Primer group for detecting fusarium graminearum and growth and development stages of fusarium graminearum and application of primer group

Abstract

The invention discloses a primer group for detecting fusarium graminearum and a growth and development stage of the fusarium graminearum and application of the primer group, and belongs to the technical field of biology. The primer group comprises an upstream primer 05737-F as shown in SEQ ID NO. 2, and a downstream primer 05737-R as shown in SEQ ID NO. 3. The primer group is characterized in that the primer group comprises an upstream primer 05737-F as shown in SEQ ID NO. A pair of special primer sequences for detecting the gene is designed by utilizing the FGSG05737 gene, and the fusarium graminearum can be identified through PCR (Polymerase Chain Reaction). According to different expression quantities of FGSG05737 in different growth stages of fusarium graminearum, RT-qPCR experiments are combined to define the hypha stage and the spore stage of fusarium graminearum, semi-quantitative detection of the number of conidia of fusarium graminearum can be achieved, the growth and development stage and the infection process of fusarium graminearum can be predicted, and the effect of giving out disease early warning in advance is achieved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a primer set for detecting Fusarium graminearum and its growth and development stages and an application thereof. Background Art

[0002] Fusarium graminearum is a filamentous fungus that can infect a variety of crops, including wheat, corn, rice, barley, oats, rye, etc., causing Fusarium diseases such as wheat fusarium head blight, corn ear rot and stem rot, and rice ear rot, which not only cause a reduction in grain crop yields, but also produce toxic secondary metabolites, such as deoxynivalenol (DON) of the trichothecenes. DON is highly toxic to both humans and animals, and can cause vomiting, diarrhea, nervous disorders, miscarriage, stillbirth and other adverse symptoms in humans and animals, causing harm to human and animal health. Therefore, scientific and green prevention and control of Fusarium diseases is of great significance to ensuring food security.

[0003] Rapid detection of Fusarium in the field is of great significance for the prevention and control of Fusarium disease epidemics. Therefore, the establishment of a rapid detection system for Fusarium graminearum is a prerequisite for the precise prevention and control of Fusarium disease. Conidia are the main source of infection for Fusarium disease. Quantitative detection of the number of conidia of Fusarium graminearum is also of great significance for predicting the trend of disease epidemics and field pesticide application. However, existing Fusarium detection technologies mostly use morphological and biochemical identification methods, which are time-consuming and labor-intensive, and cannot accurately determine the number of conidia of Fusarium, making it difficult to predict the trend of Fusarium disease epidemics. Summary of the invention

[0004] The purpose of the present invention is to provide a primer set for detecting Fusarium graminearum and its growth and development stage and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention uses the FGSG_05737 gene to design a pair of primer sequences specifically for detecting the gene, and the identification of Fusarium graminearum can be achieved through PCR reaction. According to the different expression levels of FGSG_05737 in different growth stages of Fusarium graminearum, combined with RT-qPCR experiments, the hyphae stage and spore stage of Fusarium graminearum are defined, and the number of conidia of Fusarium graminearum can be semi-quantitatively detected, and the growth and development stage and infection process of Fusarium graminearum can be predicted, so as to achieve the effect of issuing an early warning of the epidemic.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a primer set for detecting Fusarium graminearum. The primer set comprises an upstream primer 05737-F as shown in SEQ ID NO.2 and a downstream primer 05737-R as shown in SEQ ID NO.3.

[0007] The present invention also provides the use of the primer set in preparing a reagent or a kit for detecting Fusarium graminearum.

[0008] The present invention also provides a kit for detecting Fusarium graminearum, wherein the kit comprises the primer set.

[0009] The present invention also provides a method for detecting Fusarium graminearum, comprising the following steps:

[0010] The DNA of the sample to be tested is used as a template, and the above primer set is used for PCR amplification. The amplified product is subjected to gel electrophoresis to observe whether there are bands. If there are bands, it indicates the presence of Fusarium graminearum, otherwise, it indicates the absence of Fusarium graminearum.

[0011] Furthermore, the reaction system of the PCR amplification is: 1 μL of DNA template, 10 μL of 2×Rapid Taq Master Mix, 0.6 μL of upstream and downstream primers, 7.8 μL of ddH2O; the final concentration of the upstream and downstream primers is 0.3 μM;

[0012] The reaction procedure of the PCR amplification was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 4 s, 35 cycles, and finally extension at 72°C for 5 min.

[0013] The present invention also provides the use of the primer set in preparing a reagent or a kit for determining the growth and development stage of Fusarium graminearum.

[0014] The present invention also provides a kit for determining the growth and development stage of Fusarium graminearum, wherein the kit comprises the primer set.

[0015] The present invention also provides a method for determining the growth and development stage of Fusarium graminearum, comprising the following steps:

[0016] The cDNA of the sample to be tested was used as a template, and the above primer set was used for RT-qPCR reaction. Actin gene was used as an internal reference gene. -ΔΔCT Methods: The relative expression level of the gene amplified by the primer set was calculated; the higher the relative expression level, the greater the number of conidia of Fusarium graminearum.

[0017] Furthermore, the primer set for the internal reference gene includes an upstream primer Actin-F as shown in SEQ ID NO.4 and a downstream primer Actin-R as shown in SEQ ID NO.5.

[0018] Further, the reaction system of the RT-qPCR reaction is: 5 μL of cDNA, 0.6 μL of the primer set, 0.6 μL of the primer set of the internal reference gene, 10 μL of 2×RealStar Fast SYBR qPCR Mix, and 3.8 μL of ddH2O; the final concentrations of the upstream and downstream primers in the primer set are both 0.3 μM, and the final concentrations of the upstream and downstream primers in the primer set of the internal reference gene are both 0.3 μM;

[0019] The reaction procedure of the RT-qPCR reaction is: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s; annealing / extension at 60°C for 30 s; and 40 cycles.

[0020] The present invention discloses the following technical effects:

[0021] The present invention utilizes the amino acid sequence differences of the FGSG_05737 gene in multiple fungal species, designs a pair of primer sequences specifically detecting the gene, and realizes the identification of Fusarium graminearum through PCR reaction. According to the difference in the expression of FGSG_05737 in different growth stages of Fusarium graminearum, combined with RT-qPCR experiments, the mycelial stage and spore stage of Fusarium graminearum are defined, and the number of conidia of Fusarium graminearum can be semi-quantitatively detected, the infection process of Fusarium graminearum can be predicted, and the effect of issuing an early warning of the epidemic can be achieved. The primer set and the detection method of the present invention provide theoretical support for the identification of Fusarium graminearum and the anticipation and prevention of Fusarium diseases, which is of great significance for the scientific and precise prevention and control of Fusarium diseases in the field. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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 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.

[0023] Figure 1 is the position of primer 05737-F and primer 05737-R on the FGSG_05737 gene of Fusarium graminearum;

[0024] Figure 2 The gel electrophoresis diagrams after amplification of the three fungal DNAs in the specificity verification; among them, lanes 1-3 are the amplification bands of Actin-F / Actin-R primers, and lanes 4-6 are the amplification bands of 05737-F / 05737-R primers;

[0025] Figure 3 The results of the sensitivity test of primers 05737-F and 05737-R are shown;

[0026] Figure 4 The expression levels of FGSG_05737 gene in different cDNA templates in the simulation experiment of Fusarium graminearum at different growth stages. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The strains involved in the embodiments of the present invention are: wild-type strain PH-1 of Fusarium graminearum, wild-type strain Guy11 of Magnaporthe oryzae, and wild-type strain B05.10 of Botrytis cinerea.

[0033] Example 1

[0034] This embodiment designs a pair of specific primers for Fusarium graminearum and a detection method thereof for rapid detection of Fusarium graminearum.

[0035] Primers were designed based on the FGSG_05737 gene sequence of Fusarium graminearum in the fungal database (https: / / fungidb.org / fungidb / app / search?q=FGSG_05737). The protein encoded by this gene (amino acid sequence is shown in SEQID NO.1) contains an RNA recognition motif (RRM), which binds to RNA through one or more RRMs, thereby affecting RNA synthesis, shearing, degradation and other processes. Based on the differences in the sequences of this gene in different fungi, specific primers 05737-F and 05737-R for Fusarium graminearum were designed. The specific sequence information is shown in Table 1, and the position of the primers on the FGSG_05737 gene of Fusarium graminearum is shown in Table 1. Figure 1 At the same time, a set of internal reference gene primers Actin-F and Actin-R were designed, and the specific sequence information is shown in Table 1. The above primer pairs were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0036] SEQ ID NO.1:

[0037] LSSSAASCSSHFEHLSFCFVPTLIMDMNPGFSGVPANNGAPSGPMAYSNGTPPAPDMGGVAPPPGSGPDSPKTTLWMGELEPWMDENFIKGVFLSSAHENVNVKVIRDK NSGNAGYCFVEFQSPEAATKALNMNGSQVPNSSRSFKLNWASGGGLVDRRDDRGPEYSIFVGDLGPEVNEYVLVSLFQARFPSCKSAKIMTDAMSGQSRGYGFVRFSDE NDQQRALVEMQGVYCGNRPMRISTATPKNRGNHGFGGQGHQHGGPMMPGMPQQQMWNGGGMQGFPYGGFNPATQMNQFTDPNNTTVFVGGLSGYVTEDELRSFFQGFGE ITYVKIPPGKGCGFVQFVHRHAAEMAAINQMQGYPIGNSRVRLSWGRSQNNSGVGTPYRPAPPPPHYMGMPSHGGPGNYGPQHFGGPAPGPQGPPGPPPGPGPNGPPQEY.

[0038] Table 1 Primer sequences

[0039] Primer name sequence 05737-F CTGTCAAGCTCTGCAGCATCT(SEQ ID NO.2) 05737-R GGAGTAGGCCATAGGGCCAGA(SEQ ID NO.3) Actin-F GTCTTGAGAGCGGTGGTATC(SEQ ID NO.4) Actin-R GCAATGATCTTGACCTTCAT(SEQ ID NO.5)

[0040] The primers designed in Table 1 were specifically verified and tested for sensitivity using DNA of wild-type strain PH-1 of Fusarium graminearum, wild-type strain Guy11 of Magnaporthe oryzae, and wild-type strain B05.10 of Botrytis cinerea as templates:

[0041] 1. Specificity verification:

[0042] Extract fungal DNA: scrape the hyphae cultured on the PDA plate, put it into a 2mL centrifuge tube, add 500μLCTAB extraction solution and add a small steel ball to grind and shake in a tissue grinder, set the grinding frequency to 30 times / sec and run for 1min; warm in a 65℃ oven for half an hour, during which time turn the centrifuge tube upside down every 10min to mix the solution; cool the centrifuge tube to 25℃ in a fume hood, add 500μL chloroform and mix it upside down, and centrifuge it at 12000rpm in a normal temperature centrifuge for 10min; transfer the supernatant to a new 1.5mL centrifuge tube, add an equal volume of pre-cooled isopropanol, mix it upside down and place it at -20℃ for more than half an hour; centrifuge it at 12000rpm in a normal temperature centrifuge for 10min, a small amount of precipitation will be observed, and the supernatant is removed; wash the precipitate with 75% alcohol, centrifuge it at 12000rpm at room temperature for 8min, and discard the supernatant. Repeat this step twice; centrifuge the tube briefly and carefully remove excess liquid with a pipette; place the tube in a 50°C oven or clean bench until the alcohol is completely removed; add 100 μL of sterile water to the tube containing DNA to dissolve the precipitate and store at -20°C.

[0043] The three fungal DNAs extracted were used as templates, and the template concentration was 1 ng / μL. First, Actin-F / Actin-R primers were used for PCR reaction. The PCR reaction system was 20 μL: 1 μL DNA template, 10 μL 2×Rapid Taq Master Mix (Vazyme), 0.6 μL upstream and downstream primers (final concentrations were 0.3 μM respectively), and 7.8 μL ddH2O. Reaction conditions: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 4 s, 35 cycles, and finally 72°C extension for 5 min. After electrophoresis of the PCR product with 1.5% agarose in 1×TAE buffer, the sample was directly spotted and photographed. The appearance of DNA bands indicated that the DNA extraction was successful. After confirming that the DNA extraction was successful, the PCR reaction was carried out with 05737-F / 05737-R primers using the three fungal DNAs extracted as templates. The reaction system and reaction conditions were the same as above. The PCR product was electrophoresed with 1.5% agarose in 1×TAE buffer and photographed directly. Figure 2, wherein lanes 1-3 are amplified bands of Actin-F / Actin-R primers, and lanes 4-6 are amplified bands of 05737-F / 05737-R primers. It can be seen that the DNA of the three fungi was successfully extracted, but only Fusarium graminearum could be amplified with DNA bands by 05737-F / 05737-R primers, while rice blast fungus and gray mold could not be amplified. This shows that the 05737-F / 05737-R primers designed by the present invention have strong specificity.

[0044] 2. Sensitivity detection:

[0045] After the concentration of DNA extracted from the wild-type strain PH-1 of Fusarium graminearum was measured, a series of DNA samples with concentrations of 0.0005ng / μL, 0.005ng / μL, 0.05ng / μL, 0.5ng / μL, 2.5ng / μL, and 5ng / μL were obtained through gradient dilution. The DNA samples with this series of concentrations were used as templates and 05737-F / 05737-R primers were used for PCR reaction. The reaction system and reaction conditions were the same as those in Example 1. After the PCR product was electrophoresed with 1.5% agarose in 1×TAE buffer, it was directly spotted and photographed to observe whether there were bands. The results are as follows: Figure 3 As shown, when the DNA concentration is 0.5 ng / μL, an amplified band can be observed; when the concentration is lower than 0.5 ng / μL, no amplified band is observed, indicating that the 05737-F / 05737-R primers and detection method of the present invention have high sensitivity.

[0046] Example 2

[0047] This embodiment designs a method for detecting the number of conidia to infer the extent of disease occurrence and prevalence.

[0048] Preparation of mycelium and spores of Fusarium graminearum: The wild-type strain PH-1 of Fusarium graminearum was inoculated into YEPD medium and shaken for 24 hours to culture mycelium; the mycelium was transferred to CMC medium and shaken for 5 days to produce conidia. The formula of YEPD medium is: Peptone 10g / L, Yeast extract 3g / L, glucose 20g / L, and autoclaved at 121℃ for 15min. The formula of CMC medium is: (NH4)2SO40.825 g / L, KH2PO41 g / L, Yeast extract 1g / L, MgSO4·7H2O 0.5g / L, and sterilized at 121℃ for 15min.

[0049] Extraction of mycelium and spore RNA: Take mycelium and spore samples of Fusarium graminearum in a mortar, add liquid nitrogen, and grind into powder; aspirate 1 mL of supernatant, transfer to a new 1.5 mL RNase-free centrifuge tube, add 300 μL of chloroform and vortex to mix; at room temperature, mix upside down for 3 minutes, and centrifuge at 4°C and 12000 rpm for 10 minutes; aspirate 600 μL of supernatant and transfer to a new 1.5 mL RNase-free centrifuge tube, add an equal volume of chloroform: isoamyl alcohol (24:1) mixed solution, vortex to mix, and centrifuge at 4°C and 12000 rpm for 10 minutes; take 450 μL of supernatant and transfer to a new 1.5 mL Add an equal volume of pre-cooled isopropanol to the RNase-free centrifuge tube; incubate at -20°C for 20 minutes, centrifuge at 4°C, 12,000 rpm for 15 minutes, and discard the supernatant; add 75% ethanol to wash the precipitate, centrifuge at 4°C, 12,000 rpm for 10 minutes, and discard the supernatant. Repeat this step twice; place the centrifuge tube in a room temperature centrifuge at 12,000 rpm for 1 minute, and carefully remove excess liquid with a pipette; open the centrifuge tube until the alcohol is completely removed, and add 40 μL RNase-free H2O. After gently blowing the precipitate, leave it until the precipitate is completely dissolved, and RNA is obtained. After measuring the concentration, store it at -80°C.

[0050] Preparation of mycelium and spore cDNA: cDNA was obtained by reverse transcription using the Novozyme HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper). First, add 2μL 4×gDNAwiper Mix, 1μg RNA, and RNase-free H2O to 16μL in an RNase-free PCR tube. Mix by pipetting, set the PCR instrument to 42℃2min, and perform the first step of PCR; then, add 4μL 5×HiScript III qRT Supermix to the first step of PCR tube, gently pipet and mix, set the PCR instrument to 37℃15min, 85℃5s, and obtain cDNA.

[0051] The prepared mycelium and spore cDNA of Fusarium graminearum were used as templates, and primers Actin-F / Actin-R, 05737-F / 05737-R were used for RT-qPCR reaction. In order to simulate the different growth stages of Fusarium graminearum, the mycelium and spore cDNA of Fusarium graminearum were mixed in different volume ratios (2:1, 1:1, 1:2). In this experiment, a total of 5 types of cDNA templates were set up, namely mycelium (YEPD), mycelium: spore = 2:1 (2Y1C), mycelium: spore = 1:1 (1Y1C), mycelium: spore = 1:2 (1Y2C), and spore (CMC). The 20μL reaction system was: cDNA 5μL, two primers 0.6μL each (the final concentration of each primer was 0.3μM), 2×RealStar Fast SYBR qPCR Mix 10μL, ddH2O 3.8μL. The reaction conditions were: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s; annealing / extension at 60°C for 30 s; and 40 cycles. The relative expression level of FGSG_05737 at each stage was calculated using 2 -ΔΔCT Method calculation, the internal reference gene is Actin. The relative expression level of FGSG_05737 of each cDNA template type was counted.

[0052] Results Figure 4 It can be seen that under the same cNDA template concentration, the expression of FGSG_05737 gene in spores is higher than that in hyphae. As the spore content in the template increases, the expression level of FGSG_05737 also increases, thereby realizing the detection of different growth stages of Fusarium graminearum. When the expression level of FGSG_05737 is high, it means that the number of spores is large, the infection source is large, and the fusarium spore disease is prone to large-scale epidemics. Early warning of Fusarium graminearum disease can be achieved, and then prevention and control can be carried out in advance.

[0053] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer set for detecting Fusarium graminearum, characterized in that: The primer set includes an upstream primer 05737-F as shown in SEQ ID NO.2 and a downstream primer 05737-R as shown in SEQ ID NO.

3.

2. Use of the primer set according to claim 1 in preparing a reagent or a kit for detecting Fusarium graminearum.

3. A kit for detecting Fusarium graminearum, characterized in that: The kit comprises the primer set according to claim 1.

4. A method for detecting Fusarium graminearum, characterized in that: The steps include: Using the DNA of the sample to be tested as a template, the primer set described in claim 1 is used for PCR amplification, and the amplified product is subjected to gel electrophoresis to detect whether there are bands; if there are bands, it indicates the presence of Fusarium graminearum, otherwise, it indicates the absence of Fusarium graminearum.

5. The method according to claim 4, characterized in that The reaction system of the PCR amplification is: 1 μL DNA template, 10 μL 2×Rapid Taq Master Mix, 0.6 μL upstream and downstream primers, 7.8 μL ddH2O; the final concentration of the upstream and downstream primers is 0.3 μM; The reaction procedure of the PCR amplification was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 4 s, 35 cycles, and finally extension at 72°C for 5 min.

6. Use of the primer set according to claim 1 in preparing a reagent or a kit for determining the growth and development stage of Fusarium graminearum.

7. A kit for determining the growth and development stage of Fusarium graminearum, characterized in that: The kit comprises the primer set according to claim 1.

8. A method for determining the growth and development stage of Fusarium graminearum, characterized in that: The steps include: The cDNA of the sample to be tested is used as a template, the primer set of claim 1 is used to perform RT-qPCR reaction, the Actin gene is used as an internal reference gene, and 2 -ΔΔCT Method: Calculate the relative expression of the gene amplified by the primer set; The higher the relative expression level, the more conidia there are of Fusarium graminearum.

9. The method according to claim 8, characterized in that The primer set for the internal reference gene includes an upstream primer Actin-F as shown in SEQ ID NO.4 and a downstream primer Actin-R as shown in SEQ ID NO.

5.

10. The method according to claim 8 or 9, characterized in that: The reaction system of the RT-qPCR reaction is: 5 μL of cDNA, 0.6 μL of the primer set of claim 1, 0.6 μL of the primer set of the internal reference gene, 10 μL of 2×RealStar FastSYBR qPCR Mix, and 3.8 μL of ddH2O; the final concentrations of the upstream and downstream primers in the primer set of claim 1 are both 0.3 μM, and the final concentrations of the upstream and downstream primers in the primer set of the internal reference gene are both 0.3 μM; The reaction procedure of the RT-qPCR reaction is: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s; annealing / extension at 60°C for 30 s; and 40 cycles.

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