pm2, and lanes 6-9 are lanes for amplifying DNA from soybean downy mildew leaves using the specific primers designed by the present invention.

[0049] Example 2: Specific application of specific PCR primers in detecting soybean downy mildew 1. Materials and Methods 1.1 Test materials The soybean downy mildew leaves for testing were collected from parts of Jilin Province and Heilongjiang Province, as shown in Table 4.

[0050] 1.2 Test methods The sequences of specific primers WS180F and WS180R were the same as those in Example 1 1.2; DNA extraction was the same as that in Example 1 1.3; and PCR amplification was the same as that in Example 1 1.4.

[0051] 2 Results and analysis 2.2 Accuracy verification of specific primers The specific primers WS180F and WS180R designed in this study were used to detect 23 soybean downy mildew leaves collected from parts of Heilongjiang Province and Jilin Province. Bands were amplified in PCR amplification, as shown in Table 4 and Figure 3 As shown, the detection result was 100%, which further demonstrated that the specific primers designed in this study had high accuracy and could quickly and accurately identify soybean downy mildew.

[0052] Table 4 Collection sites and amplified bands of soybean downy mildew

[0053] Note: “+” indicates that the target band was amplified; “-” indicates that the target band was not amplified; “ / ” indicates that there is no such item.

[0054] Example 3: Using the specific primers to quickly classify the resistance of soybean downy mildew leaves infected with different levels 1. Materials and Methods 1.1 Test materials Table 5 shows the soybean leaves with different resistance levels 14 days after artificial inoculation with soybean downy mildew.

[0055] 1.2 Test methods The sequences of specific primers WS180F and WS180R were the same as those in Example 1 1.2; DNA extraction was the same as that in Example 1 1.3; PCR amplification was the same as that in Example 1 1.4; and the DNA concentration of all tested samples was 50 ng / μL.

[0056] 2 Results and analysis 14 days after artificial inoculation of soybean downy mildew, soybean leaves showing symptoms of level 0, 1, 3, 5, 7 and 9 were selected. The specific primers were used to detect soybean leaves of different disease levels. The results showed that the control ddH 2 No bands were detected in grade 0, and no bands were amplified in the PCR amplification of grade 0 soybean leaf DNA; bands were amplified in grade 1, grade 3, grade 5, grade 7, and grade 9 soybean leaf DNA, and the amplified bands of grade 1 were darker; the amplified bands of grades 3 and 5 were brighter, and the brightness was basically the same; the amplified bands of grades 7 and 9 were brighter, and the brightness was basically the same; the amplified bands of grades 7 and 9 were brighter than the amplified bands of grades 3 and 5, and the brightness of the bands of grades 3 and 5 was brighter than the band of grade 1, as shown in Tables 5 and Figure 4 As shown, it can be seen that with the increase of the susceptibility level, the brightness of the PCR product band also increases, indicating that the specific primers can be used to quickly grade the resistance of soybean downy mildew infected plants.

[0057] Table 5 DNA amplification bands of soybean downy mildew with different resistance levels

[0058] It should be noted that when conducting rapid initial resistance grading of soybean downy mildew leaves infected with different levels, the DNA concentration of all tested samples must remain consistent.

[0059] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0060] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A PCR primer for specific detection of soybean downy mildew, characterized in that: The specific PCR primers include WS180F and WS180R; the nucleotide sequence of WS180F is shown in SEQ ID NO.1; the nucleotide sequence of WS180R is shown in SEQ ID NO.

2.

2. Use of the PCR primers as claimed in claim 1 in the specific detection of soybean downy mildew.

3. The use according to claim 2, characterized in that The specific detection object is soybean leaves.

4. A method for specifically detecting soybean downy mildew, characterized in that: The PCR primers according to claim 1 are used for detection.

5. The method for specific detection of soybean downy mildew according to claim 4, characterized in that: The specific steps include: (1) Extracting DNA from soybean leaf samples; (2) using the sample DNA extracted in step (1) as a template, and using the PCR primers WS180F and WS180R to perform PCR amplification on the extracted sample DNA; (3) The results were determined based on the amplified band. When the size of the amplified band was 536 bp, it was determined that soybean downy mildew was present; otherwise, soybean downy mildew was not present.

6. The method for specific detection of soybean downy mildew according to claim 5, characterized in that: The concentration of the sample DNA is 10-100 ng / μL.

7. The method for specific detection of soybean downy mildew according to claim 5, characterized in that: The reaction system of the PCR amplification is 25 μL: 2×Premix Taq™ Mix 12.5 μL, WS180F and WS180R primers 0.5 μL each, DNA 1 μL, and sterile ultrapure water 10.5 μL.

8. The method for specific detection of soybean downy mildew according to claim 5, characterized in that: The reaction conditions of the PCR amplification are: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 30 cycles; and extension at 72°C for 10 min.

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